Biosynthesis Of Silver Nanoparticles Using The Extract Of .
Cancer Nano (2013) 4:137–143DOI 10.1007/s12645-013-0045-4ORIGINAL PAPERBiosynthesis of silver nanoparticles using the extractof Alternanthera sessilis —antiproliferative effectagainst prostate cancer cellsM. Jannathul Firdhouse & P. LalithaReceived: 18 March 2013 / Revised: 1 August 2013 / Accepted: 5 September 2013 / Published online: 15 September 2013# Springer-Verlag Wien 2013Abstract Green synthesis of silver nanoparticles was carriedout using the aqueous extract of Alternanthera sessilis undervarious experimental conditions. The aqueous extract ofAlternanthera sessilis showed significant potential for thequick reduction of silver ions. The synthesized silvernanoparticles were characterized with UV-visible absorptionspectrophotometer, XRD, SEM, and FTIR analysis. The average crystallite size as calculated from x-ray diffraction studies and SEM analysis was found to be less than 100 nm. Thecytotoxic activity of synthesized nanosilver was carried outagainst prostate cancer cells (PC3) by MTT assay and found toshow significant activity. The present work of biosynthesis ofsilver nanoparticles using Alternanthera sessilis appears to becost effective, eco-friendly, and an alternative to conventionalmethod of synthesis.Keywords Alternanthera sessilis . UV-visible spectroscopy .XRD . SEM . FTIR1 IntroductionUncontrolled growth and spread of abnormal cells lead togroup of diseases and finally results in death, which istermed as cancer. It can be caused by both external factors(tobacco, chemicals, radiation, and viruses) and internalfactors (hormones, immune conditions, and mutations) thatmay act together or in sequence to instigate or promotecarcinogenesis (American Cancer Society 2011). The WorldM. J. Firdhouse : P. Lalitha (*)Department of Chemistry, Avinashilingam Institute for HomeScience and Higher Education for Women University (estd u/s 3 ofUGC Act 1956), Coimbatore 641043, Tamil Nadu, Indiae-mail: goldenlalitha@gmail.comHealth Organization reported that cancer is one of the leadingdiseases which will cause global death rates up to 15 millionby 2020. In 2008, prostate cancer was the second most commonly diagnosed cancer in men. Recognizing the growingglobal cancer crisis, a smart vision is needed to implement themetal nanoparticles as a drug in the genomic era (WorldStatistical Information 2007).Nanotechnology has gained attraction in the twenty-firstcentury and grows rapidly due to the ability to manipulateand harness properties of assemblies that are at thenanosize scale of various biomolecules (Panneerselvamet al. 2011). Nanoparticles exhibit completely new or improved properties based on specific characteristics suchas size, distribution, and morphology (Linga Rao andSavithramma 2012). In recent times, the advances in thefield of nanosciences and nanotechnology has brought tofore the nanosized inorganic and organic particles whichare finding increasing applications in personal care products, industrial, medical instruments and therapeutics, synthetic textiles, and food packaging products (RavishankarRai and Jamuna Bai 2011).Phytoconstituents like flavonoids, polyphenols possessesastonishing antitumor properties; but there is no proper utilization in terms of cancer drugs due to its solubility nature, lessoral intake, and ineffective delivery. These can be overcomeby the application of nanotechnology (Tabrez et al. 2013).Biosynthesis of nanoparticles using plant extracts is the favorite method of green, eco-friendly production of nanoparticlesand exploited to a vast extent because the plants are widelydistributed, easily available, safe to handle, and with a range ofmetabolites (Kulkarni et al. 2011). The successful use of silvernanoparticles (AgNPs) in diverse medical streams as antifungal, antibacterial (Panacek et al. 2009; Singh et al. 2008), andvirucidal agents (Lara et al. 2010) has led to their applicationsin controlling phytopathogens.
