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Research Project Dissertation_Final_270815_DT

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Dominic Thum

This document discusses the cytotoxicity of two ceramic nanoparticles - Cloisite Na+ montmorillonite (MMT) and Lucentite SWN. It first defines nanoparticles and nanoclays, and describes the layered structure and properties of MMT. The aims of the study are then stated as investigating the cytotoxicity of MMT and Lucentite SWN using cytotoxicity assays, and determining the mechanism of effect using western blotting if cytotoxic. Results showed MMT had low cytotoxicity in terms of cellular metabolic activity while Lucentite SWN showed low cytotoxicity in terms of cellular uptake activity. MMT was more cytotoxic than Lucentite SWN in terms of metabolic activity, and vice versa for uptake activity. The

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DominicThum:N0625034 1 NOTTINGHAM TRENT UNIVERSITY CYTOTOXICITY OF CLOISITE NA+MONTMORILLONITE (MMT) AND LUCENTITE SWN ON TISSUE GROWTH AND DIFFERENTION by DOMINIC THUM ProjectReportsubmittedinpartialfulfilmentof therequirementsfor a M.Sc. in Biomedical Science at the School of Science and Technology, Nottingham Trent University Supervisor: Dr Jane Harper Year in which the study is completed: 2015 DECLARATIONOFOWNERSHIP Thissubmissionistheresultof mywork.Allhelpandadvice,otherthanthatreceivedfromtutors,hasbeen acknowledgedandprimaryandsecondarysourcesof informationhave beenproperlyattributed.Should thisstatementprove tobe untrue,Irecognisetherightanddutyof theBoardof Examinerstorecommend whatactionshouldbe takeninline withthe University’sregulationsonassessmentcontainedinthe Handbook. Signed................................................................................ Date .......................................................
DominicThum:N0625034 2 Acknowledgements The author wishestoexpresshisappreciationtoeverypersonthathasgiventheirsupportwhilst undergoingthisproject. Training and mentoringin laboratory, fieldorcomputational techniques: I wouldlike tothankbothDr Jane Harperand Dr Luigi de Girolamofortheirsupportandguidance offeredthroughoutmyprojectsupervision.The skillsandtechniquesthattheyhave willinglygivenwill be invaluable tome asI beginmyscientificcareer. I am alsothankful tothe laboratorytechniciansatthe RosalindFranklinlaboratoryof NottinghamTrent University.Theirwillingnesstoofferadvice andguidance throughoutmyresearchprojectwasamajor contributiontothe project’s success. I wouldalsolike tothankTianLong See fromthe Universityof Manchesterforprovidingadvice and guidance incomputingandanalyzingthe laserlightscatteringdataacquiredfromthe researchproject. Your helpwasgreatlyappreciated. Data collectedwithanother person: I wouldlike tothankmylab partner,AnkithaMishra,forhercontributionsandhelpintermsof cell culturing,proteinharvesting,BCA assaysandsoforth. Data providedby another person: I wouldlike tothankDr JohnBrameld of the Universityof Nottinghamforhiskinddonationof the C2C12 mouse myoblastcell line.Lastly,IwouldliketothankDr Fengge Gao forprovidingthe Cloisite Na+ montmorillonite (MMT) andlucentite SWN nanoparticles.These donationswere greatlyappreciated.
DominicThum:N0625034 3 Table of Contents List of Abbreviations 5 Summary 6 1) Introduction a. Nanoparticles:Definitionandapplications 7 b. Nanoclays:Definitionandapplications 9 i. Cloisite Na+ montmorillonite (MMT) 10 ii. Lucentite SWN 11 2) Aims 12 3) MaterialsandMethods a) Materials 16 b) Methods a. Cell Culture 21 b. Preparationof nanoclaysuspension 22 c. ExperimentalDesign 22 d. Cytotoxicityassays 23 e. Cell harvest 24 f. Proteinextractionandestimation 25 g. SDS-PAGEand WesternBlotting 25 h. Immunoprobingof WesternBlots 26 i. Image analysis andcell imaging 28 j. Statistical analysis 28 4) Results a) Particle size analysis 28 b) Cytotoxicityassayanalysis 34 c) The visual phenotype of C2C12cellstreatedwith: i. Cloisite Na+ montmorillonite (MMT) 44 ii. Lucentite SWN 46 d) WesternBlotanalysis 48 5) Discussion 51 6) Bibliography 59
DominicThum:N0625034 4 7) Appendices 65
DominicThum:N0625034 5 List of Abbreviations BCIP-T 5-bromo-4chloro-3-indolylphosphate BSA Bovine Serum albumin DMEM Dulbecco’s Modified Eagle Medium DMSO Dimethyl sulfoxide EDTA Ethylenediaminetetraaceticacid FBS Foetal Bovine Serum MMT Cloisite Na+ montmorillonite MTT 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan NBT NitroBlue Tetrazolium NR Neutral red PBS Phosphate BufferSaline SDS-PAGE Sodium-dodecyl-sulphate-poly-acrylamide-gel-electrophoresis TEMED Tetramethylethylenediamine
DominicThum:N0625034 6 Summary Nanomaterialsare definedasmaterialsthatrange from1 nm to 100 nm insize.Due to theirsmall size, theyare notaffectedbythe lawsof quantummechanicsandexhibitpropertiesdifferenttotheirlarger counterparts.These propertiesallowthemtobe appliedinvariousindustries,suchascatalystsin chemical industriesorreinforcingagentsinmanufacturingindustries.A classof nanomaterialsare nanoparticleswhichcanbe categorized basedontheirmethodof creationorbasedontheirchemical composition.Thisclassof nanomaterialsare primarilyusedinbiomedical andpharmaceutical industries as drug carriersor scaffoldsfortissue engineering.Due tothis,investigatingthe cytotoxicityof these nanoparticlesiscrucial.Thisresearchprojectfocusesonthe cytotoxicityof ceramicnanoparticles, specificallyCloisite Na+ montmorillonite (MMT) andLucentite SWN. Bothwere chosendue tothe discrepanciesinunderstandingthe cytotoxicityof MMT and due to the lackof informationonthe cytotoxicityof Lucentite SWN,respectively.Therefore,the primaryaimof thisresearchprojectisto investigateandclarifythe cytotoxicityof bothnanoparticles.Thiswasdone usingMTT formazanassay and neutral redassay.A secondaryaimwasto determine the mechanismbehindthe effectof the nanoparticlesonthe cell if itwasdeterminedthattheywere cytotoxic.Thiswasdone usingwestern blotting.The resultsshowedthatCloisiteNa+ montmorillonite (MMT) hadlow cytotoxicitytocell viabilityintermsof cellularmetabolicactivitywhilstlucentite SWN showedlow cytotoxicitytocell viabilityintermsof cellularuptake activity.Comparingthe twonanoparticles,itwasestablishedthat Cloisite Na+ montmorillonite (MMT) wasmore cytotoxicthanlucentite SWN intermsof cellular metabolicactivity,andvice versaforcellularuptake activity.The westernblotanalysisdidnotreveal a clearresultand couldnotbe analysed.
DominicThum:N0625034 7 1) Introduction a. Nanoparticles:Definitionandapplications The fieldof nanotechnology hasexpanded intovariousindustriesaswell asotherareasof researchin the past fewyears.Its involvementinthesesectorsrange fromthe fieldof material sciences(afield whose purpose istostudy and create new materials) tothe fieldof medicalsciences.Thiscanbe contributedtothe verynature of these nanomaterialsthatthese nanoscientistsstudy.Asdefinedby bothLine (2008) and Lӧvestamet.al (2010), nanomaterialsare materialsorpowderswhosesizesranges from1 to 100 nm.Due to theirsize andtheircomposition,thesenanomaterialsexhibitpropertiesthat are differentto theirlargersizedcounterpartsastheyare notaffectedbythe classical lawsof quantum mechanics.These propertiesinclude;(a) electrical conductivity;(b) large surface area;(c) increased mechanical strength,andmanymore (Line 2008; EdelsteinandCammaratra1996). Assuch, these nanomaterialsare usedas eitherpigmentsorcatalystsforchemical processesorusedasreinforcing agentsfor manufacturingof plastics(Line 2008; Allsopp,WaltersandSantilo2007; Edelsteinand Cammaratra 1996). These unique propertieshave alsofoundnanomaterialsbeingusedinthe fieldsof biological sciences,especiallyinthe fieldsof i) tissue engineering(wherebythese nanomaterialsare usedas scaffolds);ii) drugtargetingand delivery,andiii) diagnosticandtherapeutictools(anexampleof nanomaterialsbeingusedinthisdepartmentiscarbonnanomaterialsbeingusedinchemical biosensors) (Raoet.al 2004; Lanone and Boczkowski 2006; Liu and Guo 2012, Salata 2004). As statedinLine’s andLӧvestam’s definition,nanomaterialscanappearaseitherpowdersorother forms(e.g.tubes,fibers).Assuch,Figure 1showsthe categoriesgiventonanomaterialsaswell the field theyare mostcommonlyusedin. Table 1: Categories of nanomaterials and their primary field of application Nanomaterial Category Primary field of application Nanoparticles Medical/Pharmaceutical Nanotubes Chemicals and advanced materials Nanoporous materials Information and communication Fullerenes Energy Quantum dots Automotive Nanostructured materials Aerospace Nanofibers Textiles
DominicThum:N0625034 8 Nanocapsules Agriculture Nanowires Dendrimers (Source:Pitkethly 2004) Due to the nature of thisresearchprojectas well asits’involvementinthe medical/pharmaceutical industries,nanoparticleswill be the mainfocusof thisintroduction. Nanoparticlesare definedas particleswhichrange from1 to 100 nm insize (Abhilash2010; Lӧvestamet.al 2010). Similarlyto nanomaterials,nanoparticlescanbe furthercategorizedintogroups.Unlike nanomaterials,however, nanoparticles canbe categorizedbasedontwocriteria:a) methodof production,and;composition (Table 2). Table 2: Categories of nanoparticles based on a) method of production and b) composition Different categories of nanoparticles based on: a) Method of production b) Composition Naturallyoccurring(e.g.volcanicashandsea spray) Carbon-based(e.g.fullerenes) Incidental (i.e.createdasaresultof man-made industrial processese.g.cookingsmokeand weldingfumes) Metal (e.g.goldnanoparticles) Engineered(e.g.sunscreenpigmentsand quantumdots) Ceramic/clays(e.g.montmorillonite) Biologicallyproduced (e.g.liposomes) Quantumdots( a.k.a nanocrystalsproducedvia combinationof GroupII/IIIandGroup IV/V elementsrespectively(e.g.Cadmiumselenide (CdSe))) Polymeric(i.e.biodegradable nanoparticles consistingof syntheticpolyesters(e.g. polylactic acid (PLA)). (Source:Universityof NorthTexas(UNT) 2015)
DominicThum:N0625034 9 Evenwiththese differentcategories,nanoparticlesshare the commonfeaturesof havinglarge surface to mass ratioas well ashavingthe abilitytoadsorband carry chemical compounds(De JongandBorm 2008). This,togetherwiththe factthat theirsurfacescan be furthermodified,makesthemideal candidatesfordrugdeliveryastheirsmall size allowsthemtoeasilypenetratetissuesororgansmore effectivelywhilsttheiradsorptionabilityallowsthemtocarrythe chemical compoundsintothe affected organ. Meanwhile,the nanoparticle’ssurface canbe made to interactwithspecificbiologicalstructures furtherincreasingits’abilitytotargetspecificorgansortissues (mice injectedwithzincsulphide (ZnS)- coatedquantumdotsthat were coatedinGFE peptides (thatspecificallybindstothe membrane dipeptidaseof lungendothelial cells)were showntohave retainedthe quantumdotsintheirlungs insteadof otherorgans (Ackerman et.al 2002)). Nanoparticleshave alsobeenusedtoaidindiagnosisof cancer (e.g.goldnanoparticleshave beenusedtodetectcancerbiomarkers(Xie et.al 2009)) andas imagingtools(He et.al 2008) due to these unique properties. Thoughthe propertiesof nanoparticlesmakesthemidealdrugcarriers,theircompositionplaysa significantrole indeterminingtheirappropriatenessasdrugcarriers. This isdue to nanoparticleshaving differentdegreesof toxicityonthe tissuesororganstheyinteractwith.Thisisknownascytotoxicity. The cytotoxicityof a nanoparticle affectswhetheritisusedincancer treatments(wherebyhigh cytotoxicityisdesired) orusedintissue engineering(wherebylittle tonocytotoxicityisdesired).Thishas resultedinextensive studiesbeingdone todeterminetheircytotoxicability(e.g.Cobaltnanoparticles have beenshowntoincrease incytotoxicitywithincreasingconcentration(Amnaet.al 2013) whilst single-walledcarbonnanotubes(SWNTs) are highlycytotoxic(Cui et.al 2005)).Unfortunately,mostof these cytotoxicitystudiesfocusonmetal nanoparticlesandquantumdotswhilstothertypesof nanoparticlesare givenlittle tonoattention. One of these lesserresearched typesare ceramic nanoparticles(a.k.ananoclays). b. Nanoclays: Definitionandapplications Ceramicnanoparticlesornanoclays are nanoparticlescomposingmainlyof oxides,nitridesorcarbides (Particular® 2015). Dependingontheircomposition,these ceramicnanoparticlescaneitherexhibit electrical conductivity(e.g.tinoxide(SnO2) nanoparticleshave beenused ingassensorsdue totheir abilitytogainelectrical conductance whenexposedtoreducing gases,suchas methane orcarbon monoxide (Jainet.al 2006)) or gain photocatalyticproperties(e.g.titaniumdioxide a.k.atitania (TiO2) nanoparticlesare commonlyusedinwaterpurificationsystemsdue toits’abilitytoproduce reactive oxygenspecies(whichreactswithorganicelementsinthe watertoform carbondioxide) whenexposed
DominicThum:N0625034 10 to UV radiation(Fadeel andGarcia-Bennett2010)).Ceramicnanoparticleshave alsoseenapplicationin tissue engineeringas theyserve asstabilizingagentsinnanocompositescaffolds(Camargo, Satyanarayanaand Wypych2009) as well asdiagnostictools,suchasDNA microarrays(Caputoet.al 2007) . Lookingatthe examplesof ceramicnanoparticlesapplications,itcanbe seenthatthere are differenttypesof ceramicnanoparticles,rangingfromamorphoussilica(e.g.LUDOX® CLcolloidal silica) to fibroussilicates (e.g.Halloysite).However, thisresearchproject willbe lookingattwospecificceramic nanoparticles:i) CloisiteNa+ montmorillonite,and;ii) Lucentite SWN. i. Cloisite Na+ montmorillonite (MMT) Cloisite Na+ montmorillonite (MMT) isa white-coloured naturally-occurringclaymineralderivedfrom volcanictuffsandash and producedbyhydrothermal activity(Figure1A) (Mindat.org2015). Thisclay mineral belongs tothe large smectite claymineral group.Assuch,its’structure consistsof analuminium oxide/hydroxide layer(Al2(OH)4) sandwichedbetweensilicate layers(Figure 1B) (Uddin2008). Figure 1:A) Cloisite Na+ montmorillonite B)Layered structure of Cloisite Na+ montmorillonite (MMT) Figure 1: (A) Montmorillonitefrom Karnasurt(B) Layered structureof montmorillonite,a member of the smectite group of ceramic nanoparticles (Mis the exchangeable cation which connects the sheets of silicates [in the caseof CloisiteNa+ montmorillonite(MMT), M is Na+]) (Source: de Graaf 2015 and Pusch et. al 2012, respectively) A B
DominicThum:N0625034 11 Due to its’layeredstructure,MMT exhibitphysical propertiessuchas:i) absorbingwaterthuscausing swelling;ii) absorborganicliquids(e.g.glycerolandpolyhydricalcohols),and; iii) ahighcapacityto exchange cationsininorganicreactions(EncyclopædiaBritannica2015; Uddin2008; Hackman and Hollaway2006). These propertiesallowMMT nanoparticles tohave manyapplications rangingfrom beingcomponentsof drillingmud(Uddin2008),reinforcementof foodpackaging(Maisanabaet.al 2013) as well aslinersforirrigationditches(due toits’affinitytoswell whenexposedtowater) (Murray 2000). The biomedical applicationsof MTTnanoparticlesincludethe fieldof tissue engineeringas stabilizingagentsintheirscaffolds (due totheirease of availability)(Camargo,Satyanarayanaand Wypych2009) as well asbeingusedasdrug carriersfor anti-cancerdrugs incancer treatments(Lee et.al 2005; Fenget.al 2009). Due to its’use ina diverse range of applications,establishingthe cytotoxicityof MMT nanoparticlesisanimportanttask.However,there doesnotappeartobe a general consensusinregardsto the cytotoxicityof MMT nanoparticles.Some studieshave claimedthatMMT nanoparticlesare notcytotoxicunlessorganicallymodified(Liuet.al 2011; Sharma et.al 2010) or have anti-cancerdrugsattachedto it (Lee et.al 2005) whilstotherstudiessuggestthat regardless of modifications,MMT nanoparticlesare cytotoxic(Lordan,KennedyandHigginbotham2010; Baek,Lee and Choi 2012). ThisdiscrepanciesonMMT cytotoxicitycanbe contributedtothe lack of standardizationinrunningthe cell viability tests(Konget.al 2011). Furthermore,the cell linesusedby the studiesare alsodifferentfromone another.These range from humancells(HepG2humanhepatic carcinoma(Lordan,KennedyandHigginbotham 2010; Maisanabaet.al 2013) andCaco-2 human colon carcinoma(Jorda-Beneytoet.al 2013; Sharma et.al 2010) beingthe mostpopular) tomouse cells(e.g. NIH 3T3 mouse embryonicfibroblasts(Liu et.al 2011)).It ishopedthat thisresearchprojectwouldnot onlydecrease the discrepancyonthe cytotoxicityof MMT butalsoprovide a standardizedapproachto investigatingits’cytotoxicity. ii. Lucentite SWN Lucentite SWN,onthe otherhand,is a white odourlesspowder,composingof lithiummagnesium sodiumsilicate.Asitsharesthe same layeredstructure asMMT itis consideredtobe partof the smectite claymineral group(Co-opChemical Co.Ltd2001) (Figure 2).
