Nuclease Activity and Cytotoxicity Enhancement of the DNA

Jul 9, 2012 - In this work, we demonstrate that the nuclease activity and cytotoxicity of the small DNA intercalators can be dramatically enhanced by ...
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Nuclease Activity and Cytotoxicity Enhancement of the DNA Intercalators via Graphene Oxide Bin Zheng,†,§ Chong Wang,†,§ Congyu Wu,‡ Xuejiao Zhou,‡ Min Lin,† Xiaochen Wu,‡ Xiaozhen Xin,† Xin Chen,† Lin Xu,† Hui Liu,† Jing Zheng,† Jingyan Zhang,*,† and Shouwu Guo*,‡ †

School of Pharmacy, State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of New Drug Design, East China University of Science and Technology, Shanghai, 200237. P. R. China ‡ National Key Laboratory of Micro/Nano Fabrication Technology, Key Laboratory of Thin Film and Microfabrication of the Ministry of Education, Research Institute of Micro/Nano Science and Technology, Shanghai Jiao Tong University, Shanghai, 200240, P. R. China S Supporting Information *

ABSTRACT: The enhancement of DNA affinity of small molecules usually ensures their high nuclease activities, and may also open a new scope of their applications in biology and medicine. In this work, we demonstrate that the nuclease activity and cytotoxicity of the small DNA intercalators can be dramatically enhanced by single atomic-layered graphene oxide (GO) sheets. Through π−π stacking interaction mainly between GO and the aromatic ligands of intercalators, the conjugates of GO and a small intercalator could be formed. Because of the large planar structure of the GO sheets, the coupling of GO with the small intercalators increased their affinity to DNA. Owing to the formation of conjugates with GO, the binding site of small intercalators to DNA was also changed from a minor groove to a major groove. Notably, GO and small intercalator conjugates exhibited higher cytotoxicity than that of the small intercalator alone. The results open up potential applications of GO for new chemotherapeutic agents that work through DNA intercalation.

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INTRODUCTION Small molecules that bind DNA with high affinity would be useful in molecular biology and potentially in anticancer therapy. DNA intercalators, bound to DNA molecules by intercalating between the base pairs of the double helix, are one category of extensively studied DNA binding small molecules.1 They can stabilize, unwind, or cleave DNA molecules, and thus affect the normal functions of DNA, which make them potent antibacterials, antibiotics, anticancer drugs, and so forth.2−5 To achieve a high affinity to DNA, most organic intercalators so far discovered have aromatic frameworks.5 For the same purpose, inorganic DNA intercalators also employ aromatic heterocyclic organic compounds as ligands of the metal centers.6−10 To improve the DNA binding affinity and DNA sequence selectivity, synergistic intercalators that link the organic or inorganic intercalators with other functionalities are also developed.5,11 The small molecules with high affinity to DNA often ensure their high activities toward DNA, for instance, DNA cleavage activity.12 In spite of the significant advances, achieving effective intercalators with high affinities toward DNA and practically useful rates of DNA cleavage remains a major challenge. We have previously demonstrated by fluorescence and circular dichroism (CD) spectroscopies that single atomic © 2012 American Chemical Society

layered graphene oxide (GO) sheets, due to their unique structural property,13−15 can interact with DNA molecules through intercalation.16 The sp2-hybridized carbon backbone in GO maintains a high degree of planarity,17 which is much larger than that of most planar organic DNA intercalators or the aromatic ligand of the inorganic DNA intercalators.15,18,19 The advantage endorses GO with a higher affinity for DNA molecules than those of small molecules. This work for the first time extends the classic planar aromatic structures of DNA intercalators from organic molecules to materials with a planar structure. We also found that GO, combining with copper ions through its variety of chemically reactive functionalities on the basal plane, could effectively cleave DNA molecules.16 However, in the GO/Cu2+ system, the concentration of Cu2+ is relatively high in the millimole range, and free diffusible copper ions severally limit their potential applications as therapeutic agents. On the basis of these findings, we envisage that it would be highly advantageous if GO sheets could interact with small molecular intercalators (or intercalating ligands), and conReceived: May 23, 2012 Revised: July 4, 2012 Published: July 9, 2012 15839

