Accepted Manuscript Interaction Studies between Biosynthesized Silver Nanoparticle with Calf Thymus DNA and Cytotoxicity of Silver Nanoparticles Swarup Roy, Ratan Sadhukhan, Utpal Ghosh, Tapan Kumar Das PII: DOI: Reference:

S1386-1425(15)00062-1 http://dx.doi.org/10.1016/j.saa.2015.01.041 SAA 13209

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

6 August 2014 8 January 2015 14 January 2015

Please cite this article as: S. Roy, R. Sadhukhan, U. Ghosh, T.K. Das, Interaction Studies between Biosynthesized Silver Nanoparticle with Calf Thymus DNA and Cytotoxicity of Silver Nanoparticles, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.01.041

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Interaction Studies between Biosynthesized Silver Nanoparticle with Calf Thymus DNA and Cytotoxicity of Silver Nanoparticles

Swarup Roy*, Ratan Sadhukhan, Utpal Ghosh, and Tapan Kumar Das Department of Biochemistry and Biophysics, University of Kalyani, Kalyani – 741235, West Bengal, India

*

Correspondent author: Email: [email protected], [email protected]

Telephone: Office: (033) 25828750, 2582 8378 (Ext. 385), +91-9046546632 (Mob) Fax: +91-33-25828282

2

Abstract The interaction of calf thymus DNA (CTDNA) with silver nanoparticles (SNP) has been investigated following spectroscopic studies, analysis of melting temperature (Tm) curves and hydrodynamic measurement. In spectrophotometric titration and thermal denaturation studies of CTDNA it was found that SNP can form a complex with double-helical DNA and the increasing value of Tm also supported the same. The association constant of SNP with DNA from UV-Vis study was found to be 4.1 ×103 L/mol. The fluorescence emission spectra of intercalated ethidium bromide (EB) with increasing concentration of SNP represented a significant reduction of EB intensity and quenching of EB fluorescence. The results of circular dichroism (CD) suggested that SNP can change the conformation of DNA. From spectroscopic, hydrodynamic, and DNA melting studies, SNP has been found to be a DNA groove binder possessing partial intercalating property. Cell cytotoxicity of SNP was compared with that of normal silver salt solution on HeLa cells. Our results show that SNP has less cytotoxicity compared to its normal salt solution and good cell staining property.

Key Words: Silver nanoparticle, Interaction, Calf thymus DNA, Cytotoxicity, Fluorescence, Staining

3

1. Introduction Nanotechnology has attracted significant attention in the scientific community ever since its emergence as a powerful tool of basic and applied science [1, 2]. Simultaneously, people are increasingly exposed to various kinds of manufactured nanoparticles [3, 4]. Nanoparticles have widespread biological applications such as being employed in therapeutics, used as antimicrobial agents, drug delivery agents, biosensors and fluorescent labels [4, 5]. Due to their unique size, nanoparticles could show highly specialized physicochemical properties, and thereby it may impose extraordinary hazards to human health and the environment [3], as a result nano toxicology research should be carried out extensively during the days ahead. Deoxyribonucleic acid has an important role as it carries hereditary information and instructs molecular machinery to conduct the biological synthesis of proteins and enzymes through the process of replication and transcription in living cells. Studies on the interactive mechanism of some small molecules and DNA have been identified as one of the key topics in drug-DNA interaction now-a-days [6]. Small molecules can react with DNA via covalent or noncovalent interactions, with profound interest generally focusing on the latter. There are several sites in the DNA molecule where such binding can occur: (i) between two base pairs (full intercalation), (ii) in the minor groove, (iii) in the major groove, (iv) on the outside of the helix and (v) electrostatic binding [7]. Silver nanoparticles are widely used because of their particular optical, magnetic, electronic, and catalytic properties and the same particles are also being used increasingly in wound dressings, and various family products due to their antimicrobial activity. They also serve an important function by virtue of their anti-inflammation, antivirus, antiAIDS, and especially anticancer activities [5, 8, 9]. In spite of the wide usage of SNP, reports of toxicity of biosynthesized SNP are very scanty. The interaction of DNA with metallic nanoparticles is a topic of prime interest to researchers involved in the interdisciplinary field of nano science. Studies on interaction of metal nanoparticles with nucleic acids are remarkable in the bioinorganic field due to its possible influence on the synthesis, replication, and structural integrity of DNA and RNA [10]. There are two important principal modes by which different compounds can be bound noncovalently to DNA molecule such as- intercalation and minor groove binding [11]. Nanoparticles are of similar size to typical cellular components and proteins, and thus may bypass natural mechanical barriers, possibly leading to adverse tissue reaction [12]. For nanoparticles finding its route into the

