Evaluation of cytotoxic activity of platinum nanoparticles against normal and cancer cells and its anticancer potential through induction of apoptosis

Evaluation of cytotoxic activity of platinum nanoparticles against normal and cancer cells and its anticancer potential through induction of apoptosis I O E n i a YQ1 R a A R R 1 A A K C P A A I P h 2[.]

Available online at www.sciencedirect.com

jo u rn al h o m e p a g e :w w w i m r - j o u r n a l c o m

Evaluation of cytotoxic activity of platinum

nanoparticles against normal and cancer cells and

its anticancer potential through induction of

apoptosis

Yogesh Bendale∗, Vineeta Bendale, Saili Paul

Q1

Research and Development Section, Rasayani Biologics Private Limited, Pune 411030, India

Article history:

Received25March2016

Receivedinrevisedform

10January2017

Accepted12January2017

Availableonlinexxx

Keywords:

Cytotoxicity

Platinumnanoparticles

Anti-cancerpotential

Apoptosis

In vitro

Background:Plantmediatedgreensynthesisofnanoparticlesisaneco-friendlyand effica-ciousapproachwhichfindsimmenseapplicationinthefieldofmedicine.Thisstudyaimed

toevaluatethecytotoxicityofplatinumnanoparticles(ptNPs)synthesizedthroughgreen technologyagainstnormalanddifferentcancercelllines

Methods:Platinumnanoparticlesweresynthesizedbygreentechnologyandcharacterized earlier.Inthisstudyweexaminedthecytotoxiceffectofplatinumnanoparticles(ptNPs)

onhumanlungadenocarcinoma(A549),ovarianteratocarcinoma(PA-1),pancreaticcancer (Mia-Pa-Ca-2)cellsandnormalperipheralbloodmononucleocyte(PBMC)cellsand evalu-ateanticancerpotentialthroughinductionofapoptosisonPA-1cellsifany.Cytotoxicity wasevaluatedusingMTTassay,trypanbluedyeexclusionassayandanticancerpotential assessedthroughclonogenicassay,apoptosisassay,cellcycleanalysis

Results:WefoundthatptNPsexertedcytotoxiceffectoncancercelllines,whereasno cyto-toxiceffectwasobservedathighestdoseonnormalcells.TheresultsshowedthatptNPs hadpotentanticanceractivitiesagainstPA-1celllineviainductionofapoptosisandcell cyclearrest

Conclusion:Overall,thesefindingshaveprovedthatbiosynthesizedptNPscouldbepotent anti-ovariancancerdrugs.Furtherstudiesarerequiredtoelucidatethemolecular mecha-nismofptNPsinducedanti-tumoreffectin vivo.

©2017KoreaInstituteofOrientalMedicine.PublishedbyElsevier.Thisisanopenaccess

articleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/)

∗ Corresponding author.ResearchandDevelopmentSection,CellandMolecularBiologyLaboratory,RasayaniBiologicsPrivateLimited, Pune411030,India.Fax:+912024530995

E-mailaddresses:dr.bendale@gmail.com(Y.Bendale),vineeta.bendale@gmail.com(V.Bendale),sailipaul@rediffmail.com(S.Paul) http://dx.doi.org/10.1016/j.imr.2017.01.006

2213-4220/©2017KoreaInstituteofOrientalMedicine.PublishedbyElsevier.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/)