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growth curve (pseudo-first-order kinetics), thus Kobs was obtained. The apparent binding constants of the GO/complex conjugates to DNA were obtained by measuring the electronic absorption spectra changes of the compounds in the presence of DNA or GO. The decreases in the intensity of the absorption band of the compounds and compounds/GO in the presence of DNA were analyzed using the equation22,26

sequently improve their binding affinity to DNA. This is essential because it could be a method of improving the efficiency of the clinical extensively used anticancer drugs that work through DNA intercalation.5,12 In this work, we demonstrate conceptually that GO sheets can efficiently improve the DNA cleavage activities of small DNA intercalators through improving their affinity to DNA, using dipyridoquinoxaline-copper [Cu(dpq)2(H2O)](ClO4)2, abbreviated as Cu(dpq)2,20 and its analogues di-1,10-phenanthroline-copper (denoted as Cu(phen)2)21 and dipyridophenazine-copper (denoted as Cu(dppz)2)22 as model complexes of small DNA intercalators. Notably, we found that the cytotoxicities of the complexes were also increased in the presence of GO. These results are essential to our ongoing efforts to understand the mechanism of the GO interaction with DNA, and can possibly be used in improving the performance of the existing anticancer drugs that work through DNA intercalation.

[DNA]/(εa − εf ) = [DNA]/(εb − εf ) + 1/Kb(εb − εf )

where [DNA] is the concentration of DNA in base pairs, εa is the apparent extinction coefficient obtained by calculating A/ [complex], εf corresponds to the extinction coefficient of the complex in its free form, and εb refers to the extinction coefficient of the complex in the bound form. Each set of data, when fit to the above equation, gives a straight line with a slope of 1/(εb − εf) and a y intercept of 1/Kb(εb − εf). Thermal Denaturation Measurements. The thermal denaturation experiments were carried out according to the literature with a Cary 50 spectrophotometer (Varian, USA) connected to a temperature controller.16 The DNA solution (1 μM bp in 5 mM Tris, 50 mM NaCl, pH 7.2 buffer) was prepared, and the increase of the absorbance at 260 nm with an increase of temperature (from 40 to 94 °C at a rate of 0.5 °C per minute) was recorded as a function of temperature. For the sample in the presence of GO and GO/Cu(dpq)2, the DNA was mixed with GO (10 μg mL−1) and Cu(dpq)2 (2 μM), respectively, measured with the same method as that for DNA alone. To compare the changes of Tm for the DNA and Cu(dpq)2 in the presence and absence of GO, the (A − A0)/ (Af − A0) values for both samples were plotted versus the temperature, where A0 and Af are the absorption intensities at 40 and 94 °C, respectively; A is the intensity at a given temperature between 40 and 94 °C. Because the intensity at the total denaturation point for the sample of DNA with GO (Af(DNA + GO)) and DNA with GO/Cu(dpq)2 (Af(DNA +GO+Cu(dpq)2)) was more than 100 °C, and are practically not possible to measure, Af(DNA + GO) and Af(DNA + GO + Cu(dpq)2) were calculated as Af(DNA) − (A0(GO) + A0(DNA)) and Af(DNA) − [A0(DNA + GO) − A0(Cu(dpq)2 + GO)], respectively. In Vitro Cytotoxicity. The effect of the GO on the viability of 239T and MGC-803 cells (purchased from Shanghai Cell Bank of the Chinese Academy of Sciences) in the presence of Cu(dpq)2 was tested using WST-8 assay kit (Beyotime Institute of Biotechnology, China). Cells were plated in a 96 well microplate at a density of ∼5 × 103 cells. Background control wells containing the same volume of complete culture medium were included in each assay. The microplate was incubated for 24 h at 37 °C. Then the cells were washed with serum-free medium and incubated with Cu(dpq)2 and GO at various concentrations, and the plate was incubated further for 24 h. The optical density at 450 nm referenced with the absorption at 655 nm were recorded. The control experiments with cells and GO alone were carried out under the same conditions.