4

clinical field, it is necessary that nanotoxicology research discovers and understands how these multiple factors influence the toxicity of nanoparticles, so that their undesirable properties can be avoided [4, 13]. In the present study an attempt has been taken to investigate the interaction of SNP with calf thymus DNA by following the methods - UV spectrophotometry, fluorescence measurement, dynamic viscosity measurements, melting temperature measurement, and circular dichroism spectroscopy. We also observed cytotoxic effect of biosynthesized SNP on eukaryotic cell line (Human cervical cancer cell line, HeLa) as a model and as SNP has its own fluorescence we also take a look at the effect due to its cell staining property. 2. Materials and methods 2.1. Materials Silver nitrate, Tris-base, and EDTA were purchased from Merck, Germany and Ethidium bromide (EB), calf thymus DNA (CTDNA), MTT, and DMSO were purchased from Sigma Chemicals, USA. All the media, fetal bovine serum, antibiotics for cell culture were purchased from Himedia, India. Other fine chemicals and fluorescent dye Hoechst were purchased from SRL India, and Sigma Aldrich. Other molecular biology grade fine chemicals were purchased from SRL, India. All the other chemicals were of analytical reagent grade and double distilled water was used throughout. 2.2. Apparatus Fluorescence spectra were recorded on Agilent carry eclips fluorescence spectrophotometer well equipped with a thermostatically peltier compartment and the excitation and emission slits were considered as 5 nm. The absorption spectra were obtained from a Cary spectrophotometer (Agilent) well equipped with a thermostatically peltier compartment. Circular dichroism was recorded in JASCO J-815 CD spectrometer and viscosity was measured in Brookfield micro viscometer. All the images were taken in Carl Zeiss Axio vision 2 fluorescence microscopes. Thermo MULTISKAN ES micro plate reader was used to measure the absorbance in the study of cytotoxicity. 2.3. Methods 2.3.1. Synthesis and Characterization of SNP Silver nanoparticles have been synthesized by using the cell filtrate of the fungal species, Aspergillus foetidus and aqueous solution of silver nitrate (at a 1mM final concentration). The biosynthesized SNP were characterized by using several biophysical techniques such as UV-Vis spectra, Dynamic light scattering, Fourier transform infrared spectra, Atomic force microscopy, Transmission electron microscopy etc. as mentioned in our previous report [14].

5

Green Synthesis is an eco-friendly, cost effective as well as an alternative way of chemical synthesis. In Biosynthetic process addition of reducing agent and capping agent are not required to confer stability to the biosynthesized nanoparticles as extracellular protein alone performs the aforesaid task. Here the extracellular live cell filtrate extracted from the fungus has been used as the source of protein. After processing the estimation of concentration of biosynthesized nanoparticle has also been performed, as described in our earlier publications [15]. 2.3.2. Spectrophotometric measurements The CTDNA solution was prepared by dispersing an appropriate amount of CTDNA in Tris-EDTA buffer (0.1 M) solution (pH = 7.0) with stirring for 12 h at below 4oC. The interaction between CTDNA and SNP was studied by following the measurement of UV-VIS spectra obtained in an Agilent Cary spectrophotometer. Absorption experiments were carried out by keeping constant DNA concentration 50 µg/ml whereby varying the SNPs concentration (10-80 µM).

Spectral changes of DNA (50 µg/ml) were monitored after adding different

concentrations of SNP (10-80 µM) by recording the UV-Visible absorption in the range of 200-400 nm. All experiments were carried out in Tris-EDTA buffer (0.1 M), pH 7.0, in a conventional quartz cell thermostatted for maintenance of the temperature at 298 K. Absorption spectrum has also been studied under the same condition after adding different concentrations of CTDNA (50µg/ml) keeping constant SNP concentration (300 µM) by recording the spectra in the range of 300-600 nm. 2.3.3. Fluorescence Measurements Fluorescence measurements were carried out in an Agilent spectrofluorimeter. The solution of EB was prepared by dissolving EB in deionized water. EB was an efficient probe widely used in biochemical research for visualizing nucleic acids and it was used to assay the interaction of biosynthesized SNP with CTDNA. The fluorescence of EB is remarkably enhanced after intercalation of EB in between the base pairs of DNA [16]. The assay of ethidium bromide displacement was performed as reported in the literature [17]. At first, DNA (50 µg/ml) was added to 10 µg/ml aqueous EB solution and maximum quantum yield for EB was achieved at 270 nm, so this wavelength has been selected as the excitation radiation for samples at 293, 303, and 313 K in the emission range of 550-700 nm. To the solution containing EB and DNA different concentrations (10-70 µM) of SNP were added successively. 2.3.4. Circular dichroism (CD) spectral measurement CD spectra were recorded in a JASCO J-815 Spectrometer. Circular dichroism spectra showed changes in the structure of DNA, which were monitored in the region of 210-320 nm using 10 mm path length cells. Variable