DominicThum:N0625034 12 Figure 2: Layered Structure of Lucentite SWN However,unlikeMMT,SWN isa syntheticclaythatcan be made viatwoproductionmethods:i) reacting magnesiumsilicatesandalkali cationstogether(Jiang2007) or ii) reactingsmectite claywithorganic compounds(Co-opChemical Co.Ltd2001). The rationale behindthe productionof thissynthetic smectite wasdue tothe fact thatprevioussmectite productsshowedlow transparency, low brightness as well aslowdispersion.These propertiesare essential assmectite claysare usuallyusedasgelling agentsfor beautyproducts(e.g.lipsticks),medicinesandwater-solublepaints(Co-opChemical Co.Ltd 2001; KoboProducts,Inc 2015). Due to the demandfora betteralternatives,the propertiesof Lucentite SWN include hightransparencyandbrightnessaswell asprovide highviscositywhendispersedinwater (Co-opChemical Co.Ltd2001) as well ashavingthe sharedpropertiesof smectite clays(asdescribedin 1.b.i).Beingthatthe primaryapplicationof SWN isin cosmetics(KoboProducts,Inc2015), there would have been excessive cytotoxicstudiesdone todetermineits’appropriatenessof use inthese products. Strangelyenough,there appearstobe noexistingliterature investigatingthe cytotoxicityof SWN. 2) Aims The above sectionhasgivenan insightintothe currentknowledge inregardstothe cytotoxicityof nanoclaysandalsohighlightedthe gapswithinit, specificallyinregardstothe cytotoxicityof Cloisite Na+ montmorillonite (MMT) andlucentite SWN.Therefore,the aimsof thisresearchis: Figure 2: Layered structureof lucentite SWN (The exchangeable cation being lithium(Li+) or magnesium (Mg2+) (Source: Co-op Chemicals Co. Ltd 2001)
DominicThum:N0625034 13 i) To investigate andcompare the cytotoxicityof natural smectite Cloisite Na+ montmorillonite (MMT) andsyntheticsmectite lucentite SWN,and; ii) To investigate mechanismof cytotoxicactionof nanoclayson muscle cell growthand differentiation. For thisresearch,C2C12 mouse myoblastcellswereselectedasthe cell model.C2C12cellswere first discoveredbyYaffe and Saxel whentheyserial passagedmyoblastsderivedfrom dystrophicC3Hmouse thighmuscle (Yaffe andSaxel 1977). Beingpluripotentmesenchymal satellitecells,C2C12 cellsare are able to entertwophases:i) self-renewal(wherebytheyrapidlyproliferate),and;ii) differentiation (wherebytheydifferentiatedintomyotubesunderspecificconditions) (YaffeandSaxel 1977; Buratinni et.al 2004). These characteristicscomplimenttheirrole inmuscle growthandrepair.Whenitwas discoveredthatserumconditionswithinthe culture mediums affectedwhichphase thesecellswould enter(C2C12 cellsrapidlyproliferate andgrow inhighserumconditionswhilstlow serumconditions inducesdifferentiation)andthatthe serumconditionscouldbe controlled(LawsonandPurslow 2000), C2C12 cellswere seenasan ideal cell modeltostudyskeletalmuscle growthanddevelopment (KhodabukusandBaar2007). The fact that mice alsoshare similarmuscle physiologytohumansalso contributedtothis (The JacksonLaboratory2014). Furthermore, C2C12 cellshave alsobeenusedin cytotoxicitytestsfornanoparticles,suchasquantumdots(Rahabet.al 2012) and zincoxide nanoparticles(Panduranganet.al 2014).However,littletonocytotoxicitystudiesforclaynanoparticles have usedC2C12 cell as cell modelswhichwasone of the reasonstheywere chosenascell modelsfor thisresearchproject.Furthermore, differentiatedC2C12myotubes have distinctcellularmorphology in comparisontotheirmyoblastcounterparts(Figure 3) whichalsoplayedarole intheirselectionforthis researchproject. Figure 3: C2C12 mouse (A) myoblasts, and; (B) myotubes
DominicThum:N0625034 14 MTT formazanassay andneutral red (NR) assaywas usedto measure cell viability.MTTformazanassay isa colorimetricassaythatmeasurescell viabilityinterms of cell metabolicactivity viathe conversionof the MTT dye intopurple formazandye bythe NADPH-dependentoxidoreductase enzymesinthe cells. These can be foundon the mitochondrial membrane orthe cytosol of the cell.The presence of the purple dye once the cellsare introducedtothe MTT dye signifiesthatcellularmetabolicactivityis present,hence the cell isalive (Berridge et.al 2005; Berridge andTan 1993). Neutral redassay,on the otherhand,measurescell viabilityintermsof cellularuptake viathe stainingof lysosomes.Thisstaining representsthe abilityof the cellstotake upthe red dye and indirectly,substancesfromextracellular environment((Repettoet.al2008; Lӧwiket.al 1993). Usingthese twocell viabilityassayswouldprovide a betterperspective onhowthe nanoparticlesaffectedthe cell viabilityof the C2C12 mouse myoblast cells. In the eventthe cell viabilityassaysshowedadecrease incell viabilityof the C2C12 mouse myoblast cellsafterthe introductionof the nanoparticles,the methodbehindthe effectof the nanoparticle on the cell viabilitywasinvestigated. Itwasassumedthattwoof the followingmechanismswouldoccur: a) Necrosis,and; b) Apoptosis Necrosisisdefinedascell deathcausedbyphysical andmechanical damage tothe cells(e.g.shearingor physical puncturing) whichcausesthe cellularmembrane integritytobe lost,causingcell lysisand release of cellularcomponentstothe extracellularenvironment(Proskuryakovet.al 2003). Apoptosis, on the otherhand,is definedascontrolledcell deathwherebyspecificcell signalingcascadesare activatedandregulated whichcausescell death(Albertset.al2008). Some of the different morphological characteristicsof cellsundergoingnecrosisandapoptosisare described inTable 3below: Figure 3: (A) C2C12 myoblasts and (B) C2C12 myotubes. (Source: Gundry et.al 2009)
DominicThum:N0625034 15 Table 3: Comparison of morphological characteristics betweencellsundergoingnecrosisand apoptosis Morphological Characteristics Necrosis Apoptosis Swellingof cytoplasmdue toinflux of waterand ions Condensationof the nucleusandshrinkingof cytoplasm Loss of plasmamembrane integrity Plasmamembrane blebbing Complete lysisof vesicles Apoptoticbodies(membrane-boundvesicle) formed Swellinganddisintegrationof internal organelles Increasedpermeabilityof mitochondrial membrane Complete cell lysis Fragmentation of cell intosmallerapoptotic bodies To investigate if necrosisoccurred,cellularimagingwasusedtoidentifythe morphological characteristicsof necroticcells.Toinvestigateif apoptosisoccurred,westernblottingwasusedtodetect the presence of caspase 3. Caspase 3 (cysteinyl-aspartateproteases) are proteasesinvolvedinthe apoptoticcell signalingpathwaysandexistsasinactive pro-caspase 3(Porterand Jänicke 1993). When apoptoticsignals(e.g.release of cytochrome Cfrommitochondriaor deathsignals) are detected,the prodomainof pro-caspase 3 wascleavedintoactive caspase 3 whichisresponsible foractivating apoptosissignalingcascadesincells.Therefore,caspase 3 was a suitable apoptoticmarker. (Source: Baba 2009;University of Colorado Colorado Springs (UCCS) 2015)
DominicThum:N0625034 16 3) Materials and Methods a) Materials a. Cell lines Cell line Details Source/manufacturer C2C12 Mouse myoblastcells AmericanType culture collection(ATCC),Manasas, VA (donatedfromDr. John BrameldtoDr Jane Harper) b. Antibodies Antibody Type Description Manufacture and catalogue no. Dilutionfactor Anti-active +proCaspase 3 antibody Primary Polyclonal,Rabbit Abcam(ab47131) 1:1000 (WB) Beta-TubulinLoadingControl Antibody(BT7R) Primary Monoclonal, Mouse ThermoScientific (MAS-16308) 1:2000 (WB) MyosinHeavyChain2 antibody(EPR52801) Primary Monoclonal, Rabbit GeneTex (GTX63258) 1:10000 (WB) Anti-Mouse IgGantibody Secondary Polyclonal, Alkaline Phosphatase conjugated,Goat ThermoScientific (31322) 1:5000 (WB) Anti-RabbitIgGantibody Secondary Polyclonal, Alkaline Phosphatase conjugated, Donkey Santa Cruz Biotechnology, Inc. (sc-2315) 1:5000 (WB)
DominicThum:N0625034 17 c. Assay kits Name Details Manufacturer PierceTM BCA Assaykit Contents:BCA ReagentA (sodium carbonate, sodiumbicarbonate pierce BCA detectionreagent,and sodium tartrate in0.1N sodiumhydroxide),BCA ReagentB (4% cupric sulfate) and bovine serumalbumin(BSA) standard (sodiumchloride solutioncontaining sodiumazide) ThermoScientific(23225, 23227) d. Chemicals Name Details Manufacturer Ammoniumpersulphate Sigma-Aldrich(215589) BCIP-T 5-bromo-4chloro-3-indolylphosphate ThermoScientific(R0821) Bovine serumalbumin(BSA) ThermoScientific(B14) Biocleanse Teknon(TK210) Cloisite Na+ montmorillonite (MMT) 0.3% suspension The SouthernClay ProductsCo. USA Dulbecco’s Modified Eagle Medium(DMEM) 1X 4.5 g/L D-glucose,0.11 g/L sodium pyruvate andwithoutL-glutamine Gibco (11995-073) Dulbecco’sModifiedEagle Medium(DMEM) 1X 4.5 g/L D- glucose withoutL-glutamine Lonza (BE12-614F) Dimethyl Sulfoxide(DMSO) FisherChemical (D128-500) EDTA Ethylenediaminetetraaceticacid Sigma-Aldrich(E9884) Ethanol 70% and99.9% EZ-RunProteingel solution (12.5%) Contents:Water,acrylamide, 4- Morpholinepropanesulfonicacid, Tris (hydroxymethyl) aminomethane Fisher-Scientific(BP7712-100; BP7712-30; BP7712-500)
DominicThum:N0625034 18 EZ-RunRunningBuffer20X Fisher-Scientific(BP7700-500) Foetal bovine serum(FBS) Heat inactivated Lonza (DE14-830F) L-Glutamine Lonza (17-605) Glycine Fisher-Scientific(BP3811) HEPES buffer(1M) 1M in 0.85% sodiumchloride (NaCl) Lonza (17-737E) Laemmli buffer 5X Laemmli Sample buffer Lucentite SWN 0.3% suspension CoopCorporation,Japan Methanol Laboratory Reagent,≥99.8% FisherChemical (A412-4) Milkpowder Skimmed Marvel Monosodiumphosphate (NaH2PO4) BDH GPR MTT formazan 1-(4,5-Dimethylthiazol-2-yl)-3,5- diphenylformazan Sigma-Aldrich(M2003) NitroBlue Tetrazolium(NBT) ThermoScientific(R0841) Neutral Red 3-Amino-7-dimethylamino-2- methylphenazine hydrochloride Sigma-Aldrich(N7005) Penicillin/Streptomycin Lonza (17-602E) Phosphate buffersaline (PBS) Sterile phosphatebufferforcell culture (0.0067M PO4 withoutcalciumand magnesium) Lonza (BE17-516F) PageRulerTM PlusPrestained ProteinLadder Proteinladder ThermoScientific(26621) Protease InhibitorCocktail Contents:Aprotinin,Bestatin,E-64, Leupeptin,PepstatinA and4-(2- Aminoethyl)benzenesulfonylfluoride hydrochloride (AEBSF ) serineproteases Sigma-Aldrich(P8340) Sodiumdodecylsulphate(SDS) Sigma-Aldrich(L3771) SodiumChloride (NaCl) Melford (S0520) Sodiumorthovanadate (Na3VO4) Sigma-Aldrich(S6508) TEMED Tetramethylethylenediamine Sigma-Aldrich(T9281) Trypan blue Sigma-Aldrich(T8154)
DominicThum:N0625034 19 Tris Base Fisher-Scientific(BP154-1) Tris HCl Melford (T1513) TrypsinEDTA (ethylenediaminetetraaceticacid) solution 0.25% trypsin,1mMEDTA Lonza (CC-5012) Tween20 Polyoxyethylenesorbitonmonolaurate Fisher-Scientific(BP337-500) e. Equipment Equipment Details Manufacturer BarnsteadTM nanopureTM watersystem ThermoScientific Cell culture flask andplates 25 cm2 and 175 cm2 (red cap ventilated);6,24 and 96 well culture plates SARSTEDT AG & CO. Centrifudges 15/80 MSE HeraeusMultifuge X3 ThermoScientific Harrier ChemidocTM MP Imagingsystem Bio-Rad Disposable cell scrapers Fisher-Scientific Sterile hood ClassII microbiological safetycabinets Walker FilterPaper Standardgrade Whatman FisherbrandTM WaterBath Fisher-Scientific Haemocytometer NanoEntek Incubator MCO 18AC Panasonic Mini-PROTEAN® Tetracell SDS-PAGEkit Bio-Rad Ministartsingle use filterunit 0.2 µg and 0.4 µg Sartoriusstedimbiotech Microscope Inverted phase contrast microscope- Primovert Carl Zeiss Microscope camera AxiocamERc 5s Carl Zeiss Particle Size analyzer CoulterN4 Plus BeckmanCoulter
