Printer Friendly

Toxicity study of silver nanoparticles synthesized from Suaeda monoica on Hep-2 cell line.

Introduction

Suaeda monoica is a salt marsh herb growing in hyper saline soils that belongs to Chenopodiaceae and is distributed throughout the East and West coast mangroves in India. Traditionally, the leaf from S.monoica is known to be used as a medicine for hepatitis and scientifically it is reported to be used as ointment for wounds and possess antiviral activity because of the presence of triterpenoids, sterols (1).

Rapidly developing field of nanoscience had raised the possibility of using therapeutic nanoparticles in the diagnosis and treatment of human cancers (2). Nanoscale particles and molecules are a potential alternative for treatment of disease because they have unique biological effects based on the structure and size, which differ from traditional small molecule drugs (3).

In the last few years, several pharmaceuticals companies had obtained approval from the US Food and Drug Administration (FDA) for the development of nanotechnology based drugs. Silver had long been recognized as having an inhibitory effect towards many bacterial strains and micro organisms commonly present in medical and industrial processes (4). The most widely used and well known applications of silver and silver nanoparticles includes topical ointments and creams containing silver to prevent infection of burns and open wounds (5).

Production of nanoparticles can be achieved through different methods. Chemical approaches are the most popular methods for the production of nanoparticles. However, some chemical methods cannot avoid the use of toxic chemicals in the synthesis protocol. Since noble metal nanoparticles such as gold, silver and platinum nanoparticles are widely applied to human contacting areas, there is a growing need to develop environmentally friendly processes of nanoparticles synthesis that do not use toxic chemicals. Biological methods of nanoparticles synthesis using microorganisms (6), enzyme (7), and plant or plant extract have been suggested as possible ecofriendly alternatives to chemical and physical methods. Using plant for nanoparticles synthesis can be advantageous over other biological processes by eliminating the elaborate process of maintaining cell culture (8).

The role of silver nanoparticles as an anticancer agent should open new door in the field of medicine. Silver nanoparticles should serve as one of the best ways of treating diseases that involve cell proliferation and cell death (9). In the present study, we screened salt marsh plant species S.monoica leaf extracts for extracellular silver nanoparticles synthesis and to determine the cytotoxicity threshold in this Human Epidermoid Larynx Carcinoma (Hep-2) cell line.

Materials and Methods

Plant material

S.monoica leaves freshly were collected from the Kollidam coast (Tamil Nadu) India. The specimen was certified by Botanical Survey of India (BSI) Coimbatore, and documented in the Herbaria of C.A.S. in Marine Biology (Voucher No. AUCASMB15), Annamalai University, India.

Synthesis of silver nanoparticles

The analytical grade silver nitrate was purchased from Sigma Chemicals (Mumbai). The leaves weighing 25 g were thoroughly washed in distilled water, dried, cut into fine pieces and were crushed into 100 ml sterile distilled water and filtered through what man No.1 filter paper. The filtrate was further filtered through 0.6 m sized filters and stored at 4 [degrees]C. Ten ml suspension of leaf extract was added to 90 ml aqueous solution of silver nitrate (1 mM) solution separately for reduction in to Ag+ ions and incubated at room temperature (35 [degrees]C) for 5 hr. After 5 hr of incubation the silver nanoparticles were isolated and concentrated by repeated (4-5 times) centrifugation of the reaction mixture at 10,000xg for 10 min. The supernatant was replaced by distilled each time and suspension stored as lyophilized powder. The optical measurements, was carried out by UV-Vis spectrophotometer (UV- 2450 (Shimadzu) and scaned the spectra between 200-700 nm at the resolution of 1nm.

Atomic force microscope

Purified SNP (Silver Nanoparticles) in suspension also characterized their morphology using a VEeco diNanoscope 3D AFM (Atomic Force Microscope). A small volume of sample was spread on a well-cleaned glass cover slip surface mounted on the AFM stub, and was dried with nitrogen flow at room temperature.

Images were obtained in tapping mode using a silicon probe cantilever of 125 m length, resonance frequency 209-286 kHz, spring constant 20-80 nm-1 minimum of five images for each sample were obtained with AFM and analyzed to ensure reproducible results.