6

concentrations of SNP (0, 25, 50 and 75 µM) were added to 50 µg/ml of DNA at 303 K to study the CD spectra. All spectra were collected in triplicate in the range 210-320 nm with the back ground corrected against buffer blank. 2.3.5. DNA melting experiments The study of thermal denaturation was performed in a spectrophotometer equipped with a Peltier temperature controller with a temperature program to control the speed of the temperature change in the denaturation experiments. 50 µg/ml DNA was taken in a cell and 30 µM SNP was added. Since analyzed profiles were almost linear in the melting region, melting temperature (Tm) was determined as the average of starting and final temperatures of the melting process. 2.3.6. DNA viscosity measurements Viscosity measurements were carried out in a viscometer (Brookfield micro viscometer) which was maintained at thermostatic condition at 298 K in a constant temperature bath. The CTDNA concentration was fixed at 50µg/ml and SNP concentration was 30-300µM. 500µl of CTDNA sample in presence (30-300µM) and absence of nanoparticles was taken to monitor the viscosity in rheometer. The values of relative specific viscosity (η /η0)1/3 where η0 and η are the specific viscosity contributions of DNA in the absence (η0) and in the presence of the nanoparticles (η), were plotted against 1/R (R = [DNA]/[complex]). 2.3.7. Cell culture Human cervical cancer cell line HeLa was obtained from National Centre for Cell Sciences, Pune, India. HeLa cells were grown in DMEM supplemented with 10% bovine serum (complete medium) at 310 K in humidified atmosphere containing 5% CO2. 2.3.8. Morphology and Cell viability assay by MTT The growth inhibition was measured by means of MTT assay [18, 19]. In a 96 well plate 3000 cells (HeLa cells) per well in 100µl fresh medium were seeded on the previous day and then treated with different concentrations of nano and control silver salt (0- 100µM) for 18 hrs. After treatment, the medium was replaced by 100 µl of fresh complete medium. 10 µl of MTT solution (5 mg/ml) was added, and incubated in CO2 incubator in the dark for 4 hrs. Then medium was discarded and 100 µl of DMSO was added. Absorbance was recorded at 595 nm in a Thermo MULTISKAN ES micro plate reader. Morphology of the cell [20] upon treatment at same concentration of silver ion and SNP were monitored using visible light in a Carl Zeiss microscope.

7

2.3.9. Cell staining with the compounds The cells were processed for staining using our previous procedure [21, 22]. In brief, the cells were grown on cover slip for 24 h and washed thrice with PBS to remove the medium. Then the cells were fixed with methanol-acetone (1:1) at 4 oC for 1 h, washed with PBS and incubated for 10 min in PBS containing 1 mM of SNP and a common fluorescent dye (Hoechst dye) as standard in absence of light. After washing with PBS the cells were observed under fluorescence microscope [23]. 3. Results and discussion 3.1. UV-Vis spectral measurements Absorption spectroscopy is a useful technique to understand the binding mode of DNA with small molecules [24]. Thus, in order to provide evidence for the possibility of binding of SNP to calf thymus DNA, study of spectroscopic titration of a solution of the nanoparticle with DNA has been performed. The absorption spectra of the interaction of DNA with the SNP have been recorded at a constant DNA concentration (50 µg/ml) for different concentrations of SNP (0-80µM). UV spectra of DNA in the presence of different concentrations of SNP at 298 K are shown in Fig. 1.

Fig. 1 (color online) Changes of UV spectra of CTDNA in the presence of different concentrations of SNP (0-80 µM). The absorption intensity at 260 nm was found to be increased with almost no change of λmax. The changes observed in the absorption spectra of calf thymus DNA in the presence of SNP (the increase of the intensity at λmax 260) after mixing with SNP indicating the interaction of the nanoparticles with calf thymus DNA takes place due to a direct formation of a complex of SNP with double-helical CTDNA. This kind of binding may have caused a slight change in the conformation of DNA [24, 25].

8

The values of the binding constant, Kapp, were obtained from the DNA absorption at 260 nm according to the methods published in the literature [26]. For weak binding affinities, the data were examined using linear reciprocal plots based on the following Eq. (1) [27]

1 1 1 = + Aobs − A0 AC − A0 Kapp ( AC − A0)[ SNP]

(1)

Where A0 is the absorbance of CTDNA at 260 nm in the absence of SNP and AC is the recorded absorbance at 260 nm for CTDNA at different SNP concentrations. The double reciprocal plot[Correlation coefficient, R=0.9952] of 1/(Aobs–A0) versus 1/ [Q] is linear and the binding constant (Kapp) can be calculated which was found to be 4.1 ×103 L/mol [Fig 2] from the ratio of the intercept to the slope [26]. The intrinsic binding constant Kapp observed was comparable to other groove binders.