DominicThum:N0625034 20 Plate Reader(24 and 96 well) UVM340 Asys Pippettes Sterile Pippettes:1,2, 5, 10, 25 ml Micropippette:0.5- 10 µl,2-20 µl,20-200 µl, and 100-1000 µl Automaticpropipetter SARSTEDT AG & Co. FisherScientific FisherScientific pH/ORPmeter Hanna instruments(HI2211) Powerpack PowerPacTM Universal PowerSupply Bio-Rad Rapidsemi-dryblottersystem WSE-4020 HorizeBLOT 2M-R ATTO Flatbedorbitershaker KS 260 Basic shaker IKA Transfermembrane ProteanTM nitrocellulose Whatman Tubes Eppendorf,20 ml and 50 ml SARSTEDT AG & Co. Vortex mixer Lab Dancer IKA OxfordWeighingscale FisherScientific f. Software Software Manufacturer SPSS IBM ZEN 2012 software Carl Zeiss
DominicThum:N0625034 21 b) Methods a. Cell culture i. Growth The C2C12 mouse myoblastcellswereprocuredfrom AmericanType culture collection(ATCC), Manasas, VA (donatedfromDr.John BrameldtoDr Jane Harper).These cellswere grownin growth mediumconsistingof highglucose Dulbecco’sModifiedEagle Medium(DMEM) (4.5 g/l D-Glucose,0.11 g/l sodiumpyruvate,withoutL-Glutamine) mixedwith10% foetal bovine serum,1% penicillinand streptomycinand1%glutamine.The growthof the C2C12 cell line wasmaintainedviaincubationat 37o C withan atmosphere of 95% airand 5% carbon dioxide (CO) withamediumchange everythirdday of the week(Burattini et.al 2009; Brownet.al 2012; Fisher-AylorandWilliams2011). ii. Sub-culturing Sub-culturingwasdone whencellsreached80to 90% optical congruence.The growthmediumwas discardedandthe cellswere washedwith20ml phosphate buffersaline (PBS).AsC2C12 were adherent cells,theirdetachmentfromthe surface of the cell culture flaskwas inducedbyincubatingthe cellsin5 ml freshlythawedtrypsinEDTA (0.25%trypsin,1mM EDTA) for 5 minutes.After5 minutesincubation, cellswere observedunderaninverted phasecontrastmicroscope (Carl ZeissPrimovertInvertedphase contrast microscope) todetermine degree of detachmentfromcell flasksurface.10ml of growth mediumwasaddedtostop trypsinreaction.The mediumcontainingthe cellswere thentransferredto centrifuge tubesandcentrifugedat300 rpm for5 minutes(ThermoScientific15/80 MSE centrifuge). Supernatantwasdiscardedand2 ml of growth mediumwasusedtoresuspendthe cellsforcounting. Cellswere counted before beingseeded ataconcentrationof 2 x 103 /25 cm2 cell culture flasks. iii. Viabilityand cell counting Trypan blue wasusedtoassesscell viabilityandthe dye wasmixedwiththe cell suspensionwitha1:1 dilution(0.2ml of cell suspensionand0.2ml of trypan blue stain).Haemocytometer(NanoEntek) were usedand filledwiththisdilution.Phase contrastmicroscope wasusedtoobserve the cells.Cellsthat were stainedblue were considerednon-viablecellsastheyabsorbedthe dye.The numberof viable cells ineach of the four1X1 mm squareswere countedwiththe average countbeingusedtodetermine the numberof viable cellswithinthe cell suspension.
DominicThum:N0625034 22 b. Preparation of nanoclay suspensions The Cloisite Na+ montmorillonite (MMT) wasprocuredfrom The SouthernClayProductsCo.USA whilst the lucentite SWN wasprocuredfromCoopCorporation,Japan.The nanoclay suspensionswere preparedat 0.3% concentrationusingdeionizedwaterasa solventandhand-shaken.The suspensions were thenautoclavedat120o C to decrease the riskof biological contamination.Laserlightscattering usinga particle size analyzer(BeckmanCoulterN4Plus) wasusedonthe suspensionbeforeandafter treatingthe cells todetermine changesinparticlesize anddistribution. c. Experimental Design For the initial cell viability experiment,the cellswere grownon24-well cell culture plates(Sarstedt) and were seededattwocell densities:1x 104 cells/cm2 (low celldensity)and5 x 104 cells/cm2 (highcell density).Thiswastodetermineif cell densityaffectedthe effectof the nanoparticle suspensionsonthe viabilityof the C2C12 cells. The cellswere allowedtogrow for 24 hoursto ensure attachmentbefore additionof nanoparticle treatments.The treatmentswere asfollows: 1) 10% FBSDMEM growthmedium (i.e.control) 2) 100 µg/ml of nanoclayin10% FBS DMEM (0.1%) 3) 10 µg/ml of nanoclay in10% FBSDMEM (0.01%) 4) 1 µg/ml of nanoclayin10% FBS DMEM (0.001%) 5) 1% FBS DMEM (differentiationmedium)* The wellswere washedwithPBSgentlybeforethe treatmentswere added.The treatmentswere added intofourwells andwere randomlydistributedamongstthe 24-well plate.Inlaterstagesof the experiment,the cellswere seededat1 X 104 cells/cm2 asitwas believedthatthe highcell densitywas affectingthe growthof the cellsindependentof the treatmentsgiven.The treatedplateswere incubatedoverthe spanof three dayswithplatesbeingtakenoutat specifictime points(Day1,Day 2 and Day 3) to run the cell viability assays. For proteinestimationand extractionexperiment,asimilarexperimental setupasthe cytotoxicitytest was done ( withcellsseededat1 x 104 cells/cm2 ) exceptthe platesusedwere 6-well cellculture plates and the treatmentswere asfollows: *This treatment was used to determine if the cells were ableto differentiate
DominicThum:N0625034 23 1) 10% FBSDMEM (control) 2) 1% FBS DMEM (control) 3) 100 µg/ml of nanoclayin10% FBS DMEM (0.1%) 4) 100 µg/ml of nanoclayin1% FBSDMEM (0.1%) 5) 1 µg/ml of nanoclayin10% FBS DMEM (0.001) 6) 1 µg/ml of nanoclayin1% FBS DMEM (0.001%) The treatmentswere organizedasseeninFigure 3below: Figure 3: Organisation of treatments in 6 well plate The plateswere incubatedandharvested ontimepoints day1, 2 and 3 forfurtheranalysis. d. Cytotoxicityassay i. MTT formazan assay The MTT viabilityassayprotocol wasderivedfromImmutec Method51 withsome minordifferences. Thiscolorimetricassaymeasuresviabilitybasedon NADPH-dependentoxidoreductase enzymeactivity inconvertingthe MTT (1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan) (Sigma-Aldrich)dye into purple formazan(Berridge et.al 2005; Berridge andTan 1993). The MTT solutionwaspreparedby 10% FBS in DMEM (Primary control) 1% FBS in DMEM (Secondarycontrol) 100 µg/ml of nanoclay in 10% FBS DMEM (0.1%) 100 µg/ml of nanoclay in 1% FBS DMEM (0.1%) 1 µg/ml of nanoclay in 10% FBS DMEM (0.001%) 1 µg/ml of nanoclay in 1% FBS DMEM (0.001%)
DominicThum:N0625034 24 weighingout500 mg MTT and dissolvedin100 ml PBS. The solutionwasthensterile filteredthrough0.2 µM and 0.4 µM before beingdistributedin50ml centrifuge tubes.The tubeswere coveredin aluminiumfoiltopreventthe MTT solution beingexposed tolightandstoredat -20o C. Duringthe differenttime pointsof the treatedcell incubation (Day1,Day 2 andDay 3), MTT solutionat a concentrationof 0.5 mg/ml (i.e.100 µl perwell) wasaddedtoeachwell of the selected platesbefore theywere incubatedforanotherhour.Afterincubation,the wellswere washedgentlywithPBSbefore 500 µl of Dimethyl sulfoxide(DMSO) wasaddedtoeachwell.The plateswere thengentlyshaken ona flatbedplate shaker(IKA KS260 Basic shaker) for2 minutesat80 rpm before aplate reader (Asys UVM340) was usedto readthe absorbance at 570 nm.This wasdone within30 minutesof the addition of DMSO as the formazandye will begintodilute whenexposedtolight. ii. Neutral Red assay The protocol for the neutral redassay wasderivedfromLӧwiket.al (1993) withslightmodifications. This colorimetriccell viabilityassayserves toestimate the abilityof cellstoendocytose nutrientsviastaining lysosomesred.Neutralred(NR) solutionwaspreparedbyadding0.1g of neutral redpowderin100 ml of nanopure waterwhichwasthenfiltersterilizedusing0.2µM and 0.4 µM filters.The NRsolutionwas thenaddedto 1.8% sodiumchloride solutionat1:1 dilutiontoformthe workingsolution.0.2ml of the workingsolutionwasthenaddedtoeachwell of the selectedplatesof differenttime points.These plateswere lefttoincubate foranother1.5 hours.The plateswere thenwashedtwice usingPBSbefore 1 ml of 0.05M NaH2PO4 in50% ethanol wasaddedto the wellstoextractthe dye.A plate reader (Asys UVM340) was thenusedto readthe absorbance at 550 nm, with620 nmbeingthe reference wavelength. e. Cell harvest Cell harvestandproteinextractionmethodswere describedinBrownet.al 2011. The C2C12 cellswere resuspendedintothe mediumusingcell scrapers.Thismediumwasthentransferredintoa20 ml centrifuge tube.Wellsthatwere scrapedwasalsowashedwith 2ml PBSwhichwere alsotransferred intothe centrifuge tube.The cell suspension were thencentrifugedat500g for 5 minutes (Harrier HeraeusMultifuge X3centrifuge).The supernatantwasdiscardedand1ml of PBS wasaddedto the tubes.The tubeswere centrifugedagainat2000g for2 minutes.The supernatantwasagaindiscarded and the tubeswere air-dried.The cell pelletswere thensuspendedin0.2ml of cell lysisbuffer(10mM
DominicThum:N0625034 25 Tris Base,5mM EDTA, 150mM NaCl,1mM sodiumorthovandate,1% SDSand 0.2% Protease Inhibitor cocktail).The cell extractswere thenboiledat100o C before beingstoredin -18o C. f. Proteinextraction and estimation The bicinchoninicacidassay(BSA) protocol wasderivedfromthe manufacturer’sinstructionsforthe Pierce® BCA Assaykit (ThermoScientific).The dilutedalbuminstandards(BSA)(2000 µg/ml,1500 µg/ml, 1000 µg/ml,750 µg/ml,500 µg/ml,250 µg/ml,125 µg/ml,25 µg/ml and 0 µg/ml) were prepared followingthe instructionsgiven,usingthe cell lysisbufferasdiluent.Thiswasdue tothe presence of SDS withinthe bufferwhichcouldinterfere withthe BCA reagents.Toensure propermixing,avortex mixer was usedtostir the BCA standardsandthe cell extracts.25 µl of standardsand extractswere pipetted intoa 96-well plate,eachstandardand extractreplicatedtwice.200 µl of workingsolutionconsistingof 50:1 dilutionof ReagentA andReagentBwas thenaddedtoeach well.The 96-well plate wasshakenfor 30 secondsbefore beingincubatedfor30 minutesat37o C. Afterincubation,aplate readerwasusedto readthe absorbance at 565nm. A standardcurve wasdrawn usingthe absorbance readingsof the standardsand the concentrationof the standards.The curve was thenusedto determinethe protein concentrationof the cell extracts. g. SDS-PAGEand WesternBlotting i. SDS-PAGE Proteingelswere assembledbasedoninstructiongivenbymanufacturers(Fisher- Scientific).10ml of 12.5% EZ-Rungel solutionwasmixedwith60µl of ammoniumpersulfate(APS) and6 µl of TEMED. This mixture wasimmediatelypouredbetweenglassplatesof anassembledgel casting stand(insertproduct line here) (Bio-Rad).Combswere thenaddedtothe topof the glass platesbefore the gel wasleftto polymerise.Once polymerized,the gelswereremovedfromthe gel assemblyandplacedintoaprotein gel tank (productline here).800 ml of 20:1 dilution runningbuffer(40ml 20X EZ-RunBufferdilutedin 760 ml of deionizedwater) wasthenaddedtothe gel tank. The selectedcell extractswere thenthawedbefore 10µl of 5X Laemmli buffer(findoutreagentshere) was addedtothem.The mixture wasthenboiledbefore20µl of the mixture waspipettedintoeach well,togetherwith2.5µl of the PageRulerTM PlusPrestainedProteinLadder(Thermo-Scientific).The gel was thenallowedtorunfor1 hour at 180v or until the proteinmarkersreachedthe endof the plate.
