Nanotoxicity and Spectroscopy Studies of Silver Nanoparticle: Calf

Mar 11, 2010 - definition, are particles with lengths that range from 1 to 100 nm in two or three ... increasingly in wound dressings, catheters, and ...
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5798

J. Phys. Chem. C 2010, 114, 5798–5803

Nanotoxicity and Spectroscopy Studies of Silver Nanoparticle: Calf Thymus DNA and K562 as Targets Mahdie Rahban,† Adeleh Divsalar,*,†,‡ Ali A. Saboury,† and A. Golestani§ Institute of Biochemistry and Biophysics, UniVersity of Tehran, Tehran, Iran, Department of Biological Sciences, Tarbiat Moallem UniVersity, Tehran, Iran, and Department of Biochemistry, Tehran UniVersity of Medical Science, Tehran, Iran ReceiVed: NoVember 9, 2009; ReVised Manuscript ReceiVed: February 7, 2010

The interaction between silver nanoparticle and calf thymus DNA was studied by UV-visible, fluorescence, and far UV circular dichroism (CD) spectroscopies at a physiologic temperature of 37 °C. By the analysis of UV-visible titration and thermal denaturation studies of DNA, it was found that silver nanoparticle can form a new complex with double-helical DNA and increase the Tm value of DNA. This kind of binding may cause a slight change of the conformation of DNA. The fluorescence emission spectra of intercalated ethidium bromide (EB) with increasing concentration of silver nanoparticle at 37 °C represented a significant reduction of the ethidium intensity and quenching of EB fluorescence. Also, CD results suggested that silver nanoparticle can significantly change the helicity conformation of DNA and then induce the alteration of nonplanar and tilted orientations of DNA bases, resulting in the changes of DNA base stacking, and act as an intercalator. Spectroscopic results represented that binding of silver nanoparticle to DNA resulted in significant changes in the structure and conformation of DNA in a concentration dependent manner and act as an intercalator via increasing stability of DNA by increasing Tm, quenching of EB fluorescence intensity, and alteration of CD spectra. Also, the antitumor property of silver nanoparticle was studied by testing it on human tumor cell line K562. The 50% cytotoxic concentration (Cc50) of silver nanoparticle was determined using MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay after a 24 h incubation time. Results of the present study may provide useful information to design better anticancer compounds using metal nanoparticles with lower side effects in the future. Introduction Nanotechnology has attracted considerable attention in the scientific community ever since its emergence as a powerful basic and applied science tool.1,2 Simultaneously, people are increasingly exposed to various kinds of manufactured nanoparticles.3 Nanoparticles, according to the ASTM standard definition, are particles with lengths that range from 1 to 100 nm in two or three dimensions.4 Nanoparticles are used in bioapplications such as therapeutics, antimicrobial agents, drugdelivery agents, biosensors, imaging contrast agents, transfection vectors, and fluorescent labels.4,5 Owing to their unique nanoscale, nanoparticles are provided with many special physicochemical properties, and thereby may yield extraordinary hazards for human health and the environment;3 as a result, nanotoxicology research is now gaining attention. Nanotoxicology takes up this challenge to decipher the molecular events that regulate bioaccumulation and toxicity of nanoparticles.5 Nanoparticles are of similar size to typical cellular components and proteins, and thus may bypass natural mechanical barriers, possibly leading to adverse tissue reaction.6 For nanoparticles to move into the clinical field, it is important that nanotoxicology research uncovers and understands how these multiple factors influence the toxicity of them, so that their undesirable properties can be avoided.4 * To whom correspondence should be addressed. E-mail: divsalar@ ibb.ut.ac.ir. Phone: 0098 21 61113381. Fax: 0098 21 66404680. † University of Tehran. ‡ Tarbiat Moallem University. § Tehran University of Medical Science.