Fig. 2 (color online) Calculation of Kapp of CTDNA-SNP complex; 1/ (Aobs−A0) versus 1/ [Q] plot, Where Q is the concentration of quencher (SNP). Similarly the absorption spectra of SNP and SNP in the presence of increasing concentration of CTDNA solution (0.167-1.0 µg/ml) has been studied [S1 Figure (a)]. It was observed that as the CTDNA concentration increases, the intensity at the wavelength of 420 nm increases initially and then it becomes nearly unaltered but significant changes takes place with 8-10 nm blue shift of λmax. This result is also indicative of complex formation between SNP and CTDNA. 3.2. Fluorescence quenching Studies Ethidium bromide is well-known duplex DNA intercalator, which can cause the enhancement of fluorescence intensity due to intercalation of EB into CTDNA. By any means, in which the potential binding of EB is found to be

9

impaired may cause a decrease in fluorescence intensity [16]. Here, fluorescence titration of solutions containing the DNA and EB with SNP has been investigated. The molecular fluorophores EB, 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 [28, 29]. The fluorescence emission spectra of intercalated EB with increasing concentrations of SNP (10-70 µM) at 293, 303, and 313 K are shown in Figure 3.

Fig. 3 (color online) Fluorescence emission spectra of intercalated EB incubated with calf thymus DNA by increasing concentrations of SNP (10-70 µM) at 293, 303 and 313 K (a, b, c respectively). Figure 3 shows a significant reduction of the intercalated EB intensity in presence of different concentrations of SNP. This decrease of intercalated EB fluorescence indicates there exists a competition between SNP and EB for binding to CTDNA molecule. On the other hand, it can be concluded that the fluorescence intensity of DNA intercalated by EB is quenched when EB is removed from the duplexes by the action of SNP. These results suggest

10

that intercalation of SNP occurs in DNA molecule. The quenching efficiency of SNP is evaluated by the SternVolmer constant (KSV), which varies with the change of experimental conditions:

F0 / F = 1 + kqτ 0 [Q] = 1 + KSV[Q]

(2)

Where F0 and F are the emission intensities in absence and presence of the SNP, respectively and [Q] is the concentration of SNP (quencher). A plot of F0/F vs. [Q] yields a slope equal to Stern Volmer quenching constant (Fig. 4).

Fig. 4 (color online) Stern-Volmer plot of CTDNA-EB in the presence of different concentrations of SNP at different temperatures (293, 303 and 313 K respectively). Fig. 4 shows the Stern-Volmer plot of the SNP. The calculated KSV value of SNP has been presented in Table 1. The Stern-Volmer plot of CTDNA-EB illustrates that the quenching of EB bound to DNA by SNP is in good agreement with the linear Stern-Volmer equation (eq. 2), which proves that the partial replacement of EB from EB bound to DNA by nanoparticle results in a decrease in the fluorescence intensity. The low KSV value (9.85×10-2 L/mol at 293 K) of the SNP shows that it can be bound loosely to the CTDNA molecule [30].

T(K)

R

KSV (L/mol) ×10-2

Kq (L/mol/S)×1010

293

0.9938

9.85

9.85

303

0.9978

11.25

11.25

313

0.9975

11.71

11.71

Table 1 The Stern–Volmer constants, quenching constants of CTDNA by SNP.

11

3.3. Equilibrium binding titration The relationship between the fluorescence intensity of biomolecules and the concentration of quencher can be normally described by deriving, in order to obtain the binding constant (K) and the number of binding sites (n) can be calculated from the following Eq. (3) [31,32]

log

F0 − F = logK + n log[Q] F

(3)

Where F0 and F are the fluorescence intensities of the fluorophore in absence and in presence of different concentrations of SNP [Q] respectively. A plot of log [(F0−F)/F] vs. log [Q] gives a linear plot, whose slope equals to n (the number of binding sites of SNP on CTDNA) and the length of intercept on Y-axis equals to log K (Fig. 5).

Fig.5 (color online) Determination of binding constant and number of binding site from double logarithm plot of fluorescence quenching. The values of K and n are shown in Table 2. The binding constant of SNP with CTDNA was estimated to be 27.54×103 L/mol at 293K. Ligands that are well-known groove binders show low values of binding constants in comparison with ligands that intercalate into DNA [33, 34]. Thus fluorescence intercalators’ displacement assay favors the groove-binding mode.