138In recent years, the robust area of research focuses on thecytotoxicity study of silver nanoparticles. Several studies onthe cytotoxicity of silver nanoparticles on different cell linesare reported. Antitumor activity of Bacillus licheniformis mediated AgNPs against DLA cell line (Sriram et al. 2010)and bovine retinal endothelial cells (Sriram et al. 2012) in vitroand in vivo are reported. Sodium citrate-assisted silvernanoparticles were studied for its antiproliferation activity onhuman lung alveolar carcinoma epithelial cells (A549) (Zhouand Wang 2012). Silver and gold nanoparticles synthesizedusing guava and clove extracts showed anticancer efficacyagainst four different cancer cell lines viz. human colorectaladenocarcinoma, human chronic myelogenous leukemia,bone marrow, and human cervix (Raghunandan et al. 2011).Synthesized silver sulfide nanoworms showed good cytotoxicity against human cervical cancer cell line (HeLa) is reportedby Xing et al. 2011.Biosynthesized AgNPs from leaf extract of Vitex negundoL . proved to be an antitumor agent against human coloncancer cell line HCT15 (Prabhu et al. 2013). In vitro cytotoxicity effect was analyzed by AgNPs synthesized usingSesbania grandiflora leaf extract against human breastcancer (MCF-7) (Jeyaraj et al. 2013). The potential silvernanoparticles synthesized from calli extract of Citrulluscolocynthis was investigated on human epidermoid larynxcarcinoma cell line (Satyavani et al. 2011). Govender et al.2013, studied cytotoxic activity of Albizia adianthifolia (AA)mediated silver nanoparticles and showed mechanistically theactivation of AA AgNP in the intrinsic apoptotic pathway inA549 lung carcinoma cells.Ethanolic extract of Dioscorea membranacea rootsshowed highest cytotoxic activity than other five plants(Bridelia ovata , Curcuma zedoaria , Derris scandens ,Nardostachys jatamansi, and Rhinacanthus nasutus) againstprostate cancer cell lines (Saetung et al. 2005). Human prostate cell proliferation in vitro study was attenuated using theethanol extract of Punica granatum L. var. spinosa whichsuppress the proliferation activity at an IC50 value of250.21 μg/mL (Sepehr et al. 2012). Acetone extract ofTridax procumbens showed 82 % cytotoxic activity comparedto aqueous extract against prostate epithelial cancerous cells(PC3) by MTT assay (Vishnu Priya et al. 2011). Glochidionzeylanicum (Gaertn.) showed significant cytotoxicity activityon PC3 compared to HepG2 and HT29 cell lines was reportedby Sharma et al. 2011. Root, stem, flower, and leaf acetoneextract of Lasienthera africanum was tested for its anticanceractivity on PC3 cell lines. Acetone extract of leaf showedsignificant anticancer properties compared to the other partsof this plant (Matheen et al. 2012).Alternanthera sessilis is a weed and occurs in both wetlands and uplands and can grow on a variety of soil types. It is aweed of rice throughout tropical regions and of other cerealcrops, sugarcane, and bananas, and has many utilities. In south-M.J. Firdhouse, P. Lalithaeast Asia, young shoots and leaves are ingested as vegetables.Previous phytochemical studies have reported the isolation offlavonols, triterpenoids, steroids and tannins; β-sitosterol, stigmasterol, campesterol, and lupeol being few of its importantconstituents. The herb has been reported to have antipyretic,hepatoprotective, antiulcer, antibacterial, hematinic, and diuretic activities (Sahithi et al. 2011). Alternanthera sessilis-assistedsilver nanoparticles exhibited 100 % cell inhibition of breastcancer cells (MCF-7) at IC50 value 25 μl/mL (Firdhouse andLalitha 2013).In the present work, we have explored the green synthesisof silver nanoparticles using aqueous extract of Alternantherasessilis as an alternative to chemical methods of synthesis andstudied its antiproliferative effect against prostate cancer cellline (PC3).2 Experimental2.1 Preparation of the extractFresh leaves of Alternanthera sessilis (20 g) were weighedand washed and boiled with 100 ml of Millipore water for5 min. The extract was filtered using Whatman filter paper andrefrigerated for further studies.2.2 Synthesis of silver nanoparticlesThe aqueous extract of Alternanthera sessilis was treated with3 mM of silver nitrate solution under various conditions, i.e.,room temperature (27–30 C), higher temperature (75 C),and sonication using ultrasonic bath (PCI Ultrasonics 1.5 L(H)). The reddish brown color silver solution was centrifuged(Spectrofuge 7 M) at 13,000 rpm for 15 min. The silvernanoparticles were redispersed in water, centrifuged again,and the supernatant solutions were analyzed.2.3 Characterization of synthesized silver nanoparticlesThe synthesized silver nanoparticles were characterized byUV-visible spectroscopy, x-ray diffraction (XRD), SEM, andFourier transform infrared spectroscopy (FTIR) analysis.2.3.1 UV-visible spectroscopyThe formation of nanosilver was confirmed by UV-visibleabsorption spectra using double-beam spectrophotometer2202 (SYSTRONICS).2.3.2 XRD analysisA drop of synthesized silver nanoparticles coated on theglass substrate was examined by x-ray diffraction analysis