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MATERIALS AND METHODS Materials. The GO and chemically reduced GO sheets were prepared as described in our previous work.16,18,19,23 The aqueous suspension of GO was stored at room temperature on a lab bench and used for characterizations and DNA scission. All chemicals and reagents were purchased from SinoPharm Chemical Reagent Co. Ltd. in analytic grade and were used as received. Supercoiled PSICOR-GFP DNA was purified from a DH5α cell using an EndoFree Plasmid Kit (QIAGEN, USA). The stock solution of the DNA (2 μg μL−1) was prepared. Calf thymus DNA (CT-DNA) was purchased from SinoPharm Chemical Reagent Co. Ltd. Aqueous solutions of the compounds for strand scission experiment were prepared fresh daily and filtered with a 0.22 μm filter before use. Complexes Cu(dpq)2, Cu(phen)2, Cu(dppz)2, (dipyridoquinoxaline)(α-alanine)copper (abbreviated as Cu(dpq)(Ala)), (1,10-phenanthroline)(glycine)copper (denoted as Cu(phen)(Gly)), (dipyridoquinoxaline)(glycine)copper (denoted Cu(dpq)(Gly)), and Cu(dppz)(Gly) were synthesized and characterized following the literature.20,22−25 Instrumentation. IR spectra of GO were recorded on a Perkin-Nicolet FT-IR 200 in the range of 4000−400 cm−1. Samples were run as KBr pellets. Atomic force microscopic (AFM) images of GO were taken on a Nanoscope MultiMode V scanning probe microscopy (SPM) system (Veeco, USA). The scanning rate was usually set at 0.7−1 Hz. The samples for AFM were prepared by dropping aqueous suspension (0.02 mg mL−1) of the GO on a freshly cleaved mica surface and dried under vacuum at 80 °C. Agarose gel electrophoresis was carried out with a DYY-6C electrophoresis apparatus (Liuyi Instrumental Co., China). The agarose gels were visualized and digitized with the FR-200A gel image analysis system and analyzed by SmartView software. The UV−vis measurements were performed on a Cary 50 spectrometer equipped with a Cary temperature controller. A JASCO J815 spectropolarimeter (Jasco International Co. Ltd., Japan) equipped with a Jasco temperature controller (model PTC-423S) and controlled by a PC was used for all CD measurements at 22 °C. Each spectrum was averaged from five successive accumulations at a scan rate of 50 nm min−1. DNA Cleavage Rate and Binding Experiments. The DNA cleavage rate was obtained from the gel electrophoresis. The decrease in intensity of Band I (SC) or the increase in the intensity of Band II (NC) was plotted against reaction time, and these plots were fitted with a single-exponential decay or

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RESULTS AND DISCUSSION Effect of GO on the DNA Cleavage Activity of Cu(dpq)2. The complexes of Cu(dpq)2 and its analogues were chosen because their structures and interactions with DNA are well-understood.20,22,27 The chemical structures of Cu(dpq)2 and its analogues used in this work are shown in Figure S1 (Supporting Information). Three complexes have 15840