T(K)

R

K (L/mol)×103

n

293

0.9932

27.54

1.12

303

0.9954

6.46

0.95

313

0.9976

2.45

0.85

12

Table 2 Binding constants (K) and number of binding sites (n) of CTDNA by SNP at 293, 303, and 313 K. 3.4. Thermodynamic parameters of DNA binding The interactive forces like hydrophobic forces, electrostatic interactions, van der Waals interactions, hydrogen bonds, etc. may be involved in-between drug and biomolecules [35, 36]. According to the data obtained for changes in enthalpy (∆H) and entropy (∆S), the mode of interaction between drug and biomolecules can be concluded as [37]: (1) ∆H > 0 and ∆S > 0, hydrophobic forces; (2) ∆H < 0 and ∆S < 0, van der Waals interactions and hydrogen bonds; (3) ∆H < 0 and ∆S > 0, electrostatic interactions [38]. When there is a little change of temperature, the change of enthalpy (∆H) remains unchanged, and thereby its value and that of entropy changes (∆S) can be determined from the Van’t Hoff equation (Eq. 4).

ln k = −∆H / RT + ∆S / R

(4)

∆G = ∆H − T ∆S = −RT ln k

(5)

Where K is the binding constant at the corresponding temperature and R is gas constant. The values of ∆H and ∆S were obtained from the slope and intercept of the linear plot (Eq. (4)) based on lnK versus 1/T (Fig 6).

Fig. 6 (color online) Van’t Hoff plot for the interaction of CTDNA-EB complex and SNP. The free energy change (∆G) was estimated from Eq. (5). The values of ∆H, ∆S and ∆G for the interaction between SNP and DNA are listed in Table 3. An experimental negative value of ∆G revealed that the interaction process is spontaneous, while the negative ∆H and positive ∆S values indicated that the interaction is driven by both hydrophobic and electrostatic interactions. According to the thermodynamic data, the formation of the CTDNA-SNP complex is in favor of enthalpy, entropy and free energy for the formation of the aforesaid complex.

13

T(K)

∆H (KJ/mol)

∆S (J/mol/K)

∆G (KJ/mol)

-100.60

257.73

-25.30

303

-100.60

257.73

-22.73

313

-100.60

257.73

-20.16

293

R 0.9935

Table 3 Thermodynamic parameters for the binding reaction at 293, 303, and 313 K. 3.5. Circular dichroism spectroscopy CD spectroscopy is useful and well known in diagnosing changes in DNA structure during drug-DNA interactions, as the positive band due to base stacking (275 nm) and the negative one due to right-handed helicity (245 nm) are quite sensitive to the mode of DNA interactions with small molecules [30]. The shift in the CD signals of DNA due to interaction of CTDNA with drugs may often be designated to the corresponding changes in the DNA structure [39]. Simple groove binding and electro-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 [39]. The CD spectra of the SNP with double-stranded DNA can provide the useful information about the SNP-Nucleotide interaction.

Fig. 7 (color online) CD spectra of CTDNA incubated with SNP at different concentrations of 0, 25, 50, and 75 µM. Thus, CD spectra of calf thymus DNA incubated with SNP at different concentrations were recorded. The observed CD spectrum of DNA consists of a positive band at 275 nm due to base stacking and a negative band at 245 nm due

14

to helicity, which are characteristics of DNA in the right handed B form. When SNP were incubated with DNA, the CD spectra exhibited changes of mainly the positive bands (Figure 7). As seen in Fig. 7 after adding different concentrations of SNP, the intensities of positive and negative bands decreased. Since alterations in the positive bands of DNA are more effective compared to the negative region, so it can be assumed that SNP may not significantly change the helicity of DNA. This observation is in agreement with and indicative of groove binding of SNP to CTDNA. 3.6. Melting studies DNA denaturation, also called DNA melting, is the process by which double stranded DNA unwinds and separates into single stranded DNA through the breaking of hydrogen bonds between the bases. When solutions of DNA are exposed to extremes of pH or heat, the double helical structure of DNA undergoes a transition into a randomly single stranded form at the melting temperature (Tm). The intercalative mode of binding can increase Tm by about 13–14oC, but the non-intercalative binding causes no such apparent increase in Tm [40, 41]. The normal method of determining the melting temperatures (Tm) is UV–Vis spectroscopy. Thermal behaviors of DNA in the presence of SNP can give the information about the DNA conformational changes when the temperature is raised, and it can also predict about the strength of interactive forces existing between nanoparticles and DNA. According to the literature, [42, 43] the intercalation of natural or synthesized organic and metallo-intercalators generally results due to considerable increase in melting temperature.