Antiproliferative effect of biosynthesized silver nanoparticles139Table 1 Comparative experimental study on the biosynthesis of silvernanoparticles under different conditions2.4 In vitro cytotoxicity assay of nanosilverAqueous extract of plant silver nitrate solution (mL)2.4.1 Preparation of cell culture1 61 71 81 91 10Time for the formation of silvernanoparticles (minutes)RoomtemperatureHighertemperature(75 IMADZU Lab X XRD-6000) with a Cu Kα radiationmonochromatic filter in the range 10–80 .2.3.3 SEM analysisMorphology and size of silver nanoparticles were investigatedby scanning electron microscope using TESCAN instrumentprovided with Vega TC software for nanosilver coated onglass substrate.2.3.4 FTIR spectroscopyThe functional groups present in the synthesized nanosilverwere analyzed by FTIR spectroscopy- Tensor-27 (Bruker).PC3 (human prostate cancer cell line) was obtained fromNCCS Pune. It was maintained in Roswell Park MemorialInstitute (RPMI) supplemented with 10 % fetal bovine serum(FBS), amphotericin (3 μg/mL), gentamycin (400 μg/mL),streptomycin (250 μg/mL), and penicillin (250 units/mL) in acarbon dioxide incubator at 5 % CO2.2.4.2 Preparation of medium for cell cultureRoswell Park Memorial Institute medium The powdered media was dissolved in 900 ml of Millipore water in anautoclaved glass conical flask under sterile conditions. Theantibiotics were added in the concentration as mentionedabove and stirred well. Then, 3.7 g of sodium bicarbonatewas added into the flask and 10 % FBS was added and mixedwell. The liquid was slowly poured into the upper portion of amedia sterilization unit (Corning) and filtered through a 0.2-μfilter under negative pressure. The medium was stored at 4 Cwithout delay.Saline/trypsin/versene 10X saline A: 8-g NaCl, 0.4-g KCl,1.0-g D -Glucose, and 0.35-g NaHCO3 (tissue culture grade)were dissolved in 100-ml water and stored at 4 C.Versene: 1-g EDTA (tissue culture grade) was added into90-ml distilled water. Then 5-N NaOH was added drop wiseFig. 1 XRD patterns of silver nanoparticles synthesized using Alternanthera sessilis
140M.J. Firdhouse, P. LalithaTable 2 Determination of crystalline size of AgNPs using Debye–Scherrer's equationS.No2θFWHMβ π*FWHM/180θCosθD k λ/β. Cosθ1.32.30050.252500.0044016.150.9605332.81The crystalline size of silver nanoparticle is 32.81 nmuntil it gets dissolved. The solution was filtered and stored at4 C. One hundred milliliters of saline/trypsin/versene (STV)was prepared by adding 25 mg of trypsin in a mixture of 10 mlof 10X saline A and 2.5 ml of versene. Double distilled water(100 mL) was added, sterile filtered, liquated, and frozenat 20 to 70 C.2.4.3 Treatment of cellsPC3 cells show a steady growth rate with a doubling timeat approximately 33 h. The cells that reached confluency in3 to 4 days were stored in liquid nitrogen and used for theexperiments. The culture medium was removed from theT25 culture flask by decanting into a clean container insidethe laminar airflow chamber. The cells were rinsed withmedium to remove traces of serum, which may inhibitaction of trypsin. STV solution (2 mL) was added to theflask containing cells and incubated at 37 C for a fewminutes. As soon as cells started dislocating from thesurface, the flask was rinsed with 5 mL of serumcontaining medium to arrest the trypsinization. The suspension of cells was collected in a sterile 15-mL centrifugetube and the cells were pelleted at 1,500 rpm for 5 min. Thecell pellet was resuspended in fresh medium with serumand a part of the cells were seeded back into the flask. Theremaining cells were used for experiment and resuspendedin cryopreservative medium (Synth-a-freeze) in a cryovialFig. 