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Introduction

Inrecentyears,theinterestinthesynthesisandproperties

ofmetalnanoparticleshasbeenincreasingbecauseoftheir

uniquepropertiesandpromisingapplicationsascatalysts,

fer-rofluids,andsemiconductors.1,2Nanotechnologyisthemost

promisingfieldforgeneratingnewapplicationsinmedicine

However,onlyfewnano-productsarecurrentlyinusefor

med-icalpurposes.3

Formanyyears,platinum-basedmoleculeshavereceived

considerable attention because of their electro-catalytic

properties.4,5Forinstance,platinum-basedtherapeuticdrugs,

notably cisplatin and carboplatin, have been exploited in

chemotherapy to kill cancer cells.5 However, these drugs

are notselectivefor cancercells,because normalcells are

alsoaffected,leadingtosubstantialdose-limitingacuteand

chronictoxicities.Sincetoxicsideeffects(particularly

nephro-toxicity and gastrointestinal) and frequentdevelopment of

drugresistancerepresentthemajorchallenges inthe

clini-caloutcomeofthesepatients,itwasconceivabletosearchfor

cisplatinanalogsorothermetalcomplexesabletoofferamore

acceptableleveloftoxicityandimprovedantitumoractivity.6,7

Nanoparticlesaremakingsignificantcontributionstothe

developmentofnewapproachesofdrugdeliveryincancerand

canprovideaplatformforcombinedtherapeuticswith

subse-quentmonitoringofresponse.8Increasingevidencesuggests

thatthe specialphysicochemicalpropertiesof

nanomateri-als pose potential risks to human health.9 Therefore it is

necessarytounderstandhowcells respondto

nanomateri-alsand through what mechanisms Green nanotechnology

is generating attention of researchers toward eco-friendly

biosynthesisofnanoparticles.Withaviewtoward

develop-ingnano-therapeutics,wehaveperformedexperimentsusing

eco-friendlyplatinumnanoparticles.Platinumnanoparticles

weresynthesizedbygreentechnology,characterizeditszeta

potentialandsizebydynamiclightscattering(DLS)aswellas

scanningelectronmicroscopy(SEM)earlier.10

Thepresentstudyisthecontinuationoftheearlierwork

andiscarriedouttoassessthecytotoxicityofptNPson

nor-malandthreedifferenttypesofcancercells.Basedonhighest

cytotoxicityresults,anticanceractivitiesofplatinum

nanopar-ticlesagainstovarianterotocarcinomacellswereevaluated

Methods

Cell culturemedium reagents were purchased from

Hime-dia laboratories Fetal bovine serum (FBS) was purchased

from Invitrogen (US) An Annexin V-FITC apoptosis

detec-tion kit was purchased from BD-Bioscience (Catalogue no

556547).Cisplatin,usedasapositivecontrolwaspurchased

fromCipla(India).Platinumnanoparticlesweresynthesized

throughgreentechnologyandcharacterizedbyparticlesize,

zetapotentialandsurfacemorphology.10TherecoveredptNP

samplewasusedforcytotoxicityandanticancerstudies

Cell culture and exposure of drug

Ahumancancercells,A549,PA-1,Mia-Pa-Ca-2wereobtained

from National CenterforCell Science(NCCS), sub-cultured

and thenusedtodeterminecell cytotoxicityafterexposure

tothe drug Thecellswere culturedin minimumessential medium(MEM),Dulbecco’sModifiedEagle’smedium(DMEM) supplemented with 10% FBS at 5% CO2 and 37◦C.At 85% confluence,thecellswereharvestedusing0.25%trypsinand seededin25cm2flasks,96wellplates,6-wellplates,according

totheexperimentbeingperformed.Thecellswereallowedto 70%attachtothesurfacepriortotreatment.Astocksolutionof ptNPs10mg/mlwasmadeinvehicleanddilutedto appropri-ateconcentrationsfortreatment.Suspensionswerevortexes and aspirated10timesbeforetreatment.Cellstreatedwith vehiclecontrolweretakenascontrol

MTT cell proliferation assay

(3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) assay measures the cell proliferationrateandconversely,thereductionincell viabil-itywhenmetaboliceventsleadtoapoptosisornecrosis.The yellow compound MTT is reduced by mitochondrial dehy-drogenasestothewaterinsolubleblueformazancompound, dependingontheviabilityofthecells.11