DominicThum:N0625034 26 ii. Proteintransfer Once the run was complete,the gel wasthenplacedintotransferbuffer(48mMTRIS Base,39mM glycine,0.0375% SDS, 20% methanol).Eightfilterpapersand a nitrocellulose membrane (both Whatman) were cut at 10x8 dimensionsandwere alsosoakedintransferbuffer.A transfersandwich ( fourfilterpapers– nitrocellulose membrane –polyacrylamide gel –fourfilterpapers) was stackedon the anode surface of the rapid semi-dryblottersystem(ATTO WSE-4020 HorizeBLOT2M-R). A glass rod was usedtoremove anyair bubbleswhilstexcesstransferbufferwaswipedaway. The cathode plate of the semi-drytransferapparatuswasthenplacedandthe blotterwasallowedtorunfor 1 hour at 50 mA pergel. The nitrocellulose membrane wasthenremovedwiththe aidof tweezers. h. Immunoprobingof Westernblots The transferrednitrocellulose membrane wasthencut at70 kDa and36 kDa to formthree sections (Figure 4) in accordance to the molecularweightof the primaryantibodiesused withproteinmarkers beingusedasreference pointsforcuttingthe nitrocellulose membrane. Figure 4: Horizontal slices on nitrocellulose membrane The proteinschosentobe detectedwere caspase-3(anindicatorof apoptosis),myosin heavychain(an earlyindicatorof differentiation) andβ-tubulin(acommonhousekeepingproteinexpressedinmouse myoblaststhatservesasthe loadingcontrol).Assuch,these three antibodieswere chosen:
DominicThum:N0625034 27  Anti-active +proCaspase 3 antibody (32kDa) (Abcam)  MyosinHeavyChain2 antibody(200kDa) (Genetex)  Beta-TubulinLoadingControl Antibody (51kDa) (Thermo- Scientific) The secondaryantibodiesusedwere:  Goat anti-mouse antibodyconjugatedwithalkalinephosphatase (usedtodetect Beta-Tubulin LoadingControl Antibody) (ThermoScientific),and;  Donkeyanti-rabbitantibodyconjugatedwithalkalinephosphatase (usedtodetectboth Anti- active + pro Caspase 3 antibody andMyosinHeavyChain2 antibody) (SantaCruzBiotechnology, Inc.) The cut nitrocellulose membraneswere thenplacedintoblockingbuffer(5% Marvel milkpowderinTris buffersaline-Tween(TBST) (50mM Tris Base,200 mM NaCl at pH 7.4 and 0.1% Tween-20)) for1 hour at room temperature withcontinuousshaking.The blockednitrocellulose membraneswere then transferredintoprimaryantibodysolutions(dilutedprimaryantibodies(see3.a.b.fordilutions) in5% BSA inTBST) whichwere thenincubatedat4o C overnight. The incubatednitrocellulose membraneswere thenwashedinTBSTfor 50 minutesatroom temperature,TBSTbeingreplacedevery10minutes.The washedmembraneswere thenplacedinto secondaryantibodyblockingsolutioninrespecttothe type of primaryantibodiesusedinitially(1:5000 secondaryantibodydilutionin5%BSA in TBST) and was lefttoincubate for2 hoursin4o C. The membraneswere thenwashedwithTBSTforanother50 minutes,withTBSTbeingreplacedevery10 minutes. The nitrocellulose membrane wasthenrinsedwithdeionizedwaterbefore beingimmersed insubstrate buffer(0.75 mMTris Base pH 9.5, 0.13 mMNitroblue tetrazolium and0.29 mM5-bromo-4chloro-3- indolyl phosphate (BCIP)) whichwaskeptinthe dark.The membrane wasobservedforvisualizationof bandsat 5 minute intervalsbefore the developmentprocesswashaltedbyextensive washingin deionizedwater(Blake et.al 1984). Bio-RadChemidocTM MPImagingsystemwasusedto digitize the WesternBlots.
DominicThum:N0625034 28 i. Image analysisand cell imaging Visualisationof cell cultureswasdone usingZeissPrimoVertinverted phase contrastmicroscopewith binocularphototube,ZeissAxioCamERC5S digital camera(Carl Zeiss,Germany) whilstthe processingof the imageswasdone usingZEN 2012 software (Carl Zeiss,Germany). j. Statistical analysis Statistical analysiswasdone usingIBMSPSSStatisticsoftware. Two-wayANOVA wasusedtodetermine if there wasa statistical difference inthe effectof concentrationof nanoclayandtime of exposure to cell viabilitywhilstt-testfordifferenceswasusedtodetermine if therewere differencesinthe effects betweenthe differenttreatments.Pvalues≤0.01 were consideredstatisticallysignificant.Datavalues were presentedasMean± STD (Standarddeviation). 4) Results a) Particle size analysis i. Cloisite Na+ montmorillonite (MMT) Figure 5 belowshowsthe particle size distributionsof pre-autoclaved CloisiteNa+ montmorillonite (MMT nanoparticle suspension andthe autoclaved CloisiteNa+ montmorillonite (MMT) nanoparticle suspension.The particle size distributionswere derivedby comparingthe laserlightintensity(measured in%) to the correspondingparticle size,withnumber% representingthe numberof particlesof aspecific particle size ata specificintensityandvolume% representingthe volume of the particle ataparticular particle size ata specificintensity. InFigure 5A whichrepresentsthe particle sizedistributionsof pre- autoclavedCloisite Na+ montmorillonite(MMT) nanoparticle suspension, three peaks(apeakfor number%andtwopeaksfor the volume%) were observed.Fornumber%,the particle sizerangedfrom 1.00 nm to 215.44 nm. For volume%,the particlesize range forthe firstpeak wasmeasuredtorange from1.00 nmto 146.78 nmwhilstthe particle size range forthe secondpeak rangedfrom215.44 nm to 1000.00 nm.In Figure 5B whichrepresentsthe particle sizedistributionsof the autoclavedCloisiteNa+ montmorillonite (MMT) nanoparticle suspension, threepeaks(apeakfornumber% andtwopeaksfor the volume%) werealsoobserved.Fornumber%,the particlesize rangedfrom1.00nm to 215.44 nm. For volume the particle size range forthe firstpeakwasmeasuredtorange 1.00 nm to 215.44 nm whilst the particle size range forthe secondpeakrangedfrom 215.44 nm to3162.28 nm. Figure 5C providesa comparisonof the particle size distributionsforbothpre-autoclavedandautoclavedCloisite Na+ montmorillonite (MMT) nanoparticle suspensions,respectively.Lookingatboththeir number%,the
DominicThum:N0625034 29 peaksremainedinthe same range andat the same intensitylevels.Forvolume%,itcanbe seenthatthe firstpeaksof both pre-autoclavedandautoclaved suspensionsremainedinthe same particle range (albeitthe intensity%forthe autoclavedMMT nanoparticle waslowerthanthatof the pre-autoclaved MMT nanoparticle suspensions).Onthe otherhand,the second peakof the autoclavedMMT nanoparticle suspension showedanelongationanda decrease inintensityincomparisontothatof the pre-autoclavedMMTnanoparticle suspension. Figure 5: Particle size distributionfor A) Pre-autoclavedMMT nanoparticle suspensionand B) AutoclavedMMT nanoparticle suspension;C) Comparisonof particle size distributionbetweenpre- autoclaved and autoclaved MMT nanoparticle suspensions A
DominicThum:N0625034 30 ii. Lucentite SWN B C
DominicThum:N0625034 31 Figure 6 belowshowsthe particle size distributionsof pre-autoclavedlucentite SWN nanoparticle suspensionandthe autoclavedlucentite SWN nanoparticle suspension.The particle size distributions were derivedbycomparingthe laserlightintensity(measuredin%) tothe correspondingparticle size, withnumber%representingthe numberof particlesof aspecificparticle sizeata specificintensityand volume%representingthe volume of the particle ata particular particle size ata specificintensity.In Figure 5A whichrepresentsthe particlesize distributionsof pre-autoclavedlucentiteSWN nanoparticle suspension, twopeaks(one peakfornumber% and one peakforthe volume%)were observed. For number%,the particle size rangedfrom1.46 nmto 4.64 nm.For volume%,the peakwasmeasuredto range from 1.47 nm to 4.64 nm. InFigure 6B whichrepresentsthe particlesize distributionsof the autoclaved lucentite SWN nanoparticlesuspension, twopeaks(onepeakfornumber% andone peakfor the volume%) werealsoobserved. Fornumber%,the particlesize rangedfrom2.15nm to 10.00 nm.For volume%,the peakwasmeasuredto range from2.15 nm to46.41 nm. Figure 6C providesacomparison of the particle size distributionsforbothpre-autoclavedandautoclaved lucentiteSWN nanoparticle suspensions,respectively.Lookingatboththeir number% and volume%,itcanbe seenthatthe peaksin autoclavedsuspensions shiftedtothe leftfromthatof the pre-autoclavedsuspensions. Figure 5: Particlesizedistribution for A) pre-autoclaved MMT nanoparticlesuspension,and;B) autoclaved MMT nanoparticlesuspension;C) Comparison of pre-autoclaved and autoclaved MMT nanoparticle suspensions particlesizedistribution
DominicThum:N0625034 32 Figure 6: Particle size distributionfor A) Pre-autoclavedSWNnanoparticle suspensionand B) AutoclavedSWNnanoparticle suspension;C) Comparisonof particle size distributionbetweenpre- autoclaved and autoclaved SWNnanoparticle suspensions A
DominicThum:N0625034 33 B C
DominicThum:N0625034 34 b) Cytotoxicityassay analysis i) MTT analysis i. Initial cell viabilityanalysis on C2C12 treated withCloisite Na+ montmorillonite (MMT) at two differentcell densities:a) 1 x 104 cells,and; b) 5 x 104 cells Figure 7 depictsthe meancell viability scoresobtainedinthe treatmentsusedforthe MTT assay at two differentC2C12 cell densities overincubation time(day).Itcanbe seenthat the treatmentshadan effectonthe cell viabilities of bothcell densitiesincomparisontothe primarycontrol (10% FBS DMEM) withthe cellstreatedwith100 µg/ml (0.1%) MMT showingthe biggestdifference. A one-wayANOVA confirmedthatthe treatmentshada significanteffectoncell viability (F(4)=6.531;p=0.000) whilstthe a post-hocTukey’stestdeterminedthatcellstreatedwith 100 µg/ml (0.1%) MMT showedthe greatest difference incell viabilityrelativetocellsgrowninthe primarycontrol (Meandiff.=0.267750; p=0.000) and cellsgrown indifferentiationmedium(Meandiff.=0.253854; p=0.001). The cellswiththe other MMT treatments(10µg/ml (0.01%) MMT; 1 µg/ml (0.001%) MMT) did notshow any significant difference tocellsgrowninthe primarycontrol andthe differentiationmedium, respectively(see Appendix 1).The resultsalsoshowthatcell densityplayedarole inthe effectof the treatmentsonthe cell viabilityasdemonstratedbythe decline incell viabilityof highercell densitywhentreatedwiththe primarycontrol.Thiswas furtherconfirmedwithatwo-wayANOVA (F(4)=73.384;p=0.000). Incubation time alsoplayedapivotal factorin the effectof the treatmentsonmeancell viabilityasitcan be seen that the cell viabilitiesforlowdensitycellstreatedwith10% FBS DMEM, 1% FBS DMEM, 10 µg/ml (0.01%) MMT and 1 µg/ml (0.001%) MMT show a gradual increase fromDay 1 to Day 3. The exception are the cellstreatedwith100 µg/ml (0.1%) MMT whichshowedaninitial increase fromDay1 to Day 2 but a gradual decrease onDay 3 (Figure 5A).The highdensitycells,onthe otherhand,show anopposite effectwherebycellstreatedwith10% FBS DMEM, 100 µg/ml (0.1%) MMT and 10 µg/ml (0.01%) MMT showedagradual decrease incell viabilityfromDay1 to Day 3. Cellstreatedwith 1µg/ml (0.001%) MMT, on the otherhand,showeda slightincrease incell viabilityfromDay1 to Day 2 before showinga slightdecrease onDay3 whilstcellstreatedwith1% FBS DMEM (differentiationmedium) showedan initial decreasefromDay1 to Day 2 but increasedslightlyonDay3 (Figure 7b).Thisexperimentwas repeatedtwice. Figure 6: Particlesizedistribution for A) pre-autoclaved SWN nanoparticlesuspension,and;B) autoclaved SWN nanoparticlesuspension;C) Comparison of pre-autoclaved and autoclaved SWN nanoparticlesuspensions particlesizedistribution
DominicThum:N0625034 35 Figure 7: Mean cell viabilityscore for each treatment at a) Low cell density(1 x 104 cells),and; b) High cell density(5 x 104 cells) B A