2 SEM image of silvernanoparticles synthesized usingAlternanthera sessilisand frozen at 70 C for a day, then transferred to liquidnitrogen.2.4.4 MTT assayCell lines were maintained in RPMI supplement with 10 %FBS, amphotericin (3 μg/mL), gentamycin (400 μg/mL),streptomycin (250 μg/mL), and penicillin (250 units/mL) ina carbon dioxide incubator at 5 % CO2. Approximately 1,000cells/well were seeded in 96-well plate using culture medium,the viability was tested using trypan blue dye with the help ofhemocytometer and 95 % of viability was confirmed. After24 h, the different concentrations of silver nanoparticles (1.56,3.12, 6.25, 12.5, 25 μl/mL) were added at respective wells andkept incubation for 48 h.After 48 h of the drug treatment, the fresh medium waschanged again for all groups and 10 μl of MTT (5 mg/mLstock solution) was added and the plates were incubated for anadditional 4 h. The medium was discarded and the formazanblue crystals formed were dissolved with 50 μl of DMSO. Theoptical density was measured at 595 nm. The cell inhibition(in percentage) was determined using the following formula.Nonlinear regression graph was plotted between cell inhibition (in percentage) and Log10 concentration and IC50 wasdetermined using Graph Pad Prism software.Cell Inhibitionð%Þ ¼ 1 ½Abs ðsampleÞ Abs ðcontrolÞ 100:
Antiproliferative effect of biosynthesized silver nanoparticles141Fig. 3 FTIR spectra of silvernanoparticles synthesized usingAlternanthera sessilis3 Results and discussionThe aqueous silver ions were reduced to silver nanoparticleswhen aqueous extract of Alternanthera sessilis was added.After 6 h, the yellow-colored solution changed to reddishbrown color which indicates the formation of silvernanoparticles in room temperature. The formation of the silvernanoparticles was monitored by UV-visible spectrophotometricFig. 4 Cytomorphologicalchanges and growth inhibition ofsilver (a–b) and silvernanoparticles synthesized usingAlternanthera sessilis (d–e) onPC 3 cell line at 12.5 and 25 μl/mlconcentrations, respectivelyanalysis. The UV-visible spectra showed the maximum absorbance at 420 nm corresponding to the surface plasmon resonance of silver nanoparticles. A comparative study on variousexperimental conditions was carried out to identify the effect ofaqueous extract of Alternanthera sessilis and silver nitratesolution on the rate of bioreduction of silver ions.The results of formation of silver nanoparticles at differentconcentrations of silver nitrate (6, 7, 8, 9, and 10 ml) under
142M.J. Firdhouse, P. Lalithavarious conditions are given in Table 1. The sonication method results in easy and rapid synthesis of silver nanoparticleswithin 40 min as compared to other methods which may bedue to the effect of ultrasound, which has the ability to createclean, highly reactive surfaces on metals and thereby enhancing the rate of reactions.Figure 1 shows the XRD patterns of drop-coated silver nanoparticles synthesized using aqueous extract of Alternantherasessilis. The XRD pattern shows one intense peak of Bragg'sreflection with 2θ values of 32.30 which may be indexed to the(101) based on the face centered cubic structure of silvernanoparticles. The particle size of the silver nanoparticles wascalculated using Scherrer's equation, given in Table 2.Debye–Scherrer's equationThe Debye–Scherrer's equation is commonly used to determine the crystalline size of nanoparticles.D ¼ k λ β:Cosθwhere,DkλβθAverage crystalline size (in nanometer)Dimensionless shape factor (0.9)X-ray wavelength (0.1541 nm)Angular/line broadening at FWHM of the XRD peak atthe diffraction angleDiffraction angleFigure 2 represents the SEM image recorded from dropcoated film of the silver nanoparticles synthesized using aqueous extract of Alternanthera sessilis. The SEM image showedspherical shape of silver nanoparticles formation with diameter range 30–50 nm.Figure 3 shows the FTIR spectra of silver nanoparticlessynthesized using aqueous extract of Alternanthera sessilis.The peaks located at 3,253 and 1,634 cm 1 may be due to thepresence of NH or OH group and carbonyl stretching inproteins. The other peaks at 2,190 and 2,040 cm 1 areassigned to CC or CN triple bond, respectively.The synthesized nanosilver using Alternanthera sessiliswas studied for its cytotoxic activity against prostate cancercells (PC3) in vitro by MTT assay at different concentrations(1.56, 3.12, 6.25, 12.5, 25 μl/mL). The antiproliferation activity increases as the concentration of the nanosilver increases. It is quite obvious that the number of cancer cellsdecreases for the nanoparticles compared to that of silver ions.Figure 7a, b clearly shows the morphological changes such ascancer cell membrane lyses, coiling with the addition of silverand nanosilver synthesized using Alternanthera sessilis inFig. 