Physical parameters such as surface area, particle size, surface charge, and zeta potential are very important for providing mechanistic details in the uptake, persistence, and biological toxicity of nanoparticles inside living cells.1 Particle size may be a critical parameter for nanoparticle bioactivity, but it is difficult to ascertain which parameter plays an essential role in the biological effects when concerning various types of nanoparticles with different shapes and composition.3 With each of these nanoparticles, different data has been published about their cytotoxicity and interaction with biomacromolecules due to varying experimental conditions as well as differing nanoparticle physiochemical properties.4 The behavior of nanoparticles inside the cells is still unclear, and no metabolic and immunological responses induced by these particles are understood so far.5 Nanomaterials exist in various shapes and structures such as spheres, needle, tubes, plates, sheets, etc. The size and shape of nanomaterials contributes to the onset of cytotoxicity; for example, single-wall nanotubes are more toxic than multiwall nanotubes.7,8 Also, previous studies have demonstrated that the electromagnetic, optical, and catalytic properties of noble-metal nanocrystals are strongly influenced by shape and size.9 Elechiguerra and co-workers9 found that silver nanoparticles undergo a size-dependent interaction with human immunodeficiency virus type 1, preferably via binding to gp120 glycoprotein knobs. The size-dependent interaction of silver nanoparticles with gramnegative bacteria has also been reported by this group. Pal et al10 showed that truncated triangular nanoplates have represented high reactivity in comparison to other particles that contain fewer than three facets, like spherical or rod-shaped particles.

10.1021/jp910656g  2010 American Chemical Society Published on Web 03/11/2010

Nanotoxicity and Spectroscopy of Silver Nanoparticle Silver nanoparticles are widely utilized in material science, physics, and chemistry fields because of their particular optical, magnetic, electronic, and catalytic properties and are being used increasingly in wound dressings, catheters, and various household products due to their antimicrobial activity. They also have an important function in anti-inflammation, antivirus, anti-AIDS, and especially anticancer.5,11,12 In spite of the wide usage of silver nanoparticles, very few reports on the toxicity of silver nanoparticles are available. The interaction of DNA or its components (bases and nucleosides) with metallic nanoparticles is a subject of great interest to researchers in the interdisciplines of nanobiotechnology. The interaction of metal nanoparticles with nucleic acids is topical in the bioinorganic field due to its possible effects on the synthesis, replication, and structural integrity of DNA and RNA.13 There are two principal modes by which components can bind noncovalently to DNA: intercalation and minor groove binding.14 Metals are able to interact (covalently or not) with DNA through the electronsoffering DNA bases and phosphate groups establishing either inter- or intrastrand interactions, among others. Moreover, the coordinated groups in metal complexes contribute specific abilities, i.e., intercalation, hydrogen bonding, and electrostatic interaction, leading to a global effect.15 Molecular recognition of proteins and nucleic acids by low molecular weight compounds is an area of fundamental interest. It is a subject that exists at the interface of chemistry and biology, since interactions involving biological molecules are best understood if described in physicochemical terms.14 Nucleic acids and proteins have an important function in life processes, so they are often used as a reference for measurements of other components in biological samples. Then, in the present study, we have decided to investigate the nanotoxicology of silver nanoparticles via determination of interaction of it with calf thymus DNA and its cytotoxicity against K562, as a model cancer cell line. Experimental Section Materials. Silver nanoparticles (5-10 nm, liquid form, dispersion matrix: ethyleneglycol pH 5-8) were purchased from Biocera Co., Ltd. NaCl, Tris-base, and EDTA were prepared from Merck Co. Ethidium bromide, RPMI, MTT, streptomycin, and penicillin were obtained from Sigma Chemical Co. Methods DNA Preparations. High molecular weight DNA was purified from calf thymus according to the modified methods of Sambrook et al.16 and McGruire and Dela Granza,17 as follows. An appropriate amount of calf thymus was minced at 4 °C. It was homogenized in NET buffer (0.15 M NaCl, 10 mM EDTA, and 10 mM Trise-base, pH 7.5-8) at a ratio of 1:8 (w/v) using a blender model SM 2460 PG. Boiled RNase, 50 µg/mL (final concentration), was added and retained at 37 °C for 1 h. Sodium dodecyl sulfate (SDS) with 0.2% (w/v) final concentration and proteinase K (50 µg/mL final concentration) was added and incubated overnight at 60 °C. To obtain DNA, the crude extract was treated with an equal volume of phenol, phenol-chloroform-isoamylalcohol at a 25:24:1 ratio, and chloroform-isoamylalcohol at a ratio of 1:1, respectively. The aqueous phase was separated by centrifugation, and finally, DNA was extracted by precipitation with sodium acetate (0.3 M final concentration) and 3 vol of absolute ethanol. The DNA was pooled and washed with 70% ethanol followed by transfer of DNA using sterile forceps to 10:1 of TE buffer (1 mM EDTA