Cell culture

The Hep-2 cell line was purchased from National Cell Centre, Pune (India). Cancerous cells were seeded in flask with MEM (Minimal Essential media) medium with 2-10% Fetal Calf Serum (FCS) and incubated at 37[degrees]C in a 5% C[O.sub.2]. After 24 hr incubation period the attached cells were trypsinizated for 3-5 min and centrifuged (1,400 rpm, 5 min). The cells were counted and distributed in 96 well ELISA plate with 10,000 cells in each well. The plate was incubated 24 hr at 37 [degrees]C in a 5% C[O.sub.2] atmosphere to allow the cells attach to the bottom of the well (10).

Cell treatment with silver nanoparticles

Silver nanoparticles reduced ATP content of the cell, caused damage to mitochondria and increased production of Reactive Oxygen Species (ROS) in a dose-dependent manner (10). Hence we determined the toxicity of silver nanoparticles at different concentrations (15, 30, 62, 125, 250, 500, 1000 [micro]g/ml) and duplicates added to the wells were incubated in to as grown cell (1x[10.sup.4] cells/well) and the cell population was determined by optical microscopy at 24 and 48 hr.

MTT assay

Cell viability was evaluated by the MTT colorimetric technique with slight modification (11). Briefly, in each well 200 [micro]l of MTT [3-(4, 5-dimetheylthiazol-2)-2, 5 diphenyl tetrazolium bromide] without phenol red, yellowish in color solution (5 mg/ml in PBS), was added to each well. The plates were incubated for 6-7 hr in 5% C[O.sub.2] incubator for reduction of MTT by metabolically active cells, in part by the action of dehydrogenase enzymes, to generate reducing equivalents such as NADH and NADPH. The resulting intracellular purple formazon solubilized the MTT crystals by adding and quantified by spectrophotometric mean and then the supernatants were removed.

For solubilization of the MTT crystals, 100 [micro]l of isoproponal or DMSO was added to the wells. The plates were placed on a shaker for 15 min for complete solubilization of crystals and then the optical density of each well was determined. The quantity of formazan product, as measured by the amount of 595 nm using a scanning Multiwell spectrophotometer (Biorad, Model 680, Japan) and DMSO, served as a blank. The absorbance is directly proportional to the number of living cells in cultural.

Each experiment was done in duplicate. The relative cell viability (%) related to control wells containing cell culture medium without nanoparticles as a vechicle was calculated by [(A).sup.test]/[(A).sub.control]x100; where [(A).sub.test] is the absorbance of the test sample and [(A).sub.control] is the absorbance of control sample.

Statistical analysis

All experiments were done in duplicate, and the results were presented as mean[+ or -]standard deviation. The experimental data were analyzed by using SPSS. Statistical significance was accepted at a level of p<0.05 to calculate [IC.sub.50] values.

Results

The leaf extract of S.monoica was used for the synthesis of silver nanoparticles. The reaction started within first hr of the incubation with silver nitrate (1 mM). The primary detection of synthesized silver nanoparticles was carried out in the reaction mixture by observing the colour change of the medium from greenish to dark brown. The reaction rate was maximum after 25 hr of incubation as indicated by the formation of silver nanoparticles.

Our findings showed resemblance to the results already reported by in the case of extract of Capsicum annum (12) and in case of extract of Aloe vera (13). They reported that when the extracts of their respective test plants were challenged with silver nitrate (1 mM), it turned brown and the intensity of colour was increased with the time of incubation.

In order to verify the synthesis of silver nanopartices, the test samples were subjected to the UV-Vis spectrophotometer analysis after 5 hr of incubation. A peak specific for the synthesis of silver nanoparticles was obtained at 430 nm. AFM is used for morphological characterization of SNP (14). The shape of the SNP synthesized by leaf extract was spherical and was found to be in the range of 31 nm (Figure 1).

