Biosynthesis of gold nanoparticles using Sargassum swartzii and its cytotoxicity effect on HeLa cells

Biosynthesis of gold nanoparticles using Sargassum swartzii and its cytotoxicity effect on HeLa cells

Accepted Manuscript Biosynthesis of gold nanoparticles using Sargassum swartzii and its cytotoxicity effect on HeLa cells T. Stalin Dhas, V. Ganesh Ku...

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Accepted Manuscript Biosynthesis of gold nanoparticles using Sargassum swartzii and its cytotoxicity effect on HeLa cells T. Stalin Dhas, V. Ganesh Kumar, V. Karthick, K. Govindaraju, T. Shankara Narayana PII: DOI: Reference:

S1386-1425(14)00822-1 http://dx.doi.org/10.1016/j.saa.2014.05.042 SAA 12204

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

4 April 2014 3 May 2014 9 May 2014

Please cite this article as: T. Stalin Dhas, V. Ganesh Kumar, V. Karthick, K. Govindaraju, T. Shankara Narayana, Biosynthesis of gold nanoparticles using Sargassum swartzii and its cytotoxicity effect on HeLa cells, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa. 2014.05.042

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Biosynthesis of gold nanoparticles using Sargassum swartzii and its cytotoxicity effect on HeLa cells T. Stalin Dhasa, V. Ganesh Kumara*, V. Karthicka, K. Govindarajua, T. Shankara Narayanab a

Nanoscience Division, Centre for Ocean Research, Sathyabama University, Chennai 600 119, India.

b

Agricultural research station, Acharya N. G. Ranga Agricultural University, Kadiri-515 591, Andhra Pradesh, India

*Corresponding author: Tel: +91 44 24500646; Fax: +91 44 24500646 E-mail: [email protected] (Dr. V. Ganesh Kumar)

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Abstract In this investigation, biological synthesis of gold nanoparticles (AuNPs) using Sargassum swartzii and its cytotoxicity against human cervical carcinoma (HeLa) cells is reported. The biological synthesis involved the reduction of chloroauric acid led to the formation of AuNPs within 5 min in a water bath at 60 °C and the formation of AuNPs was confirmed using UV –vis spectrophotometer. The AuNPs were stable; spherical in shape with well–defined dimensions, and the average size of the particle is 35 nm. A zeta potential value of –27.6 mV revealed synthesized AuNPs were highly stable. The synthesized AuNPs exhibited a dose–dependent cytotoxicity against human cervical carcinoma (HeLa) cells. Furthermore, induction of apoptosis was measured by DAPI (4′, 6–Diamidino–2–phenylindole dihydrochloride) staining.

Keywords: Sargassum swartzii; gold nanoparticles; HeLa; anticancer; apoptosis.

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Introduction Nanoparticles mediate biological and chemical reactions more effectively due to their large surface areas. Nanoparticles offer a vast potential in the field of nanomedicine. Gold nanoparticles (AuNPs) provide several opportunities in imaging, diagnostics and therapeutics [1] [2, 3]. Various approaches are in practice to generate AuNPs by chemical reduction [4], photochemical [5], radiation [6] and electrochemical [7] methods. Recently, biological synthesis of nanoparticles is being used more commonly, as it is ecofriendly and these nanoparticles have superior biomedical applicability [8–10]. Biological methods of AuNPs synthesis using plants [11, 12] and algae [13, 14] have been studied in recent years. The photothermal properties of AuNPs have been exploited for localized heating for sustained release of drug thus proving its ability in drug delivery studies [15]. The possibility of nanomaterials entering the human body could be through ingestion, inhalation, penetration, and dermal contact [16]. Brown algae Sargassum species are well exploited for their medicinal value [17, 18] and have been used for the biosynthesis of AuNPs from their extracellular polysaccharides [19]. The extracts of Sargassum swartzii possess broad range of pharmacologic activities, such as anti–inflammatory, anticancer, and antioxidant properties [20–22]. Cytotoxicity depends on the type of cells, e.g. AuNPs inhibit the growth rate of HeLa cells, and their inhibitory effect is inversely proportional to the degree of aggregation of AuNPs [23]. In addition to aggregation, AuNPs–induced cytotoxicity is

size–dependent

in

several

mammalian cell

types

[24,

25].