J. Phys. Chem. C, Vol. 114, No. 13, 2010 5799 and 10 mM Tris-base, pH 7.5-8) at 4 °C until use. The DNA absorbance ratio at 260 and 280 nm was ∼1.8, and the molecular weight was confirmed by agarose (70%) gel electrophoresis. The DNA concentration was determined using an extinction of 6600 M-1 cm-1 at 260 nm and expressed in terms of base molarity.18 UV-Visible Measurements. The UV absorption spectra of calf thymus DNA solution at 258 nm were studied upon addition of different concentrations of silver nanoparticle. The UV-visible absorption spectra were measured with a Cary spectrophotometer, 100 Biomodel, with jacketed cell holders. Spectral changes of 4.00 mM DNA were monitored after adding different concentrations of silver nanoparticle (0-101 µM) by recording the UV-visible absorption (200-700 nm). All experiments were run in Tris-base buffer (0.1 M), pH 7.5, in a conventional quartz cell thermostatted to maintain the temperature at 37 °C. In temperature-scanning spectroscopy, absorbance profiles, which describe the thermal denaturation of DNA, were obtained with a CARY model 100-Bio UV/vis spectrophotometer, fitted with a temperature program to control the speed of temperature change in the denaturation experiments. The sample cells contained 4.00 mM DNA and 0 to 44 µM silver nanoparticles. The recording chart read the temperature and absorbance differences between the reference cuvettes (which were the same as the sample except without DNA) and the sample cuvettes at 258 nm. Fluorescence Measurements. The fluorescence of ethidium bromide (EB) is greatly enhanced on its intercalation between the base pairs of DNA.19 Ethidium bromide displacement assay was performed as reported in the literature.20 At first, DNA (60 µM) was added to 2 µM aqueous ethidium bromide solution and maximum quantum yield for ethidium bromide was achieved at 471 nm, so we selected this wavelength as the excitation radiation for samples at 37 °C in the range 500-720 nm. To this solution (containing ethidium bromide and DNA), different concentrations of silver nanoparticle were added (0-162 µM). Measurements were done by applying a 1 cm path length fluorescence cuvette. The fluorescence intensities of the silver nanoparticle at the highest denaturant concentration at 471 nm excitation wavelength have been checked, and the emission intensities of this nanoparticle were very small and negligible. CD Measurements. Circular dichroism spectra showed changes in the structure of DNA, which were monitored in the region (200-320 nm) using 1 cm path length cells. The DNA concentration in the experiments was 1.06 mM. Induced CD spectra resulting from the interaction of the silver nanoparticle (0, 61,123,185, 246, and 308 µM) with DNA at 37 °C were obtained by subtracting the CD spectrum of the native DNA and mixture of DNA-silver nanoparticle from the CD spectrum of the buffer and spectrum of buffer-silver nanoparticle solutions, respectively. Cytotoxic Measurements. Cell Culture. In RPMI medium, chronic myelogenous leukemia cells were grown. This medium was supplemented with L-glutamine (2 mM), streptomycin, and penicillin (5 µg/mL), and 10% heat-inactivated fetal calf serum, cells maintained in 5% CO2 and 95% air atmosphere incubator at 37 °C. MTT Assay. The silver nanoparticle inhibits the growth of the chronic myelogenous leukemia cell line, K562. This growth inhibition was measured by means of MTT assay.21 The cleavage and conversion of the soluble yellowish MTT to the insoluble purple formazan by active mitochondrial dehydrogenase of living cells has been used to develop an assay system