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possibly the result of their synergetic interaction. This phenomenon was also observed for the two analogues of Cu(dpq)2. For Cu(phen)2, the maximum cleavage was obtained with the complex-to-GO ratio of about 10 μM to 1 μg mL−1, while for Cu(dppz)2, the ratio was about 20 μM to 1 μg mL−1 (Figure S2). The GO enhancement in DNA cleavage activity of Cu(dpq)2 is also time dependent. Figure 3a displays the effect of GO on the DNA cleavage by Cu(dpq)2 as a function of incubation time. With the time increasing, the cleavage of the supercoiled DNA in the presence of GO was more complete. After 30 min of incubation, 90% of the supercoiled DNA was converted to the nicked DNA. In contrast, under the same condition, in the absence of GO, only about 60% of the supercoiled DNA was cleaved, and no linear DNA was present. Figure 3a also indicates that the DNA cleavage by Cu(dpq)2/GO varies exponentially with incubation time, from which the apparent DNA cleavage rate (Kobs) can be obtained. The concentrations of the Cu(dpq)2 versus the DNA cleavage rates is plotted in Figure 3b (black line). The maximum cleavage rate obtained from the plot (Figure 3b) is 2.48 h−1 with Cu(dpq)2 at a concentration of 150 μM. The Kobs is lower than the value obtained using pUC19 DNA plasmid, possibly because the PSICOR-GFP DNA used in this work is much larger.22 In the presence of GO, when the concentration of Cu(dpq)2 is below 50 μM, the cleavage rates are similar to that of Cu(dpq)2 alone, while the cleavage rate was dramatically increased when the concentration of Cu(dpq)2 was above 50 μM. In the presence of 75 μg mL−1 GO, Kobs reached 11.86 h−1 (Figure 3b, red line), which is significantly higher than that of the most reported metalointercalators.22,29 The greater rate of cleavage indicates that GO can dramatically improve the DNA cleavage activity of Cu(dpq)2. Under the same complexes concentration, the activity order of three complexes with GO remains the same order as that of without GO. It is thus likely that copper complexes and GO together accelerate the rate of DNA cleavage. Interaction between GO and Copper Complexes. To verify the assumption that GO and Cu(dpq)2 work together to cleave DNA, the UV−vis spectra of the Cu(dpq)2 complex in the presence of various amount of GO were recorded and are shown in Figure 3c. The addition of GO to Cu(dpq)2 solution causes the decrease of its absorption intensity at 200, 270, and 300 nm caused by the ligand π−π* and n-π* transitions of Cu(dpq)2 (the dilution of the sample caused by the addition of GO was calibrated).20,23,28 In principle, hypochromism (decrease of the absorption) of an aromatic ligand is commonly associated with the π−π stacking interactions between the molecules themselves or with other complexes.30 Figure S3 depicts that such hypochromisity is also observed for Cu(phen)2 and Cu(dppz)2 in the presence of GO. The observed hypochromisity indicates that the interaction between GO and the complexes is through π−π stacking. Through the titration method, apparent GO binding constants to Cu(dppz)2, Cu(dpq)2, and Cu(phen)2 can be estimated as 1.09, 0.378, and 0.244 mL μg−1, respectively (insets of Figure 3c and Figure S3). The order of binding constants is consistent with the orders of the sizes of planar ligands (Figure S1). Although it is possible the other interaction such as electrostatic interaction also contributes to the interaction between GO and complexes, based on the above results we conclude that the copper complexes and GO sheets together form larger conjugates mainly through π−π stacking interaction. The formed

different sizes of planar aromatic heterocyclic ligands,: phen, dpq, and dppz. They exhibited DNA cleavage activities themselves20,22 or in the presence of oxidant and reducing agent,27,28 or with photo irradiation.27 Additionally, the activity order is Cu(dppz)2 > Cu(dpq)2 > Cu(phen)2, which is consistent with the order of their planar ligand dimensions. To test whether GO can improve the DNA cleavage activity of the metallointercalators, DNA cleavage by Cu(dpq)2 in the presence of GO was first investigated using agarose electrophoresis gel. GO sheets used in this work are single layered, as shown in the AFM image in Figure 1. They were also

Figure 1. AFM image of the single layered GO sheets used in this work. Scale bar is equal to 250 nm.

characterized by IR and TEM as described in our previous work.13−16 We previously have shown that GO alone, under the same conditions, even at 10 times higher concentration, does not affect DNA plasmid, but further increasing GO concentration will lead to some smeared bands and the portion of the sample dwelling in the sample well in the gel.16 Figure 2

Figure 2. Cleavage of the DNA (38 μM bp) with 100 μM Cu(dpq)2 in the presence of different amounts of GO. Incubation time is 30 min (37 °C). %NC indicates the percentage of the nicked DNA (NC, Band II) generated. Band I and Band III refer to supercoiled and linear DNA, respectively.

shows that the supercoiled PSICOR-GFP DNA (SC, Band I) can be cleaved into nicked DNA (NC, Band II) by Cu(dpq)2 alone with a low efficiency: only 47% supercoiled DNA weas converted into the nicked DNA under this condition. In the presence of GO, the cleavage efficiency of Cu(dpq)2 was dramatically improved, and the enhancement increased with the amount of GO added. About 87% of the supercoiled DNA was converted, and also trace linear DNA (Band III) was observed when the concentration of GO was up to 18 μg mL−1. Actually, the activity enhancement by GO is closely related to the ratio of GO to the concentration of the complexes. For Cu(dpq)2, the maximum cleavage was obtained with the complex to GO ratio of 2 μM to 1 μg mL−1, similar to the GO/Cu2+ system, in which the ratio of GO to Cu2+ is critical for the system activity (see detailed discussion in cytotoxicity section).16 The fact that optimum ratio of GO to Cu(dpq)2 is required for the maximum activity enhancement suggests that the DNA cleavage is 15841