[FIGURE 1 OMITTED]

The in vitro cytotoxicity effects of silver nanoparticles were screened against human epidermoid larynx carcinoma cell lines by means of MTT assay. The silver nanoparticles were able to reduce viability of the Hep-2 cells in a dose-dependent manner, as shown in figure 2. After five hr of treatment, the silver nanoparticles were found to be cytotoxic to tumour cells at a concentration of 500 nM and higher. Silver nanoparticles at 500 nM decreased the viability of Hep-2 cells (Figure 3) to 50 % of the initial level, and this was chosen as the [IC.sub.50]. Longer exposures resulted in additional toxicity to the cells.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

These results demonstrate that silver nanoparticles mediate a dose and time dependent increase in toxicity. The result suggested that the plant mediated synthesized silver nanoparticles possess great selectivity to cancer cell and can display potential application in cancer chemoprevention and chemotherapy. Silver nanoparticles revealed to have important anti angiogenic properties (15); so are attractive for study of their potential antitumor effects. Compounds possessing antiangiogenic properties are known for their potential ability to block the activity of abnormally expressed signaling proteins (16).

Discussion

In the present study, it has been shown a simple, rapid and green synthesize of silver nanoparticles from S.monoica of size 31 nm, at a concentration of 500 nM had cytotoxic effects on Hep-2 tumor cells under in vitro conditions. Similar reports was observed by Bilberg et al, in male zebra fish with a semistatic 48 hr exposure [LC.sub.50] of 84 [micro]g [L.sup.-1] and [LC.sub.10] of 57 [micro]g [L.sup.-1] (17). Initially, a dose dependent effect of SNP on Hep-2 cell lines assessed by MTT assay showed an [IC.sub.50] value of about 500 nM that induced partial reduction in cell viability in comparison with control.

The cytotoxic effect of SNP on cell viability has a major role in antitumor activity, thereby reducing disease progression. The cytotoxic effects of silver are the result of active physiochemical interaction of silver atoms with functional groups of intracellular proteins, as well as with the nitrogen bases and phosphate groups in DNA (18).

Conclusion

In conclusion, the silver nanoparticles synthesized from S.monoica serve as an antitumor agent by decreasing progressive development of tumor cells. Our result suggests that SNP can induce cytotoxic effects on Hep-2 cells, inhibiting tumour progression and thereby effectively controlling progression without toxicity to normal cells. This may be due to their inhibitory activities in several signaling cascade responsible for the development and pathogenesis of disease which are as yet not understood.

Although, some of the researchers pointed out that the nanosilver toxicity is caused by chemical interactions, the toxic portion of nanoparticles must originate either from silver ions dissolved from the particle or from the exposed silver atoms on the particle surface (19). While the mechanism(s) by which AgNPs are toxic are unclear, their increasing use raises the concern that its release into the environment could lead to environmental toxicity.

Acknowledgement

The authors are gratefully acknowledge to the Dean, Faculty of Marine Sciences, Annamalai University, Parangipettai, Tamil Nadu, India for providing all support during the study period. The authors gratefully acknowledge the Dean, Faculty of Marine Sciences, Annamalai University, Parangipettai, Tamil Nadu and DST-PURSE program, India for financial support during the study period.

References

(1.) Bandaranayake WM. Traditional and medicinal uses of mangroves. Mangroves Salt Marshes 1998; 2(3):133-148.

(2.) Yezhelyev MV, Gao X, Xing Y, Al-Hajj A, Nie S, O'Regan RM. Emerging use of nanoparticles in diagnosis and treatment of breast cancer. Lancet Oncol 2006;7(8):657-667.

(3.) Wagner V, Dullaart A, Bock AK, Zweek A. The emerging nanomedicine landscape. Nat Biotechnol 2006;24(10):1211-1217.

(4.) Murphy CJ. Sustainability as an emerging design criterion in nanoparticle synthesis and applications. J Mater Chem 2008;18:2173-2176.

(5.) Schultz S, Smith DR, Mock JJ, Schultz DA. Single-target molecule detection with non bleaching multicolor optical immunolabels. Proc Natl Acad Sci USA 2000;97(3):996-1001.

(6.) Nair B, Pradeep T. Coalescence of nanoclusters and formation of submicron crystallites assisted by Lactobacillus strains. Cryst Growth Des 2002;2(4): 293-298.