DominicThum:N0625034 36 Due to the resultsof the statistical analysisaswell asthe initial MTTanalysis,itwas decidedthatthe nextfewMTT cell viabilitytestswere tosee the effectsof MMT treatmentsonlow cell density. Furthermore,differentiationmediumwasremovedasatreatmentasit wasonlyincludedtodetermine if the C2C12 cellswere able todifferentiate. ii. Cell Viabilityanalysison C2C12 cellstreated withCloisite Na+ montmorillonite (MMT) at 1 x 104 cell density Figure 8 showsthe pooledmeancell viabilityscores fromthe treatmentsatlow cell density(1X104 ).It can be seenthatthe MMT treatmentshadaneffectonthe cell viabilityscoresincomparisontothe cells grownin 10% FBS DMEM with100 µg/ml (0.1%) MMT showingthe biggestdifference (asseeninFigure 5A). Similarly,aone-wayANOVA testconfirmedthatthe MMT treatmentshada significanteffecton the cell viabilityscores(F(11)=21.218; p=0.000) witha post-hocTukey’stestconfirming thatcellstreated with100 µg/ml (0.1%) MMT showedthe greatestdifferenceincell viabilityrelativetocellsgrowninthe primarycontrol (Meandiff.=0.224500; p=0.000), followedbycellstreatedin10µg/ml (0.01%) MMT (Meandiff.=0.110819; p=0.002). Onthe otherhand,cellstreatedwith1µg/ml (0.001%) MMT showed no significantdifference withcellstreatedinprimarycontrol (Meandiff. =0.36319; p=0.631). Similarly, incubationtime playedacritical role inthe effectof MMT treatmentsonthe cell viability asitcanbe seenthatcellstreatedwiththatthe cell viabilitiesforlow densitycellstreatedwith10% FBS DMEM, 10 µg/ml (0.01%) MMT and 1 µg/ml (0.001%) MMT show a gradual increase fromDay 1 to Day 3. On the otherhand,cellstreatedwith100 µg/ml (0.1%) MMT whichshowedaninitial increase fromDay1 to Day 2 buta gradual decrease onDay 3 (Figure 6A).Thiswasfurtherconfirmedwithatwo-wayANOVA test(F(6)=20.669; p=0.000) and cell). Thisexperimentwasrepeatedthreetimes. Figure 7: Pooled mean cell viability scores for each treatment againstincubation timeat(A) low cell density (1 x 104 cells) and (B) high cell density (5 x 104 cells)
DominicThum:N0625034 37 Figure 8: Mean cell viabilityscore for each treatment at lowcell densityVS incubation time iii. Cell Viabilityanalysison C2C12 cellstreated withLucentite SWN at 1 x 104 cell density Figure 7 showsa representative meancell viabilityscoresfromthe treatmentsatlow cell density (1x104 ).It can be seenthatthe SWN treatmentshadan effectonthe cell viabilityscoresincomparison to the cellsgrownin10% FBS DMEM with100 µg/ml (0.1%) SWN showingthe biggestdifference (as seeninFigure 7A).Thiswasfurtherconfirmedwithaone-wayANOVAtest(F(3)=6.596;p=0.000). However,the post-hocTukey’stestshowedthat1 µg/ml (0.001%) SWN showedthe greatestdifference incell viabilityscoresrelativetocellsgrowninthe primarycontrol (Meandiff.=-0.115833; p=0.001). The otherSWN treatments(100 µg/ml (0.1%) SWN and10 µg/ml (0.01%) MMT, respectively) showedno significantdifferencetothatof cellstreatedinthe primarycontrol (10% FBSDMEM) (Appendix 2).Once again, incubationtime playedacritical role inthe effectof SWN treatmentsonthe cell viabilityasitcan be seenthat the lowdensitycellstreatedwith10% FBS DMEM, 100 µg/ml (0.1%) SWN,10 µg/ml (0.01%) SWN showeda gradual increase fromDay1 to Day 3. The exceptionbeingthe cellstreated1 Figure 8: Pooled mean cell viability scores for each treatment againstincubation time
DominicThum:N0625034 38 µg/ml (0.001%) SWN showedasharp increase fromDay1 to Day 2 before showing aslightdecrease on Day 3. The effectof incubationtime onSWN treatmenteffectswere confirmedbyatwo-wayANOVA test(F(6)=3.145; p=0.006. Thisexperimentwasrepeatedthree times. Figure 9: Mean cell viabilityscore for each treatment at low cell densityVS incubationtime ii) Neutral Red analysis i. Initial cell viabilityanalysis on C2C12 treated withCloisite Na+ montmorillonite (MMT) at two differentcell densities:a) 1 x 104 cells,and; b) 5 x 104 cells Due to circumstances,initial neutral redanalysisforMMT treatmentsonC2C12 couldonlybe done on Day 1 and Day 3. Figure 10 depictsthe pooled meancell viabilityscoresobtainedinthe treatmentsused for the neutral red(NR) assayat two differentC2C12 cell densitiesoverincubationtime(day). Itcan be seenthatthe treatmentshadan effectonthe cell viabilitiesof bothcell densitiesincomparisontothe primarycontrol (10% FBS DMEM) withthe cellstreatedwith100 µg/ml (0.1%) MMT showingthe biggest difference. A Kruskal- Wallistestconfirmedthatthe treatmentshadsignificanteffectoncell viability (X2(4)=32.536; p=0.000), However,apost-hocpairwise comparisonshowsthatthe cell viabilityscoresof Figure 9: Representative mean cell viability scores for each treatment againstincubation time
DominicThum:N0625034 39 cellstreatedwith100 µg/ml (0.1%) MMT were notsignificantlydifferentfromthe cellstreatedinthe primarycontrol (10% FBS DMEM growthmedium) (p=0.062) butrather showedsignificantdifferencesto the cell viabilityscoresof cellstreatedwith1µg/ml (0.001%) MMT and 1% FBS DMEM differentiation medium,respectively(bothp=0.000). The cell viabilityscoresof the cellstreatedinthe otherMMT treatments(10 µg/ml (0.01%) MMT; 1 µg/ml (0.001%) MMT, respectively)alsoshowednosignificant difference tothe cell viabilityscoresof cellstreatedinthe primarycontrol (see Appendix 3). Cell density alsoappearsto playan effectonthe cell viabilityasthe highdensitycellstreatedwithprimarycontrol had highercell viabilityscoresthanthatof the low densitycells. A Kruskal-Wallistestconfirmedthatcell densityhadsignificanteffectoncell viabilityscores(X2 (1)=88.599;p=0.000). Incubationtime alsoplayed a pivotal factorin the effectof the treatmentsonmeancell viabilityasitcan be seenthatthe cell viabilitiesforlowdensitycellstreatedwith10% FBS DMEM, 1% FBS DMEM, 10 µg/ml (0.01%) MMT and 1 µg/ml (0.001%) MMT showa gradual increase fromDay 1 to Day 3. The exceptionforthisiscells treatedwith100 µg/ml (0.1%) MMT as theyshow gradual decrease fromDay1 to Day 3. The high densitycellsfollowsasimilartrendwherebycells treatedwith10% FBSDMEM, 10 µg/ml (0.01%) MMT and 1% FBS DMEM showedanincrease incell viabilityfromDay1 to Day 3. . Cellstreatedwith1µg/ml (0.001%) MMT, on the otherhand, showedaslightdecrease incell viabilityfromDay1 to Day 3 whilst cellstreatedwith1µg/ml (0.001%) MMT showeda sharpdecrease fromDay 1 to Day 3 (Figure 10b). Thisexperimentwasrepeatedtwice.
DominicThum:N0625034 40 Figure 10: Mean cell viabilityscore for each treatmentat a) Low cell density(1 x 104 cells),and; b) High cell density(5 x 104 cells) A
DominicThum:N0625034 41 Due to the resultsof the statistical analysisaswell asthe initial MTTanalysis,itwasdecidedthatthe nextfewMTT cell viabilitytestswere tosee the effectsof MMT treatmentsonlow cell density. Furthermore,differentiationmediumwasremovedasatreatment asit wasonlyincludedtodetermine if the C2C12 cellswere able todifferentiate. ii. Cell Viabilityanalysison C2C12 cellstreated withCloisite Na+ montmorillonite (MMT) at 1 x 104 cell density Figure 11 showsa representativemeancell viabilityscoresfromthe treatmentsatlow cell density (1x104 ).It can be seenthatthe MMT treatmentshadan effectonthe cell viabilityscoresincomparison to the cellsgrownin10% FBS DMEM with100 µg/ml (0.1%) MMT showingthe biggestdifference (as seeninFigure 9).Thiswas furtherconfirmedwithaone-wayANOVA test(F(3)=5.349;p=0.001) witha post-hocTukey’stestconfirmingthatcellstreatedwith100 µg/ml (0.1%) MMT showedthe greatest difference incell viabilityrelativetocellsgrowninthe primarycontrol (Meandiff.=0.246292; p=0.003). The cellswiththe otherMMT treatments(10 µg/ml (0.01%) MMT; 1 µg/ml (0.001%) MMT) didnot show Figure 10: Pooled mean cell viability scores for each treatment againstincubation timeat (A) low cell density (1 x 104 cells) and (B) high cell density (5 x 104 cells) B
DominicThum:N0625034 42 any significantdifference tocellsgrowninthe primarycontrol andthe differentiationmedium, respectively(see Appendix 4).Similarly,incubationtime playedacritical role inthe effectof MMT treatmentsonthe cell viabilityasitcan be seenthatcellstreatedwiththatthe cell viabilitiesforlow densitycellstreatedwith10%FBS DMEM, 10 µg/ml (0.01%) MMT and1% FBSDMEM (differentiation medium) showagradual increase fromDay1 to Day 3. On the other hand,cellstreatedwith100 µg/ml (0.1%) MMT whichshowedaninitial increasefromDay1 to Day 2 but a small decrease onDay3 whilst cellstreatedwith1µg/ml (0.001%) MMT showeda gradual increase fromDay1 to Day 2 buta small decrease onDay 3 (Figure 9) Thiswas furtherconfirmedwithatwo-wayANOVA test(F(6)=3.464; p=0.003). Thisexperimentwasrepeated threetimes. Figure 11: Mean cell viabilityscore for each MMT treatment at low cell densityVS incubationtime Figure 11: Representative mean cell viability scores for each treatment againstincubation time
DominicThum:N0625034 43 iii. Cell Viabilityanalysison C2C12 cellstreated withLucentite SWN at 1 x 104 cell density Figure 12 showsa representativemeancell viabilityscoresfromthe treatmentsatlow cell density (1x104 ).It can be seenthatthe SWN treatmentshadan effecton the cell viabilityscoresincomparison to the cellsgrownin10% FBS DMEM with100 µg/ml (0.1%) MMT showingthe biggestdifference (as seeninFigure 9).Thiswas furtherconfirmedwithaone-wayANOVA test(F(3)=9.658;p=0.000) However,the post-hocTukey’stestshowedthatthe SWN treatments(100 µg/ml (0.1%) SWN,10 µg/ml (0.01%) MMT and 1 µg/ml (0.001%) SWN,respectively) showednosignificantdifference tothatof cells treatedinthe primarycontrol (10% FBS DMEM) (see Appendix 5).Similarly,incubationtime playeda role inthe effectof SWN treatmentsonthe cell viabilityasitcan be seenthatcellstreated with10% FBS DMEM and1 µg/ml (0.001%) SWN showedagradual increase fromDay 1 to Day 3. On the otherhand, cellstreatedwith100 µg/ml (0.1%) MMT whichshowedan small decrease fromDay1 to Day 2 buta gradual increase onDay 3 whilstcellstreatedwith10µg/ml (0.01%) SWN showeda sharpincrease from Day 1 to Day 2 buta small decrease onDay3 (Figure 10). However,the two-wayANOVA testshowed that incubationtime didnothave asignificanteffectonthe effectof the SWN treatmentsoncell viabilityscores((F(6)=2.347;p=0.033). Thisexperimentwasrepeatedthree times.
DominicThum:N0625034 44 Figure 12: Mean cell viabilityscore for each SWNtreatment at low cell densityVS incubationtime iii) The visual phenotype of C2C12 cellstreatedwith: i. Cloisite Na+ montmorillonite (MMT) Figure 11 depictstypical picturesof C2C12 cellswhenin10% FBSDMEM growthmedium(control),100 µg/ml (0.1%) MMT in 10% FBS DMEM growth medium and 1 µg/ml (0.001%) MMT in 10% FBS DMEM growth medium. Figure 12: Representative mean cell viability scores for each treatment againstincubation time
DominicThum:N0625034 45 Figure 13: C2C12 cellsin10% FBS DMEM growth medium(control),100 µg/ml (0.1%) MMT in 10% FBS DMEM growth medium and 1 µg/ml (0.001%) MMT in 10% FBS DMEM growth medium. Figure 14 depictstypical picturesof C2C12 cellswhenin1% FBS DMEM differentiationmedium (control),100 µg/ml (0.1%) MMT in 10% FBS DMEM differentiationmediumand1 µg/ml (0.001%) MMT in1% FBSDMEM differentiationmediumovertime. Figure 13: Cellular imaging of effect of 100 µg/ml (0.1%) MMT and 1 µg/ml (0.001%) MMT treatments on cells over time (Note the gaps between the cells treated with 100 µg/ml (0.1%) MMT on Day 3 in comparison to the control).
DominicThum:N0625034 46 Figure 15: C2C12 cellsin1% FBS DMEM differentiation medium(control), 100 µg/ml (0.1%) MMT in 1% FBS DMEM differentiation medium and 1 µg/ml (0.001%) MMT in 1% FBS DMEM differentiation medium. ii. Lucentite SWN Figure 16 depictstypical picturesof C2C12 cellswhenin10% FBSDMEM growthmedium(control),100 µg/ml (0.1%) SWN in10% FBS DMEM growth medium, 1µg/ml (0.001%) SWN in10% FBSDMEM growth medium. Figure 15: Cellular imaging of effect of 100 µg/ml (0.1%) MMT and 1 µg/ml (0.001%) MMT treatments on cells over time.
DominicThum:N0625034 47 Figure 16: C2C12 cellsin 10% FBS DMEM growth medium (control),100 µg/ml (0.1%) SWNin 10% FBS DMEM growth medium,1 µg/ml (0.001%) SWN in10% FBS DMEM growth medium. Figure 17 depictstypical picturesof C2C12 cellswhenin1% FBS DMEM differentiationmedium (control),100 µg/ml (0.1%) SWN in 1% FBS DMEM differentiationmedium, 1µg/ml (0.001%) SWN in 1% FBS DMEM differentiationmedium. Figure 16: Cellular imaging of effect of 100 µg/ml (0.1%) SWN and 1 µg/ml (0.001%) SWN treatments on cells over time.
DominicThum:N0625034 48 Figure 17: C2C12 cellsin 1% FBS DMEM differentiation medium(control), 100 µg/ml (0.1%) SWN in1% FBS DMEM differentiationmedium,1µg/ml (0.001%) SWNin 1% FBS DMEM differentiation medium. iv) WesternBlot analysis i. Detectionof Caspase-3expressionfollowingMMTtreatment Day 1 Westernblotsampleswere lost,leavingonlyDay2 andDay 3 C2C12 WesternBlots. Western blottinganalysisfailedtodetect the expressionof active caspase-3inanyof the Day 2 andDay 3 C2C12 cell extracts.However,there were bandspresentabove the 36 kDa proteinmarker,signifyingpresence of inactive procaspase-3(Figure 18).Unfortunately,the bandswere notsharpenoughforfurther analysis. Figure 17: Cellular imaging of effect of 100 µg/ml (0.1%) SWN and 1 µg/ml (0.001%) SWN treatments on cells over time.