Fig. 8 (color online) Melting curves of CTDNA in the absence and presence of the SNP (50 µg/ml). The melting curves of DNA in absence and presence of the SNP are presented in Fig. 8. Here, the thermal denaturation experiment carried out for DNA in absence of SNP revealed a Tm value of 66 (±0.2 °C) under the

15

experimental conditions, whereas the observed melting temperature of DNA in presence of SNP in turn increased to (Tm) 71 (± 0.2 °C). Addition of SNP to DNA resulted in a small increase in melting temperature by 5 ± 0.2°C. DNA melting studies revealed that binding of SNP to CTDNA resulted in a small increase in melting temperature, which is unlikely in case of classical intercalators; rather, it indicates external binding. Small change in melting temperature has also been observed in case of an unusual mode of binding through partial intercalation [44]. 3.7. Viscosity study The binding mode of SNP was further investigated by viscosity measurements. Hydrodynamic method, such as determination of viscidity, can be considered to be highly sensitive to the change in length of DNA. A classical intercalation model depicts lengthening of DNA helix with the separation of DNA base pairs for adapting the bound molecule, leading to the increase in DNA viscosity. In contrast, a partial, non-classical intercalation of molecule could bend (or introduce a kink) the DNA helix, reducing its length and, consequently, its viscosity. In addition, molecules that binds exclusively in the DNA grooves by partial and/or nonclassical intercalation, under the same conditions, typically cause less pronounced (positive or negative) or no change in DNA solution viscosity [45]. The effect of the addition of different concentrations of SNP (30-300µM) on the viscosity of CTDNA (50 µg/ml) is shown in Fig. 9.

Fig. 9 (color online) Effect of increasing amounts of SNP on the relative viscosity of CTDNA (50 µg/ml) at 298 K. The values of relative specific viscosity (η /η0)1/3 versus 1/R (R = [DNA]/[complex]) has been plotted in Fig. 9. The viscosity is found to decrease slowly with increasing concentration of SNP, and thus the observation supports the groove binding nature of the molecule rather than classical intercalation [46].

16

3.8. Cytotoxicity of silver nanoparticles Cytotoxicity of nanoparticles has been a robust research area in recent years. Silver nanoparticles showed different degrees of in vitro cytotoxicity [13, 47, 48]. The in vitro anticancer property of the SNP was studied by testing it on human cervical cancer cell line HeLa. Cell cytotoxicity of the SNP was studied on human cervical cancer cell line (HeLa). In this study, HeLa cells were treated with various concentrations of SNP and silver ion ranging from 0 to 100 µM for 18 h and the morphology of the cells were visualized under light microscope (Carl Zeiss). The common and easily noticeable effect following exposure of cells to toxic materials is the alteration in cell shape or morphology in a monolayer culture. The cell morphology at various concentrations are shown in Figure 10.

Fig.10 (color online) Morphology of HeLa cell after treatment with silver salt (a - d) & SNP (e - h) of different doses, 0 µM (a, e) 10 µM (b, f), 50 µM (c, g) & 100 µM (d, h) It is clear from the figure that silver ion is more toxic than the corresponding dose of SNP. For example, the micrograph of HeLa cells at 10 µM and 50 µM concentrations of silver ions seems to have undergone considerable damage, whereas HeLa cells after being treated with SNP of same doses looks healthier as shown in Figure 10. The cells are wrinkled, distorted and severely damaged at 100 µM concentration of both silver ions and nanoparticle as shown in Figure 10d & h. The cytotoxicity was detected by MTT assay and % cell survival was plotted against the concentration as shown in Figure 11.

17

Fig. 11 (color online) Cell survival of HeLa cells after treating with SNP and normal silver salt solution. The dose at which 50% cells are viable (LD50) of SNP and silver ion was determined from Fig. 11 and the values were found to be 70 µM and 30 µM respectively. The dose-dependent reduction of cell survival was seen in case of both silver ion and SNP treatment but the former has implicated magnified effect than the latter. So, it is clear that SNP imposes lesser toxicity on HeLa cells as compared to silver ion [49]. 3.9. Cell staining of nanoparticles We were also interested to investigate whether SNP can stain cultured mammalian cells since this compound has its own fluorescence property [S1 Figure (b)]. It was observed that the compounds have intrinsic fluorescence in the region around 475 nm with excitation near 410 nm. According to the method as described earlier, staining ability of these nanoparticles on HeLa cells was examined. Silver nanoparticles were found to be potent enough to stain the cultured HeLa cells after fixing. Fixing make the cells porous and the compounds were able to penetrate into cells after fixing and gave fluorescence with excitation at UV range near 410 nm as observed under fluorescence microscope.