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Figure 3. (a) Cleavage of the supercoiled DNA (38 μM bp) by 125 μM Cu(dpq)2 and Cu(dpq)2/GO (GO 62.5 μg mL−1). Symbols ∇ and ▼ indicate the percentages of the nicked DNA generated by Cu(dpq)2 and Cu(dpq)2/GO, respectively. Symbols ○ and ● indicate the percentages of the supercoiled DNA left after Cu(dpq)2 and Cu(dpq)2/GO cleavages, respectively. The solid lines are fits with exponential decay and growth functions. (b) DNA cleavage rates (Kobs) by different concentrations of Cu(dpq)2 and Cu(dpq)2/GO conjugate (the ratio of Cu(dpq)2:GO was kept constant, Cu(dpq)2, 2−150 μM, GO 1−75 μg mL−1). (c) Absorption spectra of the Cu(dpq)2 (20 μM in 5 mM Tris, 50 mM NaCl, pH 7.2 buffer) with increasing GO concentration (from 2 to 5 μg mL−1). Inset is the plot of [GO]/(εa − εf) versus GO concentration, straight line is the linear fit of the plot. The definitions of εa, εf, and εb were described in the Materials and Methods section.

for these complexes suggested that the GO/complex conjugates encompass higher binding abilities to DNA. It also should be noted that GO enhancement in binding is more pronounced in the complex with a small planar ligand. The binding of small molecules to DNA could result in a more stable double-helical DNA; the melting temperature (Tm) of the DNA duplex can therefore also be used to monitor the interaction of the Cu(dpq)2 and Cu(dpq)2/GO with DNA.32As shown in Figure 4, in the presence of GO alone, the Tm of the

conjugates play a key role in the DNA cleavage enhancement. Given the large size of GO, we believe that only part of the GO sheet is involved in DNA intercalation. However, it is hard to isolate the small size of GO. A different method is under development to obtain smaller size GO. Binding Affinity of GO/Complex Conjugates to DNA Molecules. To investigate the mechanism of the nuclease activity enhancement by GO, we first examined the binding abilities of the Cu(dpq)2 and Cu(dpq)2/GO conjugate to DNA molecules. The change in the intensity of Cu(dpq)2 spectra at 270 nm with different concentrations of DNA was recorded (Figure S4a). The apparent DNA binding constants (Kb) for Cu(dpq)2 and Cu(dpq)2/GO were determined from the plots of the concentration of DNA versus the intensity changes at 270 nm (Figure S4b). The Kb for the complexes with and without GO were summarized in Table 1.23,31 At the same Table 1. Apparent Binding Constants of the Copper Complexes (Cu(dpq)2,10 μM; Cu(dpq)(Ala), 20 μM; Cu(dppz)2, 20 μM) and Complex/GO Conjugates with DNA in 5 mM Tris-HCl, 50 mM NaCl (pH = 7.2) Buffer sample

GO, μg mL−1

Cu(dpq)2 Cu(dpq)2+GO Cu(dpq)2+GO Cu(dpq)2+GO Cu(dpq)(Ala) Cu(dpq)(Ala)+GO Cu(dppz)2 Cu(dppz)2+GO

0 1.0 2.0 3.0 0 2.0 0 2.0

Kb, M−1 2.2 2.3 5.5 1.1 7.4 1.0 9.7 3.6

× × × × × × × ×

105 106 106 107 104 106 105 106

Figure 4. Thermal melting curves of the DNA (11.5 μM bp) in the presence of GO (10 μg mL−1), Cu(dpq)2 (2 μM), or Cu(dpq)2 and GO together. Α0 and Af are the absorption intensities at 40 and 94 °C, respectively; A is the intensity at a given temperature between 40 and 94 °C.