DominicThum:N0625034 49 Figure 18: WesternBlot for caspase-3 expressioncomparedwithhousekeepingprotein β-tubulin (loadingcontrol) on (A) Day 2, and; (B) Day 3 A B Figure 18: Western Blot analysisfor β-tubulin (control) and Caspase-3 on (A) Day 2, and (B) Day 3 (Bands from left to right: Protein Marker, 10% FBS DMEM C2C12 cell extract, 1% FBS DMEM C2C12 cell extract, 100 µg/ml (0.1%) MMT in 10% FBS DMEM cell extract, 100 µg/ml (0.1%) MMT in 1% FBS DMEM C2C12 cel l extract; 1 µg/ml (0.001%) MMT in 10% FBS DMEM C2C12 cell extract; 1 µg/ml (0.001%) MMT in 1% FBS DMEM C2C12 cell extract; protein marker)
DominicThum:N0625034 50 ii. Detectionof MyosinHeavy Chain expressionfollowingMMTtreatment Day 1 Westernblotsampleswere lost,leavingonlyDay2 andDay 3 C2C12 WesternBlots. Western blottinganalysis failedtodetectthe expressionof myosinheavychain(MHC) inanyof the Day 2 and Day 3 C2C12 cell extracts(Figure 16).Due to absence of bands,furtheranalysiswasnotpossible. Figure 19: WesternBlot for myosinheavy chain expressioncomparedwith housekeepingprotein β- tubulin(loadingcontrol) on (A) Day 2, and; (B) Day 3 A B Figure 19: Western Blot analysis for β-tubulin (control) and Caspase-3 on (A) Day 2, and (B) Day 3 (Bands from left to right: Protein Marker, 10% FBS DMEM C2C12 cell extract, 1% FBS DMEM C2C12 cell extract, 100 µg/ml (0.1%) MMT in 10% FBS DMEM cell extract, 100 µg/ml (0.1%) MMT in 1% FBS DMEM C2C12 cell extract; 1 µg/ml (0.001%) MMT in 10% FBS DMEM C2C12 cell extract; 1 µg/ml (0.001%) MMT in 1% FBS DMEM C2C12 cell extract; protein marker)
DominicThum:N0625034 51 Unfortunately,due toprojecttime constraints,awesternblotanalysis forlucentite SWN cytotoxicity couldnot be done. 5) Discussion a. Cloisite Na+ montmorillonite (MMT) showslow cytotoxicitytowards C2C12 cellsat higherconcentrations As seenfromthe MTT formazanassayand neutral redassayresultsforMMT, the treatmentwiththe highestconcentrationof MMT (0.1%) showssignificanteffectonthe cell viability of the C2C12 cellsin comparisontothe othertreatmentswithdecreasingMMT concentrations(0.01% and0.001%, respectively). Thisisfurtherconfirmedlookingatthe Figure 11 whichshowsthe C2C12 cellsinin10% FBS DMEM growth medium(control),100 µg/ml (0.1%) MMT in10% FBSDMEM growthmediumand1 µg/ml (0.001%) MMT in10% FBS DMEM growthmedium.Whencomparedwiththe C2C12 cellsgrown inthe control,itcan be seenthatthe higherconcentrationof MMT affectedthe phenotypesof the C2C12 cellsgreatlyashuge gaps are observedbetweenthe cellsaswell asthe presence of roundedcells (brightspotsinthe picture).Thisisevenmore apparentwhenthe treatedcellswereincubatedtill Day3. Thisobservationappearstobe a commonobservation asLee et.al (2005) has shownthat high concentrationsof MMT had a greatereffectsoncellulargrowthandviability,statingthat Saccharomyces cerevisaetreatedwiththe highestMMT concentration(5.0g/L MMT) hadthe lowest growthrate and cell viabilityincomparison toS.cerevisaenot treatedwithMMT. Theirresultsalso agree withphenomenonabsorbedinthisresearchthatthe yeast S.cerevisaetreatedlower concentrationsof MMT (0.1 -0.5 g/l MMT) had lessof an effectoncellulargrowthandviabilityin comparisontothat of S.cerevisae treatedwith highMMT concentration(1.0-5.0 g/L). (Figure 20).
DominicThum:N0625034 52 Figure 20: (A) The Saccharomycescerevisae growth in differentMMTconcentration (B) Growth rate of S.cerevisae in various MMT concentration However,Lee et.al’sexperimentmeasuredthe S.cerevisaecell viabilityandgrowthusing standardplate countingandprobit analysis(astatistical model thatsuggestthatthe relationshipbetween individual’s tolerance totoxicmaterialsina givenpopulationisalog-normal distribution). Furthermore,Lee et.al doesnotgo intothe cellularmechanismthatcausesthe repressionof S.cerevisaegrowthandviability. Lee et.al 2005 alsoexploresthe effectof intravenousinjectionsof highMMT doses(1.42.9 mg per Kg rat bodyweightin1 ml PBS) intorats before obtaininghistopathological sectionof the heart,lung,kidney and spleen.However,the highMMTdose didnot have any effectsonthe cell morphology. Anotherphenomenonobservedwas thataftertwo daysof incubation,the MTTanalysisshowed that C2C12 cellstreatedwithhighconcentratedMMT showedaninitial increasebeforeagradual decrease on the thirdday whereasthe otherMMT treatmentsshowedagradual increase incell viability(albeit lessthancellstreatedin10% FBS DMEM growthmedium). Thisshowsthatthe nanoparticleisnotvery cytotoxiceveninhighconcentrationsalthoughitisable toimpede the cellularmetabolicactivityof Figure 20: (A) S. cerevisae growth curves in different MMT concentration (B) Growth rate of S. cerevisar in various MMT concentration. (Source: Lee et.al 2005)
DominicThum:N0625034 53 C2C12 cells(basedonthe principle workings of MTT formazanassays).MTT formazanassaysare colorimetricassaysthatmeasures NADPH-dependentoxidoreductase enzymes activityin actively convertingthe MTT (1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan)(Sigma-Aldrich) dye intopurple formazan(Berridge et.al 2005; Berridge andTan 1993). Therefore,anincrease inthe intensity of the purple dye wouldeithersuggest thatthere is highmetabolicactivityorthere are a largernumber of NADPH-dependentoxidoreductaseenzymes withinthe cell. Thisappearstoagree withpreviousMTT formazanassaysdone on MMT cytotoxicity.Maisanabaet.al (2013) useda variationof MTT formazan assayknownas MTS assay(whereby3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium(MTS) dye isutilizedinstead) todetermine if MMT wascytotoxicto HepG2 humanhepaticcells.TheirresultssuggestthatMMT doesnotcause cytotoxicityevenwhenthe HepG2 cellswere incubatedwithMMTnanoparticle suspensions from24 to 48 hours.However,the highest concentrationMaisanabe usedwas62.5 µg/ml MMT insteadof the 100 µg/ml usedinthisstudy as their rationale wasthatthe higherconcentrationwouldcause complicationwiththe measurementsystem theyused.Furthermore,Maisanabe seeminglydoesnotfurtherexpandonthisknowledge,instead focusingonMMT’s organicallymodifiedcounterpart,Cloisite® 30Bmontmorillonite (MMT). Thisis unlike Lordanet.al (2011) studieswhocomparedthe cytotoxiceffectsof Cloisite Na+montmorillonite (MMT) and Cloisite 93® montmorillonite toHepG2humanhepaticcells.Althoughhisresults suggests that MMT significantlydecreasescell viability,Lordanet.al furtherinvestigatedthe reasonforthis phenomena,statingthatMMT was causingthe necrosisof the HepG2 via productionof intracellular reactive oxidative specieswhichledtocellularmembrane damage (Lordanet.al 2011).However,the concentrationof MMT usedby Lordan et. al was significantlyhigherthanthatof the one usedinthis research,specifically500µg/ml. Previousstudieshave alsoshownthatMMT causescellularmembrane damage inP388D macrophages(Gormley et.al 1978) and N1E-115 neuroblastomacells(Murphyet.al 1993). Gormleyet.al suggeststhatthe membrane damage isdue tothe clay mineral reducingthe membrane potentialof the macrophages,leadingtocell lysis.However,inalaterstudy,Gormleyand Addison(1983) explainsthatthe variable levelsof ancillaryminerals (e.g.quartz) withinthe MMT clay mineral isresponsibleforthe mineral’scelllysisability.Thisisunderstandable consideringthatCloisite Na+ montmorillonite (MMT) isa naturallyoccurringmineral (Section1i) derivedfromvolcanicash.As such,not onlyisit not pure but(dependingonthe locationitismined) the levelsof extraneousminerals minedwithitalsovaries.Another potential contributingfactortoits cell lysisability,couldbe MMT’s affinitytoswell whenincontactwithwater(Murray 2000). Thiswouldexplain the reasonbehindthe
DominicThum:N0625034 54 mineral’sabilitytocause cell lysisasthe MMT nanoparticle wouldabsorbthe watercontentwithinthe cell,resultinginthe membrane potential reducing.However, thisisonlyaspeculation. The neutral redresultsforCloisite Na+ montmorillonite (MMT) cell viabilitytestsfollowsasimilartrend to that of the MTT formazanassayresultsin that C2C12 cellstreatedwithhighconcentrated MMT showedaninitial increase before agradual decrease onthe thirddaywhereasthe otherMMT treatmentsshowedagradual increase incell viability(albeitlessthancellstreatedin10% FBS DMEM growthmedium). This,once again,suggeststhatthe MMT nanoparticle isaffectingthe cell’sability to take up substances(basedonthe principlesof neutral redassay). However,lookingatneutral red resultsatDay 1, itcan be seenthatcell viabilityscoresforthe cellstreatedwith MMT were higherthan that of the cellstreatedinthe control (10% FBS DMEM) (withcellstreatedwith0.1% MMT havingthe highestmeancell viabilityscores).Thiscouldbe attributedtothe principle of neutral redassayswhich workby dyinglysosomesred(Repettoet.al2008; Lӧwiket.al 1993). As lysosomesare activelyprocessto allowendocytosisof nutrientsorsubstances,the rationale follows thatthe cell isalive andanincrease in the red dye suggestsmore lysosomesare beingproduced totake upthe reddye.Withthisrationale,the reasonbehindthe relativelyhighmeanviabilityscores forthe MMT treatedcellscanbe understoodthat a largeramount of lysosomeswere producedbythe cell totake up boththe neutral reddye and MMT nanoparticlesinthe growthmedium. Althoughthere isnodefinite answertohow the MMT nanoparticlesaffectedC2C12cellularuptake,Gormleyet.al(1978) providesapotential explanation. Usingmacrophages,they suggested thatthe cellulardamage beganonce the MMT mineralshadbeen phagocytosedbythe cell.However,due tothe mineralsabilitytoreduce membrane potential,the phagolysosomesrupture andreleasethe MMT mineralsintothe cytosol wherebytheywill continue reducingthe membrane potential untilthe cell lyses.Furthermore,Gormley shows thatthe cellsare still viable butare ‘leaky’ due tothe membrane beingdamagedbyMMT. Therefore,notonlyare the cells unable tocontrol the cellularintake butalsohave issuesretainingcomponentsof the cell membrane (e.g.enzymesandproteins) whichfurtherreducesthe membrane potentialandleadingtocell lysis. These suggestionscoincide withthe researchresults andcouldactas a potential explanationtoMMT’s effectoncellularuptake systems.Unfortunately, neutral redassaysare rarelyusedinthese MMT cytotoxicitystudies,withMTTassaysbeingthe preferreddue toits’reliability. Maisanabaet.al (2013) alsoutilizedneutral redassaytodetermineMMT cytotoxicityonHepG2but similarlytothe MTS assay, didnot furtherexpandonit.