Fig.12 (color online) HeLa cells stained with Hoechst dye (a) and SNP (b)

18

We observed that SNP can stain the HeLa cells and at 50 µM concentration it gives comparable fluorescence as that of Hoechst dye under fluorescence microscope (Axio Scope A1, Carl Zeiss) with UV filter. The image of HeLa cells using both Hoechst dye as standard and our synthesized nanoparticle is shown in Fig. 12. 4. Conclusion From the above results it can be concluded that binding of SNP to CTDNA occurs and here SNP acts as a groovebinder showing possibilities of partial intercalation as evident from the spectroscopic, hydrodynamic, DNA melting experiments. The HeLa cell growth in presence of SNP was found to be comparatively less toxic than the silver ion. To design anticancer drug with lower side effects in future this investigation, where SNP inhibits human cancer cell growth will be relevant. Acknowledgement One of the authors SR thanks Department of science technology (DST) of India, for his DST INSPIRE fellowship. UG thanks Department of Biotechnology (DBT) of India for partial fulfillment of consumables required for this work. UG is also grateful to DST (New Delhi) for providing Axio Vision 2 (Carl Zeiss) under DST-FIST program. This work was also supported by research funds from University of Kalyani, UGC and DST–PURSE, Govt. of India. References [1] A. S. Karakoti , L. L Hench, S. Seal, Journal of the Minerals, Metals and Materials Society 58 (2006) 77-82. [2] C. F. Jones, D. W. Grainger, Adv Drug Deliv Rev. 61(2009) 438-456. [3] H. Yang, C. Liu, D. Yang, H. Zhang, Z. Xi, J Appl Toxicol. 29 (2009) 69–78. [4] N. Lewinski, V. Colvin, R. Drezek, Small 4 (2008) 26-49. [5] P. V. AshaRani, G. L. K. Mun, M. P. Hande, S. Valiyaveettil, ACS Nano 3 (2009) 279–290. [6] Q. Y. Chen, D. H. Hi, Y. Zhao, J. X. Guo, Analyst 124 (1999) 901–906. [7] Y. Ni, D. Lin, .S Kokat, Anal Biochem. 352 (2006) 231–242. [8] J. Zheng, X. Wu, M. Wang, D. Ran, W. Xu, J. Yang, Talanta 74(2008) 526-32. [9] H. Zhou, X. Wu, J. Yang, Talanta 78 (2009) 809-13. [10] S. Basu, S. Jana, S. Pande, T. Pal, J Colloid Interface Sci. 321 (2008) 288-93. [11] I. Hag, Arch. Biochem. Biophys. 403 (2002) 1-15.

19

[12] Y. Pan, S. Neuss, A. Leifert, M. Fischler, F. Wen, U. Simon, G. Schmid, W. Brandau, W. J. Dechent, Small 3 (2007) 1941-1948. [13] N. Asare, C. Instanes , W. J. Sandberg, M. Refsnes, P. Schwarze, M. Kruszewski, G. Brunborg, Toxicology 291 (2012) 65– 72. [14] S. Roy, T. Mukherjee, S. Chakraborty, T. K. Das, Dig. J. Nanomater. Biostruct. 8 (2013) 197-205. [15] S. Roy, T. K. Das, Nanosci. Nanotechnol. Lett. 6 (2014) 181-189. [16] A. G. Krishna, D. V. Kumar, B. N. Khan, S. K. Rawal, K. N. Ganesh, Biochim Biophys Acta. 1381(1998) 104112. [17] L. Pérez-Flores, A. J. Ruiz-Chica, J. G. Delcros, F. M. Sánchez-Jiménez, F. J. Ramírez, Spectrochim Acta A Mol Biomol Spectrosc. 69 (2008) 1089-1096. [18] F. M. Freimoser, C. A. Jakob, M. Aebi, U. Tuor, Appl. EnViron. Microbiol. 65 (1999) 3727-3729. [19] K. Bishayee, S. Ghosh, A. Mukherjee, R. Sadhukhan, J. Mondal, A. R. Khuda-Bukhsh, Cell Prolif. 46 (2013) 153–163. [20] U. Ghosh, M. Nagalakshmi, R. Sadhukhan, A. Girigoswami, Adv. Sci Eng. Med. 5 (2013) 783-788. [21] K. C. Majumdar, S. Ponra, T. Ghosh, R. Sadhukhan, U. Ghosh, Eur. J. Med. Chem. 71 (2014) 306-315. [22] U. Ghosh, N. P. Bhattacharyya, Mol. Cell Biochem. 320 (2009) 15-23. [23] S. Mukherjee, C. Basu, S. Chowdhury, A. P. Chattopadhyay, A. Ghorai, U. Ghosh, H. Stoeckli-Evans, Inorg. Chim. Acta 363 (2010) 2752-2761. [24] G. S. Son, J. A. Yeo, M. S. Kim, S. K. Kim, A. Holmen, B. Akerman, B. J. Norden, J. Am. Chem. Soc. 120 (1998) 6451-6457. [25] T. M. Kelly, A. B. Tossi, D. J. McConnell, T. C. Strekas, Nucl. Acids Res. 13 (1985) 6017-1634. [26] J. J. Stephanos, J. Inorg. Biochem. 62 (1996) 155–169. [27] R. Marty, C. N. N'soukpoé-Kossi, D. Charbonneau, C. M. Weinert, L. Kreplak, H. A. Tajmir-Riahi, Nucleic Acids Res. 37 (2009) 849–857. [28] J. L. Butour, J. P. Macquet, Eur J Biochem. 78 (1977) 455-63. [29] A. J. Geall, I. S. Blagbrough, J Pharm Biomed Anal. 22 (2000) 849-59. [30] G. Psomas, A. Tarushi, E. K. Efthimiadou, Polyhedron 27 (2008) 133-138. [31] M. Jiang, M. X. Xie, D. Zheng, Y. Liu, X. Y. Li, X. Chen, J. Mol. Struct. 692 (2004) 71–80.