A549,PA-1,Mia-Pa-Ca-2cells(2×104cells/ml)wereseeded

in96-wellplatesandexposedtodifferentconcentrations(50,

100 and200␮g/ml)ofptNPsand 10␮g/mlofcisplatinfora periodof48hours.Afterthetreatmentperiod,thecellswere allowedtoreactwithMTTforaperiodof3–4hoursindark

at 37◦C At the end of the incubation period, dark purple formazancrystalswereformed.Thesecrystalswere solubi-lizedwithanorganicsolvent(e.g.isopropanol)andabsorbance

at595nmwasmeasuredspectrophotometrically.The experi-mentwasrepeatedatleastthreetimes.Cisplatinwasusedas positivecontrolforthisexperiment.Todeterminethecell via-bility,wecalculatedpercentviabilityas%viability=[(Optical density{OD}oftreatedcell−ODofblank)/(ODofvehicle con-trol−ODofblank)×100]

Preparation of PBMC and assessment of cytotoxicity using trypan blue assay

Peripheralbloodmononuclearcells(PBMC)wereisolatedfrom healthy humanvolunteerbyFicoll-Paque(Histopaque1077, Himedialaboratories)densitygradientcentrifugationasper standardprocedure.12PBMC(2×105cells/well)werecultured

incomplete RPMI-1640mediaas usualand incubatedwith ptNPs(200␮g/ml),cisplatin(10␮g/ml)toevaluatecytotoxicity for48hoursusingtrypanblueexclusionassay.Thismethods yieldsapproximatelymorethan95%viablePBMC

Clonogenic survival assay

Clonogenicassayorcolonyformationassayisanin vitrocell survivalassaybasedontheabilityofasinglecelltogrowintoa colony.Thecolonyisdefinedtoconsistofatleast50cells.The assayessentiallytestseverycellinthepopulationforits abil-itytoundergo“unlimited”division.13Clonogenicassayisthe methodofchoicetodeterminecellreproductivedeathafter treatmentwithptNPs.Afterharvestingwith0.05%trypsin,200 (dependingonthetreatment)cellswereplated24hoursbefore treatmentinMEMat37◦C.Culturedcellsweretreatedwith

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thetreatment,cellswereincubatedin5%CO2atmosphereat

37◦Cfor14daystoallowcolonyformation.Colonieswerefixed

withmethanolandstainedwith1%crystalviolet.Coloniesof

morethan50cellscountedandtheplatingefficiency(PE)was

calculated

Observation of morphological changes with Acridine

orange/Ethidium bromide (AO/EB) staining

Cellswereseededataconcentrationof2×105cell/mlin6-well

tissuecultureplates.Followingincubation,themediumwas

removedandreplacedwithphosphate-buffersaline(PBS)and

supplementedwithptNPs(100and200␮g/ml).Afterthe

treat-mentperiod,monolayercellswerestainedwithAO/EBstain

(1mg/ml).14Afterstaining,thecellswerevisualized

immedi-atelyunderthefluorescencemicroscope(Axiovert,CarlZeiss)

at20×magnification

Annexin V and propidium iodide (PI) staining for

apoptosis assay

Apoptosiswasassessedviaflowcytometricanalysisofcontrol

andptNPstreatedcellsthatwerestainedwithFITC-Annexin

Vand PIusingthe Annexin V-FITCapoptosis detection kit

according to the manufacturer’s protocol (BD Bioscience)