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Figure 2. The melting curves of calf thymus DNA in the absence ([) and presence (]) of the silver nanoparticle (44 µM) in Tris-base buffer (0.1 M), pH 7.5. The inset shows variations of ∆G° against temperature. Figure 1. Changes of UV spectra of calf thymus DNA in the presence of different concentrations of silver nanoparticle (0, 4, 9, 13, 22, 27, 35, 40, 44, 48, 53, 57, 63, 70, 82, 95, and 101 µM) in Tris-base buffer (0.1 M), pH 7.5 at 37 °C. The inset represents the alterations in maximum absorbance (at 258 nm) of calf thymus DNA in the presence of different concentrations of silver nanoparticles.

alternative to other assays for measurement of cell proliferation. Harvested cells were seeded into a 96-well plate (2 × 104 cell/ mL) with varying concentrations of the sterilized silver nanoparticle (0, 3.1, 7.7, 15.5, 31.0, 62.0, and 124.0 µM) and incubated for 24 h. Four hours to the end of incubations, 25 µL of MTT solution (5 mg/mL in PBS) was added to each well containing fresh and cultured medium.22 At the end, the insoluble formazan produced was dissolved in a solution containing 10% SDS and 50% DMF (left for 2 h at 37 °C under dark conditions) and optical density (OD) was read against reagent blank with a multiwell scanning spectrophotometer (ELISA reader, Model Expert 96, Asys Hitchech, Austria) at a wavelength of 570 nm. Absorbance is a function of the concentration of converted dye. The OD value of study groups was divided by the OD value of untreated control and presented as a percentage of control (as 100%). Results and Discussion UV-Visible Studies. Electronic absorption spectroscopy is an effective method to examine the binding mode of DNA with metal complexes.23 Thus, in order to provide evidence for the possibility of binding of silver nanoparticle to calf thymus DNA, spectroscopic titration of a solution of the nanoparticle with DNA has been performed. The absorption spectra of the interaction of DNA with the silver nanoparticle have been recorded for a constant DNA concentration in different [silver nanoparticle]/[DNA] mixing ratios. UV spectra of DNA in the presence of different concentrations of silver nanoparticle at 37 °C are shown in Figure 1. In general, hyperchromism and hypochromism are the spectral features of DNA concerning its double-helix structure; hyperchromism means the breakage of the secondary structure of DNA, and hypochromism means that the DNA-binding mode of the complex is electrostatic effect or intercalation, which can stabilize the DNA duplex, while the existence of a red-shift is indicative of the stabilization of the DNA duplex.24 The changes observed in the absorption spectra of calf thymus DNA in the presence of silver nanoparticle (the

increase of the intensity at λmax ) 258 nm up to 31% without any shift in λmax of 258 nm) after mixing with silver nanoparticle indicate that the interaction of the complexes with calf thymus DNA takes place by a direct formation of a new complex with double-helical calf thymus DNA (inset of Figure 1). The absorption intensity at 258 nm is increased (inset of Figure 1) due to the fact that the purine bases and pyrimidine bases of DNA are exposed because of the binding of the complex to DNA. This kind of binding may have caused the slight change of the conformation of DNA.23,24 DNA Thermal Denaturation Studies. Thermal behaviors of DNA in the presence of silver nanoparticle can give insight into DNA conformational changes when the temperature is raised, and offer information about the interaction strength of nanoparticles with DNA. It is well-known that, when the temperature in the solution increases, the double-stranded DNA gradually dissociates to single strands, and generates a hyperchromic effect on the absorption spectra of DNA bases. In order to identify this transition process, the melting temperature (Tm), which is defined as the temperature where half of the total base pairs are unbonded, is usually introduced. According to the literature,25-27 the intercalation of natural or synthesized organicand metallo-intercalators generally results in a considerable increase in melting temperature. The melting curves of DNA in the absence and presence of the silver nanoparticles are presented in Figure 2. Here, the thermal denaturation experiment carried out for DNA in the absence of the silver nanoparticles revealed a Tm value of 75.2 ( 0.2 °C under our experimental conditions, whereas the observed melting temperature of DNA in the presence of silver nanoparticles successively increased (Tm ) 80.3 ( 0.2 °C). The increases in Tm of DNA in the presence of silver nanoparticles are comparable to those observed for classical intercalators.27 In order to evaluate the quality of our pseudoequilibrium measurements, we estimate the standard Gibbs free energy (∆G°) of thermal unfolding from the Pace analysis.24 The amounts of ∆G°25, Gibbs free energy of DNA denaturation at 25 °C for native and denatured in the presence of silver nanoparticles, can be obtained from the Pace analysis of thermal denaturation curves (Figure 2). Determination of ∆G°, as a criterion of conformational stability of a DNA, is based on the two-state theory as follows:28,29