20

[32] S. Roy, T. K. Das, J Nanosci. Nanotechnol. 14 (2014) 4899-4905. [33] D. L. Boger, B. E. Fink, M. P. Hedrick, J Am Chem Soc. 122 (2000) 6382–6394. [34] M. Lee, A. L. Rhodes, M. D. Wyatt, S. Forror, J. A. Hartley, Biochemistry 32 (1993) 4237–4245. [35] D. P. Ross, S. Subramanian, J. Biochemistry 20 (1981) 3096–3102. [36] S. Roy, T. K. Das, Nanosci. Nanotechnol. Lett. 6 (2014) 547-554. [37] D. B. Nike, P. N. Moorty, K. I. Priyadarsini, Chem. Phys.Lett. 168 (1990) 533–538. [38] N. Shahabadi, A. Fatahi, J. Mol. Struct. 970 (2010) 90–95. [39] D. M. Kong, J. Wang, L. N. Zhu, X. Z. Li, H. X. Shen, H. F. Mi, J. Inorg. Biochem. 102 (2008) 824-832. [40] G. A. Neyhart, N. Grover, S. R. Smith, W. A. Kalsbeck, T. A. Fairley, M. Cory, H. H. Thorp, J Am Chem Soc. 115 (1993) 4423–4428. [41] C. V. Kumar, R. S. Turner, E. H. Asuncion, J. Photochem. Photobiol. A: Chem. 74 (1993) 231-238. [42] S. S. Jain, M. Polak, N. V. Hud, Nucleic Acids Res. 31 (2003) 4608-15. [43] J. L. Mergny, G. Duval-Valentin, C. H. Nguyen, L. Perrouault, B. Faucon, M. Rougee, T. MontenayGarestier, E. Bisagni, C. Helene, Science 256 (1992) 1681-1684. [44] A. Rajendran, B. U. Nair, Biochim Biophys Acta 1760 (2006) 1794–1801. [45] S. Satyanarayana, J. C. Dabrowiak, J. B. Chaires, Biochemistry. 31 (1992) 9319–9324. [46] G. L. Eichhorn, Y. A. Shin, J Am Chem Soc. 90 (1968) 7323-28. [47] Y. H. Hsin, C. F. Chen, S. Huang, T. S. Shih, P. S. Lai, P. J. Chueh, Toxicol. Lett. 179 (2008) 130–139. [48] S. Park, Y. K. Lee, M. Jung, K. H. Kim, N. Chung, E. K. Ahn, Y. Lim, K. H. Lee, Inhal. Toxicol. 19 (2007) 59–65. [49] N. Pernodet, X. Fang, Y. Sun, A. Bakhtina, A. Ramakrishnan, J. Sokolov, A. Ulman, M. Rafailovich, Small 2 (2006) 766–773.

21

Interaction Studies between Biosynthesized Silver Nanoparticle with Calf Thymus DNA and Cytotoxicity of Silver Nanoparticles

Swarup Roy*, Ratan Sadhukhan, Utpal Ghosh, and Tapan Kumar Das Department of Biochemistry and Biophysics, University of Kalyani, Kalyani – 741235, West Bengal, India

Interaction between biosynthesized silver nanoparticles and Calf thymus DNA and study of silver nanoparticles toxicity as well as cell staining ability has been carried out in this work.

22

Highlight

► Interaction between CT-DNA and biosynthesized SNP. ► Groove binding with partial intercalation of SNP to CT-DNA. ► Cytotoxicity and cell staining of SNP on HeLa cell line.