PA-1 cells were seeded onto 6 well plates and allowed to

adhere.Aftercellsbecome70%ofconfluent,cellweretreated

with200␮g/ml ofptNPsfor 48hours at37◦C and 5%CO2

Subsequently,the cells were collected,washed in PBS and

resuspendedin500␮lof1XAnnexin-bindingbuffer.Cellswere

thenincubatedatroomtemperaturewithAnnexinV-FITCand

PIstainintheabsenceoflight.Followingthe10minute

incuba-tion,sampleswereimmediatelyanalyzedviaflowcytometry

AnnexinVstainingwasdetectedasgreenfluorescenceandPI

asredfluorescence

Cell-cycle analysis

Cellcycleperturbationswereassessedusingflowcytometryto

measuretheproportionofcellsindifferentphases.Cellcycle

perturbationsinducedbyptNPswereanalyzedusing

propid-ium iodideDNA staining.14 Approximately 2×105 cells per

wellwereplatedinsix-wellplatesandallowedtoattach.After

cellsbecomes70%confluent,treatedwith100and200␮g/ml

ptNPsfor48hoursandthencollectedandfixedinice-cold70%

ethanolfor4hoursandstoredat4◦CuntilPIstaining

Ethanol-suspendedcellswerethencentrifugedat1000rpmfor5min

andwashedtwiceinPBStoremoveresidualethanol.Pellets

weresuspendedin1mlofPI/RNaseAreagentandincubated

at37◦C for30min Cell cycle profiles were obtainedusing

aBDFACScan Cell flowCytometer(BectonDickinsonUSA)

Debrisandaggregatesweregatedoutduringdataacquisition

and5000-10,000eventswerecollectedfromeachsample.Data

wereanalyzedwiththeCellQuestProsoftware

0 20 40 60 80 100 120

VC ptNPs 50 ptNPs 100 ptNPs 200 Cisplatin 10

Dose (µg/ml)

567A5 PA-1 Mia-Pa-Ca-2

***

***

*

**

Fig 1 – Effect of ptNPs on viability of lung adenocarcinoma (A549), ovarian teratocarcinoma (PA-1), pancreatic cancer (Mia-Pa-Ca-2) cells A Cells were treated with vehicle, different concentrations of ptNPs and positive control (10 ␮g/ml cisplatin) for 48 hours Cell viability was analyzed using the MTT assay Data represented as mean ± SE of three independent experiments made in three replicates.

*P < 0.05, **P < 0.01, ***P < 0.001 versus vehicle control (VC) group.

Statistical analysis

Statistical comparisons were made using Student’s t-test Resultswereexpressedasmeans±standarderrors(SEs) P-valuesoflessthan0.05wereconsideredsignificant

Results

Cytotoxicity of ptNPs on cancer cells

Differentcancercelllineswereusedtoscreenforthein vitro

cytotoxicactivityofptNPs.A549,PA-1,Mia-Pa-Ca-2cellswere incubatedwithdifferentconcentrationofptNPsfor48hours CellviabilitywasdeterminedbyMTTassay.Weobservedthat ptNPssuppressedgrowth ofcancercells(Fig.1)andgrowth inhibitionwere28.52-34.85%inA549cells,33.16-46.06%in

PA-1cellsand11.12-41.18%inMia-Pa-Ca-2cellsaftertreatmentof ptNPs(50–200␮g/ml).TheresultsshowninFig.1indicatedthat ptNPs(200␮g/ml)causedasignificantdecreaseincell viabil-ityofA549(**P<0.01),PA-1(***P<0.001),Mia-Pa-Ca-2(*P<0.05) respectivelyascomparedtocontrol.PtNPsshowedthehighest growthinhibitoryeffectsonPA-1cells.AspresentedinFig.1, somecytotoxiceffectwasalreadyobservedat50␮g/mldose

ofptNPswhereasmaximaleffectwasobtainedata concen-trationof200␮g/mlinPA-1.Basedonthesedata,thepresent studyfocusedonPA-1cellsforsubsequenttests

Cytotoxicity of ptNPs on normal cells

ToexaminethecytotoxiceffectoftheptNPsagainstnormal PBMCcells,trypanbluedyeexclusionassaywasperformed PBMC cells were treated with highest dose (200␮g/ml) of the ptNPsfor 48hours No significant cytotoxic effect was observedinthe normalPBMCatthehighestconcentration (200␮g/ml) (Fig 2) of ptNPs that significantly affected the