Nanotoxicity and Spectroscopy of Silver Nanoparticle

Native (N) S Denatured (D)

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(1)

By assuming a two-state mechanism for biomacromolecule denaturation by temperature, one can determine the process by monitoring the changes in the absorbance and hence calculate the denatured fraction of DNA (Fd) as well as determine the denaturation equilibrium constant (KD):

YN - Yobs YN - YD

(2)

Fd YN - Yobs ) 1 - Fd Yobs - YD

(3)

Fd )

KD )

where Yobs is the observed variable parameter (e.g., absorbance) and YN and YD are the values of Y characteristics of a fully native and denatured conformation, respectively. The ∆G° change is given by the following equation:

∆G° ) -RT ln KD

(4)

where R is the universal gas constant and T is the absolute temperature. ∆G° varies linearly with T over a limited region. The simplest method of estimating the conformational stability at 25 °C, ∆G°25, is to assume that ∆G° versus temperature in the transition range of temperatures up to 70 °C is a linear plot (∆G° ) ∆G°25 - aT, where a is the slope of the linear plot).30 From the inset of Figure 2, the ∆G°25 values in the absence and presence of silver nanoparticles are 14.4 and 14.6 kcal/ mol, respectively. Fluorescence Studies. Fluorescence titration of solutions containing the DNA and ethidium bromide (EB) with silver nanoparticle has been investigated. It is known that the fluorescence intensity of EB grows when it goes from a polar to a nonpolar medium because of the decrease in the intersystem crossing lifetimes.19 The displacement of DNA intercalated EB by groove binding molecules has been used as a standard technique to assay DNA binding agents.16 The molecular fluorophore EB, a phenenthridine fluorescence dye, forms soluble complexes with nucleic acids and emits intense fluorescence in the presence of DNA due to the intercalation of the planar phenenthridinium ring between adjacent base pairs on the double-helix EB.31 Previous studies have shown that there are two binding sites on DNA for EB: the primary site, which has been interpreted as intercalation between base pairs, and the secondary site, which is thought to be electrostatic between the cationic EB and the anionic phosphate groups on the DNA surface. The secondary mode of binding is most evident at low salt and high dye concentrations.32 No fluorescence has been observed for the silver nanoparticles at room temperature in solution or in the presence of DNA. Thus, the binding of the nanoparticles and DNA cannot be directly predicted through the emission spectra. Hence, competitive EB binding studies have been undertaken to gain support for the extent of binding of silver nanoparticles with DNA. EB does not show any appreciable emission in a buffer solution due to fluorescence quenching of the free EB by the solvent molecules.33 On addition of DNA, its fluorescence intensity is highly enhanced due to its strong intercalation between the adjacent DNA base pairs. The fluorescence emission spectra of intercalated ethidium with increasing concentrations of silver nanoparticle at 37 °C are shown in Figure 3 and the inset of Figure 3. Figure 3 shows a