DominicThum:N0625034 55 Lookingat Figure 12, it can be observedthatthe cellstreatedwith100 µg/ml (0.1%) MMT in 1% FBS DMEM differentiationmediumshowedthe greatesteffectonthe C2C12 phenotype incomparisonto the cellstreatedwiththe differentiationmedium(1% FBSDMEM) and 1 µg/ml (0.001%) MMT in 1% FBS DMEM differentiationmedium,therebydepictingthatMMT had an effectoncell differentiation. However, MMT concentrationisnotthe onlyfactorcontributingtothe treatedcells’phenotype.Asthe cellsare beinggrowninlowserumconditions,the cellsare inducedtodifferentiate insteadof growing. Notall the cellswoulddifferentiate andthe low serumconditionscancause themto die.Assuch,this lowserumconditioncanfurtheraggravate the effectsof highMMT concentrationoncell growth,a phenomenonconfirmedbyLordanand Higginbotham(2013) wherebylow concentrationof MMT (1 mg/ml) significantlyinhibitedthe cell growthof humanmonocyte U937 growninlow serumconditions (Lordanand Higginbotham2013). Thisisfurtherconfirmedfromthe westernblotdetectionformyosin heavychain.Asstated,myosinheavychainisa proteinexpressedduringthe earlystagesof muscle differentiation.Withthe absenceof MHC proteinbandsonthe westernblotsfromboththe Day 2 and Day 3 cell extracts,itcan be assumedthatnone of the C2C12 cellswere differentiatingatthe time of the treatments. b. Lucentite SWNdecreases cellularmetabolicactivity but has drastic effectoncellular uptake system Looking at Figure 7, it can be seen that lucentite SWN does not have much effect on the cellular metabolic activity of the C2C12 cells as the mean cell viabilities for each of the SWN treatments gradually increase over time (albeit at a slower rate than the cells treated in 10% FBS DMEM). This can also be further confirmed in Figure 13 which showsthe C2C12 cellsinin10% FBS DMEM growthmedium(control), 100µg/ml (0.1%) SWN in 10% FBS DMEM growthmediumand1 µg/ml (0.001%) SWN in10% FBS DMEM growthmedium.Whencomparedwiththe C2C12 cellsgrowninthe control, there appearstobe nodifference betweenthe cellstreatedin0.1% SWN and 0.001% SWN, respectivelyevenamongstthe differenttimepoints. Itisspeculatedthatdue toits’syntheticnature,the SWN nanoparticle ismore pure andhasa lowerriskof carryingtoxicchemical particles.Unfortunately, thisisonlya speculationasnopreviousstudiesonlucentite SWN cytotoxicityexists. The neutral redanalysis,onthe otherhand,showsthat at 0.1% concentration,lucentiteSWN wasable to haltcellularuptake fromDay1 to Day 2 before increasingagaininDay3. Anotherphenomenon observedwasthe sharpincrease inmeancell viabilityscoresof cellstreatedin0.01% SWN from Day 1
DominicThum:N0625034 56 to Day 2 before asmall decrease inDay3. Cellstreatedwith0.1% SWN showedgradual increase asseen inthe MTT analysis.The phenomenonseenat0.1% SWN and 0.01% SWN showsthat lucentite SWN has an effectonthe cellularuptake system.Thiscouldbe attributedtothe nanoparticlesabilitytoincrease the viscosityof the solutionthe nanoparticle issuspendedin.Thisrationale couldexplainthe haltin lysosomal uptake of the neutral reddye incellstreatedwith0.1% SWN as the large amountof nanoparticleswouldcause the growthmediumto become more viscous,makingitmore difficultforthe cellstoendocytose the reddye.However,thisisonlyaspeculation. Unfortunately,due totime constraints,awesternblotanalysiscouldnotbe done forlucentite SWN, therefore the mechanismbehindits’effectoncellularuptake couldnotbe analyzed. c. ComparisonbetweenMMT and SWNshows that SWNis lesscytotoxic than MMT from a metabolicactivity standpointand MMT is lesscytotoxicto SWNfrom a cellular uptake standpoint Lookingat the cell viabilityresults,itcanbe seenthat whilstboth Cloisite Na+ montmorillonite (MMT) and lucentite SWN nanoparticlesimpedecellularmetabolicactivityandcellularuptake atmiddle orlow concentrations,the effectsof highconcentrationof the nanoparticlesdiffersoncell viability. Itcanbe seenthathighconcentrationof MMT has a greatereffectoncellularmetabolicactivitywhilsthigh concentrationof SWN has a greatereffectoncellularuptake system.Withthese results,the taskof determiningwhichismore nanoparticleismore cytotoxicisnotas simple asthe questionboilsdownto whichaspectof cell viabilitytoconsider.If cellularmetabolicactivityisconsidered,then CloisiteNa+ montmorillonite (MMT) wouldbe consideredmore cytotoxicandvice versaif cellularuptake is consideredinstead.Tofurtheraddto the confusion,the definitionof cell viabilityisratherambiguous and there existsothercell viabilityassaysthatmeasure otheraspects (e.g.lactate dehydrogenase leakage assay(LDH),(acolorimetricassayaimedatmeasuringcellularmetabolicactivityof lactate dehydrogenaseinconvertingNAD+ toNADH) (Deckerand Lohmann-Matthes1988); . Assuch, previous studiesdone oncytotoxicityof nanoparticles,have alwaysusedatleasttwocell viabilityassaysto give a betterperspectiveoncell viability. d. Conclusionand recommendationfor future research The current researchshowsthatCloisite Na+ montmorillonite (MMT) showsrelativelylow cytotoxicity towardsC2C12 cellsat highconcentrationasit hasan effecton NADPH-dependentoxidoreductase enzymes metabolicactivitywhilsthighconcentrationof lucentiteSWN hasan effectonthe cellular
DominicThum:N0625034 57 uptake systemof the C2C12 cells.However, due tothe limitedknowledge onCloisiteNa+ montmorillonite (MMT) cytotoxicityonC2C12 and SWN cytotoxicityingeneral,itisstill tooearlyto conclude if these canbe consideredfact as there were areasthatwere notexploredinthis research projectdue to time constraints. An example of anareato explore in future researchcould be the effectof layerexfoliationon Cloisite Na+ montmorillonite (MMT) andlucentite SWN cytotoxicity. Asseenin3.b., bothCloisite Na+ montmorillonite (MMT) andlucentite SWN nanoparticles were suspended indeionizedwaterviahand- shakinginsteadof the more conventionalmethodof using ultrasonicvibrations. Thiscouldaffectthe nanoparticles’effectoncellsas nanoparticles have atendencytoaggregate withone anotherviaVan derWaals forces overa periodof time due theirlarge surface area(Othmanet.al 2012). Thisalso affectstheirdispersionwithinthe aqueoussolution. Therefore,ultrasonicvibrations are usedto prevent the nanoparticlesfromaggregatingandformingclumps,andensuringthattheymore dispersed within the aqueoussolutiontheyare in (Changet.al 2004; Satoet. al 2008). The appearance of three peaks insteadof twofor the particle size distributionsof Cloisite Na+ montmorillonite(MMT) nanoparticle suspensions beforeandafterautoclaving(Figure 5) confirmsthisaggregationphenomenon.The appearance of a lone peakfornumber%suggeststhat,numerically,mostof the nanoparticleswithinthe suspensionare atthat size range.Therefore,logicallythereshouldbe onlyone peakforthe volume%as mostof the nanoparticlesshouldbe the same size andvolume.However, asFigure 5has shown, volume%hastwopeaksat differentparticle size ranges.Asthe firstvolume% peakcoincideswiththe lone number%peak, itissuggestedthatthe sharedparticle range corresponds tothe particle size distributionof one MMT nanoparticle.The appearance of asecondvolume% peakatadifferentparticle size range suggestthe appearance of aggregatedparticlesand corresponds tothe particle size distributionof aggregated Cloisite Na+ montmorillonite(MMT) nanoparticles. LordanandHigginbotham (2013) alsoaddressesthe implicationsof nanoparticle dispersiononcytotoxicity astheystate thatinlow serumconditions,claynanoparticlesare more evenlydispersedwhereashighserumconditionscauses the nanoparticlestoaggregate intolarge bundles,therebyreducingits’cytotoxicability. Furthermore, the effectof autoclavingonthe nanoparticle size anddispersionshouldbe investigatedthoroughly as Figure 5 and 6 have shownthatautoclavinghadsome effectof the aggregated Cloisite Na+ montmorillonite (MMT) nanoparticle size andthe lucentite SWN nanoparticle size(asevidencedinthe shiftinboththe number%andvolume% peaksof the autoclavedSWN nanoparticlesuspension, suggestinganincrease inparticle size).
DominicThum:N0625034 58 FollowingGormleyandAddison(1983) suggestion,the mineral compositionof MMT and SWN should alsobe analysed beforeacytotoxicity testtodetermine the levelsof ancillarymineralswithinthe clay. Thiscan be done usinglaser-lightscatteringandparticle distribution,andprovidesfuture researchersa bettercomparisonbetweenthe cytotoxiceffectsof Cloisite Na+ montmorillonite(MMT) withvarying levelsof ancillaryminerals.Anotherareaof researchcouldbe to compare the cytotoxicityof purified Cloisite Na+ montmorillonite (MMT) andnatural Cloisite Na+ montmorillonite(MMT). Anotherthingtohighlightinthisresearchwasthat the cell viabilitieswere measuredusingtwodifferent cell viabilityassayswhoworkondifferentprinciples andthere are othercell viability assaysthatworkon differentprinciplesinmeasuringcellviability.Thisisbecause of the ambiguityof whatisconsidereda measure of cell viability.Therefore,atleasttwodifferentcell viabilityassaysare usedtomeasure cell viabilityasitprovidesabetterperspective oncell. Althoughneutral redassay andMTT assayare both consideredthe more sensitive of the optionsavailable(FotakisandTimbrell2006), future researchcould use a biggercombinationof cell viabilityassaystoprovide abetterunderstandingof Cloisite Na+ montmorillonite (MMT) andlucentite SWN cytotoxicityasmostof the previousstudiesdone showan inclinationtowardsMTTformazanassays. As thisresearchisthe firstinvestigation(tothe author’sknowledge) onlucentiteSWN cytotoxicity, there are still areasyetto be explored. Due totime constraints,the mechanismbehindthe effectof lucentite SWN oncellulargrowthandcellularuptake systemwasnotfullyexplored.This,togetherwith the fact that there existsnopreviousresearchon lucentite SWN cytotoxicity,providesanopportunityto investigatethisareaof research. As there appearstobe no literature inregardsto Cloisite Na+ montmorillonite (MMT) andlucentite SWN cytotoxicitytowardsC2C12 mouse myoblastcells,acomparisonstudycouldalsobe done comparingthe effectsof MMT and SWN on C2C12 cellsandotherwell-documentedcellmodels(Caco-2andHepG2 to name a few) toinvestigate if the phenomenaobservedinthisresearchisexclusivetoC2C12 cellsor these observationsare sharedinothercell models.
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DominicThum:N0625034 64 62. Universityof NorthTexas(2015) Nanoparticle Safetytraining:Nanoparticle Types.Available at https://web3.unt.edu/riskman/index.php?section=onlinetraining&group=nanoparticlesafety&m odule=3#content(Accessedon01 August 2015) 63. Universityof ColoradoColoradoSprings(UCCS) (2015).1.2 Differencesbetweennecrosisand apoptosis.Availableathttp://www.uccs.edu/Documents/rmelamed/apoptosis_003_004.pdf (Accessedon13 August2015) 64. Xie C.;Xu F.; Huang X.;Dong C.; RenJ. (2009) Single goldnanoparticlescounter:anultrasensitive detectionplatformforone-stephomogenousimmunoassayandDNA hybridizationassays, J. Am.Chem.Soc.,131(35), p.g.12763-12770 65. Yaffe D. and Saxel O.(1977) Serial passaginganddifferentiationof myogeniccellsisolatedfrom dystrophicmouse muscle, Nature,270(5639), p.g.725-727 Picture references 1. GundryR.L.; Raginski K.;Tarasova Y.; TchernyshyovI.;Bausch-FluckD.;ElliotS.T.;BohelerK.R.; VanEyk J.E.; WollscheidB.(2009) The Mouse C2C12 myoblastcell surface N-linked glycoproteome, Mol.Cell.Proteomics,8(11), p.g.2555-2569 2. De Graaf M. (2015) Montmorillonite.Available at http://maurice.strahlen.org/minerals/montmorillonite.htm(Accessedon01 August2015) 3. PuschR,; KnutssonS.;Al-Taie L.;MohammedM.H. (2012) Optimal waysof disposal of highly radioactive waste, NaturalScience,4(11A), p.g.906-918
DominicThum:N0625034 65 6) Appendices a. Appendix1: Post-hoc Tukeytests results for initial MTT formazan assay analysisfor Cloisite Na+ montmorillonite (MMT) Multiple Comparisons DependentVariable: Absorbance Tukey HSD (I) Treatment (J) Treatment Mean Difference (I-J) Std. Error Sig. 99% Confidence Interval Lower Bound Upper Bound 10% FBS DMEM 100 microgram/ml MMT .267750* .064720 .000 .05466 .48084 10 microgram/ml MMT .131333 .064720 .255 -.08176 .34443 1 microgram/ml MMT .001875 .064720 1.000 -.21122 .21497 1% FBS DMEM .013896 .064720 1.000 -.19920 .22699 100 microgram/ml MMT 10% FBS DMEM -.267750* .064720 .000 -.48084 -.05466 10 microgram/ml MMT -.136417 .064720 .220 -.34951 .07668 1 microgram/ml MMT -.265875* .064720 .001 -.47897 -.05278 1% FBS DMEM -.253854* .064720 .001 -.46695 -.04076 10 microgram/ml MMT 10% FBS DMEM -.131333 .064720 .255 -.34443 .08176 100 microgram/ml MMT .136417 .064720 .220 -.07668 .34951 1 microgram/ml MMT -.129458 .064720 .269 -.34255 .08364 1% FBS DMEM -.117438 .064720 .368 -.33053 .09566 1 microgram/ml MMT 10% FBS DMEM -.001875 .064720 1.000 -.21497 .21122 100 microgram/ml MMT .265875* .064720 .001 .05278 .47897 10 microgram/ml MMT .129458 .064720 .269 -.08364 .34255 1% FBS DMEM .012021 .064720 1.000 -.20107 .22511 1% FBS DMEM 10% FBS DMEM -.013896 .064720 1.000 -.22699 .19920 100 microgram/ml MMT .253854* .064720 .001 .04076 .46695 10 microgram/ml MMT .117438 .064720 .368 -.09566 .33053 1 microgram/ml MMT -.012021 .064720 1.000 -.22511 .20107 *. The mean difference is significantatthe 0.01 level.
DominicThum:N0625034 66 7) Appendix2: Post-hocTukey testsresults for MTT formazan assay analysison Lucentite SWN Multiple Comparisons DependentVariable: Absorbance Tukey HSD (I) Treatment (J) Treatment Mean Difference (I-J) Std. Error Sig. 99% Confidence Interval Lower Bound Upper Bound 10% FBS DMEM (0 microgram/ml SWN) 100 microgram/ml SWN -.007896 .029628 .993 -.10139 .08560 10 microgram/ml SWN -.061521 .029628 .165 -.15501 .03197 1 microgram/ml SWN -.115833* .029628 .001 -.20933 -.02234 100 microgram/ml SWN 10% FBS DMEM (0 microgram/ml SWN) .007896 .029628 .993 -.08560 .10139 10 microgram/ml SWN -.053625 .029628 .272 -.14712 .03987 1 microgram/ml SWN -.107938* .029628 .002 -.20143 -.01445 10 microgram/ml SWN 10% FBS DMEM (0 microgram/ml SWN) .061521 .029628 .165 -.03197 .15501 100 microgram/ml SWN .053625 .029628 .272 -.03987 .14712 1 microgram/ml SWN -.054313 .029628 .261 -.14780 .03918 1 microgram/ml SWN 10% FBS DMEM (0 microgram/ml SWN) .115833* .029628 .001 .02234 .20933 100 microgram/ml SWN .107938* .029628 .002 .01445 .20143 10 microgram/ml SWN .054313 .029628 .261 -.03918 .14780 *. The mean difference is significantatthe 0.01 level.
DominicThum:N0625034 67 8) Appendix3: Post-hoc pairwise comparisontests resultsfor neutral red assay analysis for Cloisite Na+ montmorillonite (MMT)
DominicThum:N0625034 68 9) Appendix4: Post-hoc Tukeytests results for neutral red assay analysis on Cloisite Na+ montmorillonite (MMT) Multiple Comparisons DependentVariable: Absorbance Tukey HSD (I) Treatment (J) Treatment Mean Difference (I-J) Std. Error Sig. 99% Confidence Interval Lower Bound Upper Bound 10% FBS DMEM (0 microgram/ml MMT) 100 microgram/ml MMT .246292* .069124 .003 .02817 .46442 10 microgram/ml MMT .020896 .069124 .990 -.19723 .23902 1 microgram/ml MMT .051917 .069124 .876 -.16621 .27004 100 microgram/ml MMT 10% FBS DMEM (0 microgram/ml MMT) -.246292* .069124 .003 -.46442 -.02817 10 microgram/ml MMT -.225396* .069124 .007 -.44352 -.00727 1 microgram/ml MMT -.194375 .069124 .028 -.41250 .02375 10 microgram/ml MMT 10% FBS DMEM (0 microgram/ml MMT) -.020896 .069124 .990 -.23902 .19723 100 microgram/ml MMT .225396* .069124 .007 .00727 .44352 1 microgram/ml MMT .031021 .069124 .970 -.18710 .24914 1 microgram/ml MMT 10% FBS DMEM (0 microgram/ml MMT) -.051917 .069124 .876 -.27004 .16621 100 microgram/ml MMT .194375 .069124 .028 -.02375 .41250 10 microgram/ml MMT -.031021 .069124 .970 -.24914 .18710 *. The mean difference is significantatthe 0.01 level.