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Control ptNPs (200µg/ml) Cisplatin (10µg/ml)

**

Fig 2 – Cytotoxic effect of ptNPs (200 ␮g/ml) and cisplatin (10 ␮g/ml) on normal PBMC cells Data represented as mean ± SE

of three independent experiments **P < 0.01 versus control group.

cancercellsFig.1,suggestingthattheeffectoftheptNPswas

selectiveforcancercells

Clonogenic survival assay

The clonogenic assay showed the effect of ptNPs on the

colony-formingcapacityofexponentiallygrowingPA-1cells

Weperformedaclonogenicassayforconfirmingthegrowth

inhibitionresultsofptNPs.Platinumnanoparticlestreatment

enhancescelldeathandalsoinhibitscolonyformation

capa-bilityinthePA-1cellpopulationinaconcentrationdependent

manner.Aftertreatmentswithdifferentconcentrations(50,

100,200␮g/ml),theplatingefficiencyofPA-1cellsdeclines,as

evidencedbythereductioninthenumberofcoloniesformed

(Fig.3B-D).ExposureofptNPs(50,100and200␮g/ml)showsa

declineincolonysurvivalandplatingefficiencywasfoundto

be36,31and11respectively(Fig.3F).Thisresultindicatesthat ptNPsat200␮g/mlsignificantly(p<0.05)inhibit thecolony formationcapabilitiesofPA-1cells

Morphological changes using AO/EB staining

Apoptosiswasalsoconfirmedbyexaminingthenuclear mor-phology by AO/EB staining As shown in Fig 4A, control PA-1cellswerestainedwithuniformgreenfluorescenceand

no apoptotic features were observed Following treatment

of PA-1 cells withptNPsfor 48hours, obvious morphologi-calchangesandapoptoticcellswithchromatincondensation were observed (Fig 4B-C) The results suggest that ptNPs induced PA-1 cell apoptosis Cells stained green represent

Fig 3 – Effect of platinum nanoparticles on colony forming capacity or clonogenic survival of exponentially growing PA-1

cells studied by a clonogenic assay PA-1 cells were treated with ptNPs (50, 100, 200 ␮g/ml) and cisplatin (10 ␮g/ml), allowed

to form colonies in fresh medium for 14 days A = Control; B = ptNPs (50 ␮g/ml); C = ptNPs (100 ␮g/ml); D = ptNPs (200 ␮g/ml);

E = Cisplatin (10 ␮g/ml) F: Representative histogram showing percentage of platting efficiency in PA-1 cells Data are

expressed as mean ± SE (n = 3) *P < 0.05, ***P < 0.001 versus control group.

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Fig 4 – PA-1 cells were stained by AO/EB and observed under fluorescence microscope PA-1 cells were treated with (A)

vehicle control, (B) 100 ␮g/ml and (C) 200 ␮g/ml of ptNPs for 48 hours L indicates live cells, EA indicates early apoptotic cells and LA indicates late apoptotic cells.

viablecells,whereasyellowstainingrepresentsearly

apop-totic cells and reddish or orange staining represents late

apoptoticcells

Effect of ptNPs on apoptosis in PA-1

Apoptosis, autophagy and necrosis are the major types of

celldeath.15 To determinethepercentageofapoptotic and

necrotic cells, the cells were treatedwith 200␮g/ml ptNPs

for48hoursandstained withAnnexinVandPIusingflow

cytometry.Here,wefoundthatthatptNPsiscapableof induc-ingapoptosisinPA-1cells.Theflowcytometryanalysisresults showedthattherateofapoptosiswas8%inthecellstreated with200␮g/mlofptNPsafter48hours(Fig.5

Cell-cycle analysis

Cell flow cytometry was used to determine the effect of ptNPs on the cell cycle progression.A significant increase

inthe percentageofcells insub-G1phasewasfoundafter

Fig 5 – Apoptosis induced by ptNPs in PA-1 cells PA-1 cells were treated with (A) vehicle control and (B) 200 ␮g/ml of ptNPs for 48 hours Then cells were stained with FITC-conjugated Annexin V and PI for flow cytometric analysis The flow

cytometry profile represents Annexin V-FITC staining inxaxis and PI inyaxis (C) Results showing the percentage of early apoptotic cells, late apoptotic cells and necrotic cells Data are expressed as Mean ± SE (n = 3) **P < 0.01 versus control group.