(7.) Willner I, Baron R, Willner B. Growing metal nanoparticles by enzymes. Adv Mater 2006;18(9): 1109-1120.

(8.) Taleb C, Pettai M, Pileni P. Nanoparticles and nanostructured films: preparation, characterization and applications. Chem 1998;22:1203.

(9.) Vaidyanathan R, Kalishwaralal K, Gopalram S, Gurunathan S. Nanosilver-the burgeoning therapeutic molecule and its green synthesis. Biotechnol Adv 2009;27(6):924-937.

(10.) AshaRani PV, Low Kah Mun G, Hande MP, Valiyaveettil S. Cytotoxicity and genotoxicity of silver nanoparticles in human cells. ACS Nano 2009;3(2): 279-290.

(11.) Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65(1-2):55-63.

(12.) Gurunathan S, Lee KJ, Kalimuthu K, Sheikpranbabu S, Vaidyanathan R, Eom SH, Antiangiogenic properties of silver nanoparticles. Biomaterials 2009;30(31):6341-6350.

(13.) Li S, Han Q, Qiao C, Song J, Lung Cheng C, Xu H. Chemical markers for the quality control of herbal medicines: an overview. Chin Med 2008;28;3:7.

(14.) Chandran SP, Chaudhary M, Pasricha R, Ahmad A, Sastry M. Synthesis of gold nanotriangles and silver nanoparticles using Aloe vera plant extract. Biotechnol Prog 2006;22(2):577-583.

(15.) Song JY, Kim BS. Rapid biological synthesis of silver nanoparticles using plant leaf extracts. Bioprocess Biosyst Eng 2009;32(1):79-84.

(16.) Martins D, Frungillo L, Anazzetti MC, Melo PS, Duran N. Antitumoral activity of L-ascorbic acid-poly-D,L-(lactide-co-glycolide) nanoparticles containing violacein. Int J Nanomedicine 2010;5:77-85.

(17.) Bilberg K, Hovgaard MB, Besenbacher F, Baatrup E. In vivo toxicity of silver nanoparticles and silver ions in Zebrafish (Danio rerio). J Toxicol 2012; 2012:293784. Epub 2011 Dec 1.

(18.) Blagoi Yu P, GalkinVL, Gladchenko GO, Kornilova SV, Sorokin VA, Shkorbatov AG. Metal complexes of nucleic acids in solutions. Naukova Dumka 1991:272. (Russian)

(19.) Morones JR, Elechiguerra JL, Camacho A, Holt K, Kouri JB, Ramirez JT, et al. The bactericidal effect of silver nanoparticles. Nanotechnology 2005;16(10):2346-2353.

Kaliyamurthi Satyavani, Selvaraj Gurudeeban, Thiruganasambandam Ramanathan *, and Thangavel Balasubramanian

Marine Medicinal Plant Biotechnology Laboratory, Faculty of Marine Sciences, Annamalai University, Parangipettai, Tamil Nadu, India

* Corresponding author: Thiruganasambandam Ramanathan, Ph.D., Marine Medicinal Plant Biotechnology Laboratory, Faculty of Marine Sciences, Annamalai University, Parangipettai, Tamil Nadu, India

E-mail: marinepharmacology@gmail.com

Received: 19 Nov 2011

Accepted: 11 Jan 2012
COPYRIGHT 2012 Avicenna Research Institute
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2012 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Original Article
Author:Satyavani, Kaliyamurthi; Gurudeeban, Selvaraj; Ramanathan, Thiruganasambandam; Balasubramanian, Than
Publication:Avicenna Journal of Medical Biotechnology (AJMB)
Date:Jan 1, 2012
Words:2476
Previous Article:In silico evaluation of crosslinking effects on denaturant [m.sub.eq] values and [DELTA]Cp upon protein unfolding.
Next Article:Construction and stable expression of a truncated human receptor tyrosine kinase Ror1 (Ror1-ECD).
Topics:

Terms of use | Copyright © 2018 Farlex, Inc. | Feedback | For webmasters