Figure 3. Fluorescence emission spectra of intercalated ethidium bromide incubated with calf thymus DNA by increasing concentrations of silver nanoparticle at 37 °C.

significant reduction of the ethidium intensity in different concentrations of silver nanoparticle at physiologic temperature (37 °C). This decrease of EB fluorescence (up to 25% of the initial EB-DNA fluorescence intensity) (inset of Figure 3) indicates the competition of silver nanoparticle with EB in binding to DNA. On the other hand, it can be concluded that the fluorescence intensity of DNA intercalated by ethidium is quenched when ethidium is removed from the duplexes by the action of silver nanoparticle. These results suggest that the silver nanoparticles intercalate in DNA. The quenching efficiency of silver nanoparticle is evaluated by the Stern-Volmer constant (KSV), which varies with the experimental conditions:

F0 ) 1 + [silver nanoparticle]KSV F

(5)

where F0 and F are the emission intensities in the absence and the presence of the silver nanoparticle, respectively. Figure 4 shows the Stern-Volmer plot of the silver nanoparticle. The KSV value calculated for the silver nanoparticle has been given in Table 1. The Stern-Volmer plot of DNA-EB illustrates that the quenching of EB bound to DNA by silver nanoparticle is in good agreement (R ) 0.99) with the linear Stern-Volmer equation (eq 5), which proves that the partial replacement of EB bound to DNA by nanoparticle results in a decrease in the fluorescence intensity. The high KSV value of the silver nanoparticle shows that it can be bound very tightly to the DNA.34 CD Studies. CD spectroscopy is useful in diagnosing changes in DNA morphology during drug-DNA interactions, as the positive band due to base stacking (275 nm) and the negative one due to right-handed helicity (246 nm) are quite sensitive to the mode of DNA interactions with small molecules.34,35 The changes in the CD signals of DNA observed on interaction with drugs may often be assigned to the corresponding changes in the DNA structure.36 Thus, simple groove binding and electro-

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Figure 4. Stern-Volmer plot of DNA-EB in the presence of different concentrations of silver nanoparticle.

TABLE 1: Various Parameters of DNA upon Interaction with Silver Nanoparticle sample

Tma (°C)

∆G°25b (kcal/mol)

Ksvc (mM-1)

DNA DNA + silver nanoparticle

75.2 ( 0.2 80.3 ( 0.2

14.4 14.6

2.2

a

Figure 5. CD spectra of calf thymus DNA incubated with silver nanoparticles at different concentrations of 0, 60, 123, 185, and 246 µM.

b

The melting temperature (Tm). The amounts of Gibbs free energy of DNA denaturation at 25 °C. c Value of the Stern-Volmer constant.

static interaction of small molecules show less or no perturbation on the base-stacking and helicity bands, while intercalation enhances the intensities of both of the bands, stabilizing the right-handed B conformation of DNA, as observed for the classical intercalator methylene blue.36 The CD spectra of the silver nanoparticles with double-stranded DNA can provide us with useful information concerning the silver nanoparticlenucleotide interaction. Thus, CD spectra of calf thymus DNA incubated with silver nanoparticles at different concentrations were recorded. The observed CD spectrum of DNA consists of a positive band at 277 nm due to base stacking and a negative band at 247 nm due to helicity, which are characteristics of DNA in the righthanded B form. Silver nanoparticles have no CD spectrum when they are free in the solution but have an induced CD spectrum when they interact with DNA. When silver nanoparticles were incubated with DNA, the CD spectra displayed changes of both positive and negative bands (Figure 5). As seen in Figure 5 after adding different concentrations of silver nanoparticles, the intensities of positive and negative bands increase significantly; this observation is a strong indicator of classical intercalation. Since alterations in negative bands of DNA are more than positive region, then it can be concluded that silver nanoparticle may significantly change the helicity of DNA. These conformational changes caused alterations in the orientations of DNA bases and tilted them, resulting in the changes of DNA base stacking and act as an intercalator. This data shows good agreement with increasing of Tm and decreasing of EB fluorescence intensity. Cytotoxicity Studies. The in vitro antitumor property of the silver nanoparticle was studied by testing it on human tumor cell line K562. In this study, various concentrations of silver nanoparticles ranging from 0 to 124 µM were used to culture the tumor cell lines for 24 h (Figure 6). The 50% cytotoxic concentration (Cc50) of silver nanoparticle was determined from

Figure 6. Growth suppression activity of the silver nanoparticle on the K562 cell line assessed using MTT assay. The tumor cells were incubated with varying concentrations of the silver nanoparticle ranging from 0 to 120 µM for 24 h.