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Fig 6 – Flow cytometry analysis of cell cycle phase distribution in PA-1 cells Histogram representing propidium iodide

staining of control (A) and ptNPs (100 and 200 ␮g/ml) (B-C) treated PA-1 cells for 48 hours D: Bar diagram showing the cell

distribution in the subG1, G0/G1, S and G2/M phases for PA-1 cells treated with vehicle control and ptNPs (100 and

200 ␮g/ml) Data are expressed as mean ±SE (n = 3) *P < 0.05 versus control group.

treatmentwithptNPs,comparedwithcontrolcells(Fig.6 As

showninFig.6,whencellswereexposedtoptNPs(200␮g/ml)

for48hours,thepercentageofsub-G1populationshoweda

markedincreaseof42%(p<0.05)comparedwiththecontrol

(12%).G0/G1phaseweredecreasealongwiththeincreaseof

sub-G1phasecomparedwiththatofcontrolcells.Allthese

dataindicatethatinhibitionofcellgrowthcausesincreased

cellsenteringinsub-G1phase,subsequentlyinducescell

apo-ptosis

Discussion

Dependenceofhumanlifeonnanotechnologyemerged

nat-urally from ayurveda, a 5000-year-old system of Indian

medicine.Thoughthemodernsciencehasstartedexploring

the term “Nano” in 21stcentury, ayurvedicmedicinal

sys-tems used noble metals such as gold, silver etc., in nano

formas bhasmasfor various medicalapplications.16 Since

nanoparticles(NPs)aremorebiocompatiblethanthe

conven-tionaltherapeutics,theyplayanimportantroleinimproving

theirbioavailability aswell ascompatibilityfor

therapeuti-calapplicationsindiseaseslikecancer.17Thereisagrowing

list of reports indicating that NPs might be medically and environmentallytoxic,astheirhighsurface-to-volumeratio makestheparticlesofsomemetalsveryreactiveorcatalytic.18 There is also evidence that NPs pass through cell mem-branes andinteractwithcellularstructures,and thushave

a direct impact on cell functioning and consequently on cell viability Therefore, in this study,we have determined the cytotoxicity of ptNPs using cancer cell lines and nor-mal humanperipheralblood mononucluocyte (PBMC) cells forunderstandingtoxicityunderin vitroconditions.We car-riedoutscreeningofptNPsindifferentcancercellsoflung, ovarian,pancreatic,breast,colon,renal,leukemiatypes Inter-estingly, the cytotoxicityofptNPstowardmammalian cells depends on thecell type.Our in vitrostudies showed that ptNPsinducedcelldeathinovarian,lungandpancreatic can-cercelllines(dataofbreast,colon,renal,leukemiacancercell lines were notshown).In contrast,nosignificant cytotoxic effectwasobservedinthenormalhumanPBMCcells(Fig.2)

atthehighestconcentration(200␮g/ml)ofthenanoparticles that significantlyaffected the lung, ovarianand pancreatic cancer cells (Fig 1 suggestingthat the effect ofthe plat-inumnanoparticleswascytotoxicfortheselectedcancercell lines.AsptNPsexhibitinggreatercytotoxicityonPA-1cells,