DNA was increased from 80 to 87 °C.16 In the presence of Cu(dpq)2, the ΔTm of the DNA is only 3 °C, which is consistent with the fact that Cu(dpq)2 is a minor groove binder.20,33 In the presence of Cu(dpq)2/GO conjugate, ΔTm is about 12 °C. ΔTm caused by the Cu(dpq)2/GO conjugate is larger than that of the individual components, indicating that Cu(dpq)2/GO conjugates can stabilize the DNA duplex by interacting with it in an intercalation mode. The results are also supported by the decrease of the intensities of both positive and negative peaks in the CD spectra of the DNA in the presence of Cu(dpq)2/GO (Figure S5).34 Mechanism of the GO Enhancement. On the basis of the above results, we propose a mechanism for the interaction

concentration, the binding constant of the complex is dependent on the concentration of GO; increasing the GO concentration, Kb is increased. For instance, in the presence of 0−3.0 μg mL−1 of GO, Kb increased from 2.2 × 105 to 1.1 × 107 M−1. This trend is consistent with the aforementioned result of the GO concentration dependence of cleavage activity enhancement (Figure 2). The Kb enhancement by GO was also investigated for the complex Cu(dpq)(Ala),24 that has only one dpq ligand, and the complexes with other planar ligands, such as Cu(dppz)2. The increased Kb values in the presence of GO 15842

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Figure 5. The effect of GO on the DNA (38 μM bp) cleavage by the complexes with one and two planar ligands at concentration of 20 μM, GO 10 μg mL−1, 37 °C for 30 min in 50 mM Tris, 18 mM NaCl, pH 7.2 buffer. NC% indicates the percentage of the nicked DNA.

changed in the presence of GO. Figure S8 compares the effect of methyl green to the cleavage of DNA by Cu(dpq)2 and Cu(dpq)2/GO conjugate. The DNA cleavage by Cu(dpq)2 alone was slightly affected by the presence of methyl green (lane 3 vs lane 2 in Figure S8), which is consistent with the literature results.20,33 However, for the Cu(dpq)2/GO system, in the presence of methyl green, the nicked DNA is much less (87% for lane 4 vs 75% for lane 5). The result reveals that the combination of GO with Cu(dpq)2 changed the interaction site of Cu(dpq)2 with DNA from the minor groove to the major groove. It is possibly because the formed conjugate has a much larger dimension than that of Cu(dpq)2 ligand, hence it intercalates more likely to DNA at the major groove. The collective aforementioned results with Cu(dpq)2 and its analogues point to a hypothesis that DNA cleavage activity of any complexes with a planar ligand could be improved in the presence of GO or chemically reduced GO. Complexes with different planar ligands were synthesized,16,20,22,24,25 and DNA cleavage experiments of GO with these complexes were carried out. Figure 5 exhibits the GO enhancements for the copper complexes with one or two planar ligands. Remarkable activity enhancements for the complexes with planar ligand(s) suggest that GO can expand the intercalating plane of the complexes to DNA as we proposed. Comparably, in the presence of GO, the DNA cleavage capabilities of the complexes with two planar ligands are much higher than that of the complexes with one planar ligand, presumably, they cleave DNA with the same mechanism. For example, the enhancement for Cu(phen)2 is about 8-fold, while for Cu(phen)(Gly),25 which has one phen ligand, it is only about 5-fold. Also, under the same conditions, supercoiled DNA was completely cleaved into nicked DNA by GO/Cu(dpq)2 conjugates, but only 37.7% of the supercoiled DNA was converted to the nicked DNA by GO/Cu(dpq)(Gly) conjugates. It is likely that when the two ligands are in one complex, they both are possibly involved in the interaction with DNA molecules. However, the crystal structures of Cu(phen)2, Cu(dpq)2, and Cu(dppz)2 showed that the two planar ligands in each complex are not coplanar, and they have certain dihedral angles (56.58, 42.95, and 52.94°, respectively).21,22,28 We thus infer that in addition to the alteration of the interaction site with DNA as aforementioned, GO possibly also could twist the outer planar ligand of the biligand complexes through π−π stacking, hence leading to a more effective intercalating system. For the complexes without a planar ligand, their ligand cannot interact with GO effectively through π−π stacking, thus there no DNA cleavage activity enhancement was observed (data not shown). Cytotoxicity Study. The cleavage enhancement of the copper complexes by GO is potentially applicable for cancer therapy drugs. To this end, the cell toxicity of the GO/copper complex system was examined with human stomach cancer MGC-803 and embryonic kidney 293T cells with WST-8