Figure 6. As shown in Figure 6, the cell growing after incubation time was significantly reduced by various concentrations of silver nanoparticle. Also, it is clear that the silver nanoparticles produced dose-response suppression on growing of the K562 leukemia cell line. Conclusion Binding of a small molecule to DNA is assumed to stabilize the helix against its thermal denaturation, and the usual mark of stabilization is a rise in the transition temperature, Tm, for the double- to single-stranded form morphing of DNA. Experimentally, this is accomplished by comparing Tm for DNA in solution with and without the intercalator while monitoring some property dependent on the DNA helix. Due to the difference between the extinction coefficients of DNA bases in the doublestranded form versus the single-stranded form at 260 nm, the absorbance increases sharply at Tm as the DNA strands separate. The helix denaturation of DNA was thus monitored as a function of temperature by recording absorbance at 258 nm.37 As shown

Nanotoxicity and Spectroscopy of Silver Nanoparticle in Figure 2, the DNA melting experiment reveals that Tm of the calf thymus DNA was 75.2 °C and in the presence of silver nanoparticle increased to 80.3 °C, thus emphatically confirming heightened helix stability as a result of intercalation into DNA of the silver nanoparticle. The addition of a DNA binding agent induces a progressive decrease in fluorescence of ethidium due to its displacement from the duplex. This also allows distinguishing nonintercalative binding agents from intercalating agents. From Figure 3, it might be concluded that silver nanoparticle can remove EB from the duplexes of DNA by the action of silver nanoparticle as an intercalator. This result shows good agreement with increasing of the Tm value of DNA in the presence of silver nanoparticle from thermal denaturation studies. CD spectra of DNA in the presence of silver nanoparticle show that both the negative and positive peaks change, but the change of the negative peak is bigger than that of the positive peak when silver nanoparticle is added into the DNA system. This suggests that silver nanoparticle can significantly change the helicity conformation of DNA and then induce the alteration of nonplanar and tilted orientations of DNA bases, resulting in the changes of DNA base destacking and act as an intercalator. From the above results, it can be concluded that binding of silver nanoparticle to DNA resulted in significant changes in the structure and conformation of DNA and act as an intercalator via increasing the stability of DNA by increasing Tm, quenching of EB fluorescence intensity, and alteration of CD spectra. Structural and conformational changes of DNA due to the silver nanoparticle binding results and anticancer power of this nanoparticle obtained here may provide useful information to design better anticancer compounds using metal nanoparticles with lower side effects in the future. Acknowledgment. The financial support of Research Council of University of Tehran is highly appreciated. References and Notes (1) Karakoti, A. S.; Hench, L. L.; Seal, S. JOM 2006, 58, 77. (2) Jones, C.; Grainger, D. W. AdV. Drug DeliV. ReV., in press. (3) Yang, H.; Liu, C.; Yang, D.; Zhang, H.; Xi, Z. J. Appl. Toxicol. 2009, 29, 69. (4) Lewinski, N.; Colvin, V.; Drezek, R. Small 2008, 4, 26. (5) AshaRani, P. V.; Mun, G. L. K.; Hande, M. P.; Valiyaveettil, S. ACS Nano 2009, 3, 279. (6) Pan, Y.; Neuss, S.; Leifert, A.; Fischler, M.; Wen, F.; Simon, U.; Schmid, G.; Brandau, W.; Dechent, W. J. Small 2007, 3, 1941.

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