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PA-1 cell line was selected to find out mode of action of

platinumnanoparticlesandevaluateditsanticancer

poten-tial

Thepresentstudyrevealedthatpotentialcytotoxiceffect

ofptNPsinPA-1cellsaftershort(48hours)andlongterm(14

days)exposure.Clonogenicassay,usedtoevaluatetheeffects

inducedafterprolongedexposure(14days).Ourfindingshows

thatptNPsdosedependentlyinhibitedthePA-1colony

forma-tionascomparedtothecontrolcells(Fig.3 Thisindicates

thatptNPsdecreasesthepotentialofindividualcellstoform

acolonyandtherebyactsasananti-cancerdrug.Thisfinding

wellcorroboratedwithourcellproliferationstudies

Themajordrawbacksofmanyeffectivecancer

chemother-apeutic agentsare systemic toxicityand drug resistance.19

Cisplatinisthefirstapprovedplatinumdrugthathasbeen

usedformorethanthreedecadesinstandardchemotherapy

regimens.However,theuseofcisplatinisrestrictedbecauseof

itsseveresideeffects,includingnephrotoxicity,neurotoxicity,

ototoxicityandmyelo-suppression,aswellastheintrinsicand

acquiredresistancedevelopedbyvariouscancers.Thus,the

newtherapeuticagentsshouldbemoreactivewhileproducing

fewerside effects Theyalso shouldact through a

mecha-nismdifferentfromthatofcytotoxicagentsalreadyused.A

varietyofnanomaterialsarebeingevaluatedinclinicaltrials

asdrugcarrierandforimagingofspecifictargets,withaim

ofincreasingtheefficiencyofdrugbioavailability, reducing

sideeffectsand preventiondamagetoother tissues.20,21 At

presenttherehasbeenaconsiderableinterestinthe

biologi-calsynthesisofnanoparticlesbecauseofitssimple,safeand

eco-friendlyprinciplesanditdoesnotrequireelaborate

pro-cess.Thebiosafetyandbiocompatibilityofanybiomaterialare

vitalconcernsthatshouldbeaddressedbeforesuchmaterials

areappliedtobiologicalsystems.Weinvestigatedtheacute

toxiceffect ofptNPsindifferent dosesand no toxiceffect

wasfoundupto5000mg/kgbwafteroraladministrationto

mice(datanotpublished).Nocytotoxicitywasalsofoundafter

treatmentofdifferentdosesofptNPs(25-100␮g/ml)against

normalPBMC.10OurpresentPBMCresultsalsoshowedthat

nocytotoxicitywasobservedathighest200␮g/mldose.The

activityofbiosynthesizedptNPsinhumancancercellsin vitro

and in vivoprovides the rationale for clinical use oforally

administeredagentinpatientswithsolidtumorasmostof

thechemotherapeuticdruglikecisplatinadministered

intra-venouslyandhavesideeffects.Inthisregard,platinumnano

particles synthesized by green technology with anticancer

activityandnotoxicitytonormaltissueshasbeensuggested

aspossiblecandidatesfortheircapabilitytoimprovethe

effi-cacyofanticancerdrugs

In conclusion, biosynthesized ptNPs exhibited cytotoxic

activity against ovarian, lung and pancreatic cancer cells

without showing toxicity against normal peripheral blood

mononucleocytecells.ResultsalsodemonstratedthatptNPs

induce apoptosis and cell cycle arrest in PA-1 Based on

our observation, we suggest that ptNPs may represent an

emergingnoveltherapeuticagentforthetreatmentofhuman

ovariancancerwithoutshowingcytotoxicitytowardsnormal

cells.Furtherstudiesarerequiredtosupportourobservations

oftheanti-tumorpotentialoftheseplatinumnanoparticles

in vivo.

Conflict of Interest

Theauthorsdeclarethattherearenoconflictofinterest

Acknowledgments

This study was supported by grants from Department Q2

(BT/BIPP0446/13/11) We also would like to acknowledge

Dr.UlhasWaghfortechnicaladviceduringtheexperiments

WethankDr.PadmaShastri oftheNationalCentreforCell Science(NCCS)forrevisingthemanuscript

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