between Cu(dpq)2/GO conjugate and DNA molecule. With the formation of Cu(dpq)2/GO conjugate, the planar aromatic region of ligand in the complex was expanded. As a result, the conjugates can bind more strongly to DNA through the intercalation mode, and the corresponding DNA cleavage activity is improved. The extent of the intercalation of Cu(dpq)2/GO conjugate to DNA molecule is unknown, because the dimensions of the single layered GO used in this work is from few to hundreds of nanometer (Figure 1). For the GO sheets with dimensions much larger than the dimension of DNA molecule (∼2 nm), the intercalation could be weak. That could be improved if the size distribution of the GO sample is more uniform and the size of GO is smaller, which is under investigation. According to the proposed mechanism, removing the surface functional groups of GO should be unfavorable to the electrostatic interaction, but favorable to the π−π stacking between GO and the ligand of the complexes,35 thus it could lead to a higher cleavage activity. The conjugate of Cu(dpq)2 with chemically reduced GO,18,28 in which some functional groups were removed but the planar structure was retained, exhibited the expected higher cleavage activity than that of GO/ Cu(dpq)2 under the same conditions (Figure S6). This result further supports the mechanism. On the basis of the interaction mechanism of GO with complexes, the GO sheet can be regarded as a larger planar ligand, thus DNA cleavage by GO/ complex conjugates should follow the same pathway as that of the complexes alone. Cu(dpq)2 and its analogues have been reported to cleave DNA through hydrolytic and oxidative mechanisms under different reaction conditions.20,22,24,27 When the DNA cleavage reaction with GO/complex conjugates was performed in the presence of hydroxyl radical scavengers, such as dimethyl sulfoxide (DMSO), visible activity inhibition was observed, suggesting that the cleavage by GO/complex conjugates is through the oxidative process too (Figure S7). Neither NaN3 nor KI was observed to inhibit the DNA cleavage reaction. To ascertain the nature of the oxidative cleavage, we also carried out the DNA cleavage reaction in the presence of either ascorbate or H2O2, and the cleavage was dramatically improved. We tried to religate the nicked DNA, but no supercoiled form was observed. These results further indicate that the nuclease activity enhancement by GO for the complexes originates from the high affinity of GO/complex to DNA molecules, instead of changing the cleavage mechanism. It has been reported previously that Cu(dpq)2 tends to bind the minor groove of the DNA.20,33The increased binding constants Kb and Tm, CD spectral variations, and higher nuclease activity suggested that GO/Cu(dpq)2 conjugate could possibly interact with DNA at a different site. Methyl green, a DNA major groove binder, was used to examine whether the interaction mode between Cu(dpq)2 and DNA had been 15843

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Figure 6. (a) Cell viability of human stomach cancer MGC-803 and embryonic kidney 293T cells after 24 h of exposure to different concentrations of GO and Cu(dpq)2 determined by WST-8 assay. (b) Cleavage of DNA (38 μM bp) with 10 μM Cu(dpq)2 in the presence of different amounts of GO. Incubation time is 30 min (37 °C). (c) Proposed mechanism of the nuclease activity and cytotoxicity decreases for Cu(dpq)2/GO conjugates at higher GO-to-Cu(dpq)2 ratio.

assay.36,37 The 293T cells were used, because they are easy to work with and are straightforward to culture. As a control, the cytotoxicity of GO alone was measured first. As shown in Figure S9, the GO alone showed weak cytotoxicity at lower concentrations, and was almost nontoxic at higher concentration (100 μg mL−1). This is consistent with the literature results that GO has low toxicity to human fibroblast cells below 50 μg mL−1,38 and no obvious cytotoxicity on A549 and NIH3T3 fibroblast cells.39,40 Although some reported that GO has cytotoxicity,37,41 we believe the difference was caused by the quality of GO sample used. The cytotoxicity of Cu(dpq)2 as a control was also examined. Similar to many Cu(dpq)2 analogues that exhibited cytotoxicity under irradiation,42,43 Cu(dpq)2 exhibited cytotoxicity (black lines in Figure 6). By contrast, in the presence of 10 μg mL−1 GO, the cell toxicity of Cu(dpq)2 was higher than that of it alone (red lines in Figure 6a). The most notable observation is that when the concentration of GO was increased from 10 to 100 μg mL−1, the cytotoxicity of the GO/complex conjugates decreased for both cell lines (blue lines in Figure 6a), and the cytotoxicity is even lower than that of Cu(dpq)2 alone at some compound concentrations. The cytotoxicity of some Cu(dpq)2 analogues was believed to be through the apoptotic pathway.41 The interaction of GO with a cell is not fully understood yet. Nevertheless, the cytotoxicity of Cu(dpq)2/GO conjugate agrees with the result obtained in DNA cleavage activity measurements (Figure 6b). When Cu(dpq)2 was at a relatively low concentration (10 μM), the percentage of the nicked DNA was increased with the GO concentration increase, and reached the maximum when GO was about 30 mg mL−1 under that