DAPI

(4′, 6–Diamidino–2–phenylindole dihydrochloride), a fluorescent chromophore binds to double–stranded DNA in the nuclei of all cells, helps to analyze the nuclear damage [26, 27], and is known to form fluorescent complexes with natural double–stranded DNA, showing fluorescence specificity for adenine-thymine (AT), and adenine–uracil (AU) clusters. When 3

DAPI binds to DNA, its fluorescence is strongly enhanced [28]. Because of this property, DAPI is used as a powerful tool in various cytochemical investigations. In the current study, the cytotoxicity effect of AuNPs (synthesized using S. swartzii) against human epidermoid larynx carcinoma (HeLa) cells is addressed. Materials and methods Preparation of seaweed extract and synthesis of gold nanoparticles S. swartzii seaweed was collected from Mandapam, Rameswaram, Tamilnadu, India, and was cleaned with double distilled water and shade-dried for a week at room temperature, S. swartzii seaweed was ground to powder and stored for further study. The extract was prepared by dissolving 5 g of dry seaweed powder in 50 mL of distilled water in a conical flask, and the flask was kept on an orbital shaker for 3 h and filtered using Whatman No.1 filter paper. Chloroauric acid (HAuCl4) was purchased from Loba Chemie, India and used as received. For the synthesis of AuNPs, 200 µL, 400 µL, 600 µL, 800 µL and 1000 µL of the extract were added to 5 mL of 1 mM chloroauric acid solution, and it was placed in a water bath at 60° C for the reduction of Au3+ to Au0. Characterization of gold nanoparticles The light absorption pattern of AuNPs was kinetically monitored in the range of 200 to 800 nm and recorded using UV–vis spectrophotometer (Shimadzu UV–1800). Fourier transform infrared (FTIR) spectra of AuNPs were recorded by using the KBr pellet technique on a Perkin Elmer 983/G detector double beam spectrophotometer to identify the possible functional groups. For High Resolution Transmission Electron Microscopy (HR–TEM) imaging, the aqueous solutions of AuNPs were dropped on carbon-coated copper grids. The films were dry prior to the measurement of the AuNPs, and operated at an accelerating voltage of 300 kV using 4

JEOL 3010. X–ray diffraction (XRD) pattern of the AuNPs was analyzed out using Rigaku SMART lab instrument operated at a voltage of 40 kV and a current of 30 mA with Cu Kα1 (λ=1.5406 Å) radiation. A semi contact mode atomic force microscopy study was carried out using titanium nitride coated tips with different frequencies (NTEGRA PRIMA–NTMDT). The effective surface charges on the AuNPs were measured using zeta–potential and the particle sizes were as determined by dynamic light scattering (DLS) in back scattering mode, using a laser particle zetasizer (Malvern 110, Zetasizer) at 25 ºC. Cytotoxicity assay HeLa cell lines were obtained from National Centre for Cell Science (Pune, India) and were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with antibiotic agents (penicillin 120 IU/mL and streptomycin 100 IU/mL) and 10% fetal bovine serum (FBS). The cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C. Effect of AuNPs on the proliferation of cells was determined by MTT (3–(4, 5–dimethylthiazol–2–yl)–2, 5– diphenyltetrazolium bromide) assay. Briefly, the cells were grown to 1 × 105 cells/well in 96 –well plates and then incubated with various concentrations of AuNPs (15.63, 31.25, 62.5, 125, 250 and 500 μg/mL) at 37 ◦C with 5% CO2 for 24 h, cell viability was measured by MTT assay [29] and 50 % inhibitory concentration (IC50) was calculated. DAPI Staining HeLa cells (1x105) were washed with PBS and fixed with 3.7% paraformaldehyde in PBS for 20 min at room temperature. Fixed cells were washed with PBS and stained with DAPI (Sigma) solution for 10 min at room temperature and subsequently examined by epifluorescence microscopy (Eclipse 80i, Nikon, Japan).

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Results and Discussion The appearance of ruby red color within 5 min confirmed the formation of AuNPs and no further color change indicated the completion of reaction. UV–vis spectroscopy technique was used to ascertain the formation of AuNPs. Figure 1 shows that 1000 µL of S. swartzii extract with 5 mL of chloroauric acid gave a sharp intense peak at 527 nm compared to that of other concentrations. It is well known that AuNPs exhibit a ruby red color in mixture of solution due to surface plasma resonance (SPR) of AuNPs [30]. Fourier transform infrared spectroscopy (FTIR) was used to identify the possible biomolecules present in S. swartzii extract which are responsible for capping and lead to efficient stabilization of AuNPs. Figure 2 shows the FTIR spectra recorded for AuNPs synthesized using S. swartzii where strong bands were observed at 3360, 2346, 1638, 1424, 1130, 854 and 613 cm−1. The peak at 3360 cm−1 indicates the involvement of –OH (alcohol) or –COOH (carboxylic) groups in reduction. The peak at 1638 cm−1 indicates the role amide I group from biomolecules, probably from proteins present in the algae [31, 32]. Similarly, the peak at 1424 cm−1 indicates the C–H bend. Furthermore, the reduction is evident from the alcoholic (C–O) stretched at 1130 cm−1 confirmed the reduction of AuNPs. HR–TEM micrograph indicates the nanoparticles were spherical and few particles were hexagonal in shape (Fig. 3A & B). The synthesized AuNPs were formed in the range of 20–60 nm in size. The phase purity of AuNPs was analyzed using XRD where four diffraction peaks were observed in the 2θ range 20–80°, with reference to the relative intensity and position the peaks can be indexed as (111), (200), (220) and (311) reflections of face centered cubic (fcc) structure of metallic gold (JCPDS No: 04–0784) revealing that synthesized AuNPs were of pure