4d, e after 48 h. The reduction in the number of PC3cancer cells is evidently observed at the highest concentrations(12.5 and 25 μl/mL) of PGAG-AgNPs compared to that ofcontrol.Fig. 5 Antiproliferative effect of silver (AG) and silver nanoparticlessynthesized using Alternanthera sessilis (PGKAG) on prostate cancer(PC3) cell lineThe IC50 value of AgNPs was observed at 6.85 μg/mLcompared to silver ions (14.62 μg/mL). The percentagegrowth inhibition of PC3 cell lines at different concentrationsof silver (AG) and nanosilver (PGAG) is depicted in Fig. 5and Table 3. The apoptosis rate was more in nanosilvercompared to that of silver ions as shown in Fig. 4.The results suggested that the Alternanthera sessilis assisted nanosilver exerts its cytotoxic effect on prostate cancer cells possibly via an apoptosis-dependent pathway.Intracellular suicides program possessing morphologicalchanges like cell shrinkage, oxidative stress, coiling, andbiochemical response lead to apoptosis. The reason may bedue to the interaction of silver nanoparticles with the functioning of cellular proteins which lead to the consequentchanges in the cells. Otherwise, the deionization of silverions may take place before entering the tumor cells due tothe low stability and high reactive nature of Ag ions. Thecomplete apoptosis (95 %) was observed at 25 μl/mL forprostate cancer cell (PC3); whereas 100 % growth inhibition was obtained for breast cancer cells (MCF-7). Theresults demonstrated that the antiproliferative effect ofPGAG-AgNPs mainly depends on the time of exposureand its concentration. Thus, Alternanthera sessilis -mediated silver nanoparticles may provide as promising drug forchemotherapeutic treatment.Table 3 Results of cytotoxicstudies with silver and nanosilversynthesized using the extract ofAlternanthera ll 8120.0842.7361.53
Antiproliferative effect of biosynthesized silver nanoparticles4 ConclusionThe biosynthesis of silver nanoparticles using the extract ofAlternanthera sessilis was economical, non-toxic, and environmentally benign. The formation of silver nanoparticles wascharacterized by UV-visible spectrophotometer. The synthesized silver nanoparticles were stable due to the reducing andcapping nature of phytoconsti
In vitro cytotox-icity effect was analyzed by AgNPs synthesized using Sesbania grandiflora leaf extract against human breast cancer (MCF-7) (Jeyaraj et al. 2013). The potential silver nanoparticles synthesized from calli extract of Citrullu
Nano? Silver containing substance (SCAS) CAS No. Elemental silver a) Particulate silver 7440-22-4 b) Silver ionisation systems 7440-22-4 Silver adsorbed to silica dioxide Not yet allocated Silver chloride adsorbed to titan dioxide Not yet allocated Silver nitrate 7761-88-8 Silver sodium hydrogen zirconium phosphate 265647-11-8 Silver zinc .
into metallic silver nanoparticles.18 Aquatic humic acids of various origins were responsible for forming metallic silver nanoparticles from dissolved ionic Ag.19 Dissolved organic matter reduced dissolved Agþ in the presence of sunlight to metallic nano-silver.20 Superoxide has been shown to reduce Agþ to silver
Alginate nanoparticles were the most stable in the salivary environment, while chitosan nanoparticles were the most cytocompatible. Alginate nanoparticles and pectin nanoparticles revealed possible cytotoxicity due to the presence of zinc. This knowledge is important in the early design of polymer-based nanoparticles for oral
the biosynthesis of chlorophylls (phytyl side-chain) and carotenoids, and differentiates it from the cytosolic ac-etate/mevalonate pathway of sterol biosynthesis. The light-induced accumulation and final functional con-centration of carotenoids and chlorophylls in green leaves is presented. Moreover, the biosynthesis of isoprene and
Isoprenoids Fig. 1 The relationship between alginate biosynthesis and the cellular metabolism in P. fluorescens. a The proteins and metabolites needed for alginate biosynthesis. b A simplified model of the cell's metabolism highlighting the processes identified in the present study as being important for full alginate biosynthesis levels.
2.5 FTIR analysis of silver nanoparticles . For FTIR measurements, the synthesized silver nanoparticles solution was centrifuged at 10000 rpm for 30 minutes. The pellet was washed thrice with 5 ml of deionised water to get rid of the free proteins or enzymes that are not capping the silver n
The most conventional metallic nanoparticles for LSPR apparatus are gold and silver nanoparticles. On the whole, the safety and surface stability of AuNPs are strongly better than silver nanoparticles (7, 13). In general, LSPR sensing technique occurs in colorimetric sensing as a result of absorption band shift (14).
The silver nanoparticles were synthesized by using a rapid, single step and completely green synthetic method. The synthesized silver nanoparticles were characterized by using various instrumental techniques such as ultraviolet-visible spectroscopy (UV-Vis), Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD) and scanning .