condition. Further increasing GO concentration led to the decrease of the percentage of nicked DNA. This result may be rationalized on the basis of the conjugates formed by the GO and Cu(dpq)2 complex. When the ratio of GO to Cu(dpq)2 is low, the complex will interact with GO, thus the size of their planar ligands are effectively expanded by GO as we proposed earlier, hence its binding ability to DNA will be increased, and the cleavage activity will be enhanced. While in the presence of excess GO, GO sheets could superpose on the top and/or underneath of the conjugates, or interact with the other ligand of the complexes, much larger conjugates could be formed as schematically shown in Figure 6c. The formation of larger conjugates by excess GO with Cu(dpq)2 was also supported by the observations that the large portion of the sample dwelled in the sample well during the gel electrophoresis with the higher percentage of GO in the sample, and the precipitation was also observed in the sample at the higher GO concentration. The dimension of the large conjugates will definitely hinder the cell uptake, as well as the interaction with DNA if they are in the nucleus. Hence the enhancement in the nuclease activity and cytotoxicity will be lost, and is even worse than the complex alone when GO concentration is too high. We cannot declare that the cytotoxicity of the GO/complex is the sole result of their DNA cleavage activity from the current results, although it was reported that similar carbon materials (single-walled carbon nanotubes) were detected in nucleus.44 The cellular distribution and location of the GO and GO/complex are under investigation. This work may open up applications for new chemotherapeutic agents in the common class that work through DNA intercalation. 15844

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CONCLUSIONS We demonstrated that small DNA intercalators and GO could form conjugates mainly through π−π stacking interactions. The spectroscopic and gel electrophoresis results showed that the as-generated GO/complex conjugates improved the binding affinities of the small DNA intercalators, which suggested that GO can expand the dimension of the planar aromatic ligands of small DNA intercalators. The nuclease activities of the small intercalators were dramatically improved. The cleavage mechanism of as-generated GO/complex conjugates remains the same oxidative pathway as that of the complexes alone. In addition, the interaction site for the minor groove binder was changed into the major groove binder. The high cytotoxicity of the GO/complex conjugates further manifests their potential for pharmaceutical applications. Although we do not fully understand the correlation between the nuclease activity and the cytotoxicity of the conjugate systems at the moment, if this behavior is proved to be general for other small DNA intercalators, GO would be a promising candidate for combination anticancer drug design. Moreover, the nature in which GO can be chemically modified renders its possibility to combine with potential anticancer drugs leading to conjugates with high affinity and selectivity toward DNA molecules.

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ASSOCIATED CONTENT

S Supporting Information *

The chemical structures of the copper complexes, the rate constants of the DNA cleavage reactions with other complexes, CD spectra of the DNA in the presence of the GO and Cu(dpq)2, argarose gel electrophoresis of DNA in the presence of different additives and with different reduced GO, and cell viability of 239T cells exposed to different concentrations of GO. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(J.Z.) Tel: 0086-21-64253846; Fax: 0086-21-64253846; e-mail :[email protected]. (S.G.) Tel: 0086-21-34206915; Fax: 008634206915; e-mail: [email protected]. Author Contributions §

Bin Zheng and Chong Wang contributed to this work equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Science foundation of China (Nos. 31070742, 91123011, 90923041), the state key laboratory of bioreactor engineering (No. 2060204), 111 Project (No. B07023), the Shanghai Committee of Science and Technology (No. 11DZ2260600), and the National “973 Program” (No. 2010CB933900).

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