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crystalline gold (Figure 4). The particle size was calculated using Debye–Scherrer equation [33], which was found to be 37 nm and the result, agrees with TEM. AFM analysis was carried out to analyze the surface morphology of AuNPs (Fig. 5 A–C). Supplementary data S1A shows AuNPs ranging from 30 to 70 nm with spherical and triangular shapes. The height profile analysis of AuNPs indicates that the thickness of the particles is in the range 13–35 nm (Inset Supplementary data S1A). The zeta potential measurement of synthesized AuNPs showed the surface charges of particles exhibited a value of –27.6 mV suggesting higher stability of AuNPs (Fig. 5A). The size distribution of synthesized particles was examined by DLS analyzer (Fig. 5B). DLS data suggest that the size of AuNPs in aqueous solution range from 14–70 nm. Evidently, the particles size by DLS analysis is larger than the particles size by TEM analysis. The larger DLS values are usually attributed to the hydrodynamic radius probed with DLS [34]. Figure 6 shows the concentration–dependent toxicity effect on cells upon exposure to AuNPs. The inhibitory concentration (IC50) after 24 h exposure of AuNPs, which reduces the mitochondrial function by 50 % was found to be 41.10 µg/mL for HeLa cells. The cytotoxicity attributed to cells by AuNPs is based on the ability of mitochondrial dehydrogenase enzyme from viable cells to cleave the tetrazolium rings of the pale yellow MTT and form dark blue formazan crystals, largely impermeable to the cell membranes [35]. The toxicology effects (nuclear damage) of AuNPs were also analyzed by studying the morphology of cells using DAPI staining. The fluorescence image (Fig. 7A) of untreated cells shows no double–strand breaks in nuclei whereas, after treatment of HeLa cells with AuNPs image shows condensed and fragmented chromatin (Fig. 7B). Fascinatingly, previous studies reported nuclear fragmentation in AuNPs treated cells [36, 37]. This result clearly indicates that synthesized AuNPs induced 7

apoptosis in HeLa cells. It should be noted that the DAPI staining clearly indicates the cytotoxicity of AuNPs of cancer cells. Conclusions A simple biological synthesis of AuNPs using marine brown alga S. swartzii has been discussed. The synthesized AuNPs were characterized using spectroscopic and microscopic techniques. Further, the synthesized AuNPs exhibited well defined and colloidal stability dimensions observed in HR–TEM and zeta potential analysis. The cytotoxicity effect of AuNPs on HeLa cell lines was assessed using cell viability and staining techniques. Thus proving that AuNPs exert cytotoxicity effect on human cervical carcinoma (HeLa) cell lines. Acknowledgements We thank DST–Nanomission, Government of India for its financial support to the project (SR/NM/NS-06/2009) and the management of Sathyabama University, Chennai for their stanch support in research activities. SAIF–IIT Madras and CNSNT Sathyabama University are gratefully acknowledged for characterization studies.

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Legends Figure 1

UV–vis spectrum obtained for gold nanoparticles synthesized using various concentrations of seaweed extract.

Figure 2

FT–IR spectra of gold nanoparticles synthesized using S. swartzii.

Figure 3

HR–TEM images indicating the presence of spherical and hexagonal gold nanoparticles (A & B).

Figure 4

X–ray diffraction pattern of gold nanoparticles obtained from S. swartzii.

Figure 5

Zeta potential determination (A) and Size distribution of synthesized gold nanoparticles (B).

Figure 6

Cytotoxicity effects of gold nanoparticles on HeLa cells.

Figure 7

Fluorescence microscopic study of DAPI staining of control cells (A) and gold nanoparticles treated cells (B).

i

1.0

1000 L

Absorbance (a.u.)

800 L 0.8

600 L 400 L 200 L

0.6

0.4

0.2

0.0 400

500

600

Wavelength (nm)

Figure 1

ii

700

Figure 2

iii

Figure 3 iv

350

(111)

Intensity (a.u.)

300 250 200 150

(200) (220)

100

(311)

50 0 20

30

40

50

2 theta (degree)

Figure 4

v

60

70

80

Figure 5 vi

Figure 6

vii

Figure 7 viii

GRAPHICAL ABSTRACT

+

HIGHLIGHTS

 Synthesis of AuNPs using marine seaweed resulted in 35 nm sized particles  The role of biochemical components present in the AuNPs is causing cytotoxicity  Gold nanoparticles induced cytotoxicity in term of mitochondrial damage