DominicThum:N0625034 69 10) Appendix5: Post-hoc Tukeytests results for neutral red assay analysis on Lucentite SWN Multiple Comparisons DependentVariable: Absorbance Tukey HSD (I) Treatment (J) Treatment Mean Difference (I-J) Std. Error Sig. 99% Confidence Interval Lower Bound Upper Bound 10% FBS DMEM (0 microgram/ml SWN) 100 microgram/ml SWN .163979 .105845 .410 -.17002 .49798 10 microgram/ml SWN -.302021 .105845 .025 -.63602 .03198 1 microgram/ml SWN -.305604 .105845 .022 -.63960 .02839 100 microgram/ml SWN 10% FBS DMEM (0 microgram/ml SWN) -.163979 .105845 .410 -.49798 .17002 10 microgram/ml SWN -.466000* .105845 .000 -.80000 -.13200 1 microgram/ml SWN -.469583* .105845 .000 -.80358 -.13559 10 microgram/ml SWN 10% FBS DMEM (0 microgram/ml SWN) .302021 .105845 .025 -.03198 .63602 100 microgram/ml SWN .466000* .105845 .000 .13200 .80000 1 microgram/ml SWN -.003583 .105845 1.000 -.33758 .33041 1 microgram/ml SWN 10% FBS DMEM (0 microgram/ml SWN) .305604 .105845 .022 -.02839 .63960 100 microgram/ml SWN .469583* .105845 .000 .13559 .80358 10 microgram/ml SWN .003583 .105845 1.000 -.33041 .33758 *. The mean difference is significantatthe 0.01 level.

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Research Project Dissertation_Final_270815_DT

  • 1. DominicThum:N0625034 1 NOTTINGHAM TRENT UNIVERSITY CYTOTOXICITY OF CLOISITE NA+MONTMORILLONITE (MMT) AND LUCENTITE SWN ON TISSUE GROWTH AND DIFFERENTION by DOMINIC THUM ProjectReportsubmittedinpartialfulfilmentof therequirementsfor a M.Sc. in Biomedical Science at the School of Science and Technology, Nottingham Trent University Supervisor: Dr Jane Harper Year in which the study is completed: 2015 DECLARATIONOFOWNERSHIP Thissubmissionistheresultof mywork.Allhelpandadvice,otherthanthatreceivedfromtutors,hasbeen acknowledgedandprimaryandsecondarysourcesof informationhave beenproperlyattributed.Should thisstatementprove tobe untrue,Irecognisetherightanddutyof theBoardof Examinerstorecommend whatactionshouldbe takeninline withthe University’sregulationsonassessmentcontainedinthe Handbook. Signed................................................................................ Date .......................................................
  • 2. DominicThum:N0625034 2 Acknowledgements The author wishestoexpresshisappreciationtoeverypersonthathasgiventheirsupportwhilst undergoingthisproject. Training and mentoringin laboratory, fieldorcomputational techniques: I wouldlike tothankbothDr Jane Harperand Dr Luigi de Girolamofortheirsupportandguidance offeredthroughoutmyprojectsupervision.The skillsandtechniquesthattheyhave willinglygivenwill be invaluable tome asI beginmyscientificcareer. I am alsothankful tothe laboratorytechniciansatthe RosalindFranklinlaboratoryof NottinghamTrent University.Theirwillingnesstoofferadvice andguidance throughoutmyresearchprojectwasamajor contributiontothe project’s success. I wouldalsolike tothankTianLong See fromthe Universityof Manchesterforprovidingadvice and guidance incomputingandanalyzingthe laserlightscatteringdataacquiredfromthe researchproject. Your helpwasgreatlyappreciated. Data collectedwithanother person: I wouldlike tothankmylab partner,AnkithaMishra,forhercontributionsandhelpintermsof cell culturing,proteinharvesting,BCA assaysandsoforth. Data providedby another person: I wouldlike tothankDr JohnBrameld of the Universityof Nottinghamforhiskinddonationof the C2C12 mouse myoblastcell line.Lastly,IwouldliketothankDr Fengge Gao forprovidingthe Cloisite Na+ montmorillonite (MMT) andlucentite SWN nanoparticles.These donationswere greatlyappreciated.
  • 3. DominicThum:N0625034 3 Table of Contents List of Abbreviations 5 Summary 6 1) Introduction a. Nanoparticles:Definitionandapplications 7 b. Nanoclays:Definitionandapplications 9 i. Cloisite Na+ montmorillonite (MMT) 10 ii. Lucentite SWN 11 2) Aims 12 3) MaterialsandMethods a) Materials 16 b) Methods a. Cell Culture 21 b. Preparationof nanoclaysuspension 22 c. ExperimentalDesign 22 d. Cytotoxicityassays 23 e. Cell harvest 24 f. Proteinextractionandestimation 25 g. SDS-PAGEand WesternBlotting 25 h. Immunoprobingof WesternBlots 26 i. Image analysis andcell imaging 28 j. Statistical analysis 28 4) Results a) Particle size analysis 28 b) Cytotoxicityassayanalysis 34 c) The visual phenotype of C2C12cellstreatedwith: i. Cloisite Na+ montmorillonite (MMT) 44 ii. Lucentite SWN 46 d) WesternBlotanalysis 48 5) Discussion 51 6) Bibliography 59
  • 5. DominicThum:N0625034 5 List of Abbreviations BCIP-T 5-bromo-4chloro-3-indolylphosphate BSA Bovine Serum albumin DMEM Dulbecco’s Modified Eagle Medium DMSO Dimethyl sulfoxide EDTA Ethylenediaminetetraaceticacid FBS Foetal Bovine Serum MMT Cloisite Na+ montmorillonite MTT 1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan NBT NitroBlue Tetrazolium NR Neutral red PBS Phosphate BufferSaline SDS-PAGE Sodium-dodecyl-sulphate-poly-acrylamide-gel-electrophoresis TEMED Tetramethylethylenediamine
  • 6. DominicThum:N0625034 6 Summary Nanomaterialsare definedasmaterialsthatrange from1 nm to 100 nm insize.Due to theirsmall size, theyare notaffectedbythe lawsof quantummechanicsandexhibitpropertiesdifferenttotheirlarger counterparts.These propertiesallowthemtobe appliedinvariousindustries,suchascatalystsin chemical industriesorreinforcingagentsinmanufacturingindustries.A classof nanomaterialsare nanoparticleswhichcanbe categorized basedontheirmethodof creationorbasedontheirchemical composition.Thisclassof nanomaterialsare primarilyusedinbiomedical andpharmaceutical industries as drug carriersor scaffoldsfortissue engineering.Due tothis,investigatingthe cytotoxicityof these nanoparticlesiscrucial.Thisresearchprojectfocusesonthe cytotoxicityof ceramicnanoparticles, specificallyCloisite Na+ montmorillonite (MMT) andLucentite SWN. Bothwere chosendue tothe discrepanciesinunderstandingthe cytotoxicityof MMT and due to the lackof informationonthe cytotoxicityof Lucentite SWN,respectively.Therefore,the primaryaimof thisresearchprojectisto investigateandclarifythe cytotoxicityof bothnanoparticles.Thiswasdone usingMTT formazanassay and neutral redassay.A secondaryaimwasto determine the mechanismbehindthe effectof the nanoparticlesonthe cell if itwasdeterminedthattheywere cytotoxic.Thiswasdone usingwestern blotting.The resultsshowedthatCloisiteNa+ montmorillonite (MMT) hadlow cytotoxicitytocell viabilityintermsof cellularmetabolicactivitywhilstlucentite SWN showedlow cytotoxicitytocell viabilityintermsof cellularuptake activity.Comparingthe twonanoparticles,itwasestablishedthat Cloisite Na+ montmorillonite (MMT) wasmore cytotoxicthanlucentite SWN intermsof cellular metabolicactivity,andvice versaforcellularuptake activity.The westernblotanalysisdidnotreveal a clearresultand couldnotbe analysed.
  • 7. DominicThum:N0625034 7 1) Introduction a. Nanoparticles:Definitionandapplications The fieldof nanotechnology hasexpanded intovariousindustriesaswell asotherareasof researchin the past fewyears.Its involvementinthesesectorsrange fromthe fieldof material sciences(afield whose purpose istostudy and create new materials) tothe fieldof medicalsciences.Thiscanbe contributedtothe verynature of these nanomaterialsthatthese nanoscientistsstudy.Asdefinedby bothLine (2008) and Lӧvestamet.al (2010), nanomaterialsare materialsorpowderswhosesizesranges from1 to 100 nm.Due to theirsize andtheircomposition,thesenanomaterialsexhibitpropertiesthat are differentto theirlargersizedcounterpartsastheyare notaffectedbythe classical lawsof quantum mechanics.These propertiesinclude;(a) electrical conductivity;(b) large surface area;(c) increased mechanical strength,andmanymore (Line 2008; EdelsteinandCammaratra1996). Assuch, these nanomaterialsare usedas eitherpigmentsorcatalystsforchemical processesorusedasreinforcing agentsfor manufacturingof plastics(Line 2008; Allsopp,WaltersandSantilo2007; Edelsteinand Cammaratra 1996). These unique propertieshave alsofoundnanomaterialsbeingusedinthe fieldsof biological sciences,especiallyinthe fieldsof i) tissue engineering(wherebythese nanomaterialsare usedas scaffolds);ii) drugtargetingand delivery,andiii) diagnosticandtherapeutictools(anexampleof nanomaterialsbeingusedinthisdepartmentiscarbonnanomaterialsbeingusedinchemical biosensors) (Raoet.al 2004; Lanone and Boczkowski 2006; Liu and Guo 2012, Salata 2004). As statedinLine’s andLӧvestam’s definition,nanomaterialscanappearaseitherpowdersorother forms(e.g.tubes,fibers).Assuch,Figure 1showsthe categoriesgiventonanomaterialsaswell the field theyare mostcommonlyusedin. Table 1: Categories of nanomaterials and their primary field of application Nanomaterial Category Primary field of application Nanoparticles Medical/Pharmaceutical Nanotubes Chemicals and advanced materials Nanoporous materials Information and communication Fullerenes Energy Quantum dots Automotive Nanostructured materials Aerospace Nanofibers Textiles
  • 8. DominicThum:N0625034 8 Nanocapsules Agriculture Nanowires Dendrimers (Source:Pitkethly 2004) Due to the nature of thisresearchprojectas well asits’involvementinthe medical/pharmaceutical industries,nanoparticleswill be the mainfocusof thisintroduction. Nanoparticlesare definedas particleswhichrange from1 to 100 nm insize (Abhilash2010; Lӧvestamet.al 2010). Similarlyto nanomaterials,nanoparticlescanbe furthercategorizedintogroups.Unlike nanomaterials,however, nanoparticles canbe categorizedbasedontwocriteria:a) methodof production,and;composition (Table 2). Table 2: Categories of nanoparticles based on a) method of production and b) composition Different categories of nanoparticles based on: a) Method of production b) Composition Naturallyoccurring(e.g.volcanicashandsea spray) Carbon-based(e.g.fullerenes) Incidental (i.e.createdasaresultof man-made industrial processese.g.cookingsmokeand weldingfumes) Metal (e.g.goldnanoparticles) Engineered(e.g.sunscreenpigmentsand quantumdots) Ceramic/clays(e.g.montmorillonite) Biologicallyproduced (e.g.liposomes) Quantumdots( a.k.a nanocrystalsproducedvia combinationof GroupII/IIIandGroup IV/V elementsrespectively(e.g.Cadmiumselenide (CdSe))) Polymeric(i.e.biodegradable nanoparticles consistingof syntheticpolyesters(e.g. polylactic acid (PLA)). (Source:Universityof NorthTexas(UNT) 2015)
  • 9. DominicThum:N0625034 9 Evenwiththese differentcategories,nanoparticlesshare the commonfeaturesof havinglarge surface to mass ratioas well ashavingthe abilitytoadsorband carry chemical compounds(De JongandBorm 2008). This,togetherwiththe factthat theirsurfacescan be furthermodified,makesthemideal candidatesfordrugdeliveryastheirsmall size allowsthemtoeasilypenetratetissuesororgansmore effectivelywhilsttheiradsorptionabilityallowsthemtocarrythe chemical compoundsintothe affected organ. Meanwhile,the nanoparticle’ssurface canbe made to interactwithspecificbiologicalstructures furtherincreasingits’abilitytotargetspecificorgansortissues (mice injectedwithzincsulphide (ZnS)- coatedquantumdotsthat were coatedinGFE peptides (thatspecificallybindstothe membrane dipeptidaseof lungendothelial cells)were showntohave retainedthe quantumdotsintheirlungs insteadof otherorgans (Ackerman et.al 2002)). Nanoparticleshave alsobeenusedtoaidindiagnosisof cancer (e.g.goldnanoparticleshave beenusedtodetectcancerbiomarkers(Xie et.al 2009)) andas imagingtools(He et.al 2008) due to these unique properties. Thoughthe propertiesof nanoparticlesmakesthemidealdrugcarriers,theircompositionplaysa significantrole indeterminingtheirappropriatenessasdrugcarriers. This isdue to nanoparticleshaving differentdegreesof toxicityonthe tissuesororganstheyinteractwith.Thisisknownascytotoxicity. The cytotoxicityof a nanoparticle affectswhetheritisusedincancer treatments(wherebyhigh cytotoxicityisdesired) orusedintissue engineering(wherebylittle tonocytotoxicityisdesired).Thishas resultedinextensive studiesbeingdone todeterminetheircytotoxicability(e.g.Cobaltnanoparticles have beenshowntoincrease incytotoxicitywithincreasingconcentration(Amnaet.al 2013) whilst single-walledcarbonnanotubes(SWNTs) are highlycytotoxic(Cui et.al 2005)).Unfortunately,mostof these cytotoxicitystudiesfocusonmetal nanoparticlesandquantumdotswhilstothertypesof nanoparticlesare givenlittle tonoattention. One of these lesserresearched typesare ceramic nanoparticles(a.k.ananoclays). b. Nanoclays: Definitionandapplications Ceramicnanoparticlesornanoclays are nanoparticlescomposingmainlyof oxides,nitridesorcarbides (Particular® 2015). Dependingontheircomposition,these ceramicnanoparticlescaneitherexhibit electrical conductivity(e.g.tinoxide(SnO2) nanoparticleshave beenused ingassensorsdue totheir abilitytogainelectrical conductance whenexposedtoreducing gases,suchas methane orcarbon monoxide (Jainet.al 2006)) or gain photocatalyticproperties(e.g.titaniumdioxide a.k.atitania (TiO2) nanoparticlesare commonlyusedinwaterpurificationsystemsdue toits’abilitytoproduce reactive oxygenspecies(whichreactswithorganicelementsinthe watertoform carbondioxide) whenexposed
  • 10. DominicThum:N0625034 10 to UV radiation(Fadeel andGarcia-Bennett2010)).Ceramicnanoparticleshave alsoseenapplicationin tissue engineeringas theyserve asstabilizingagentsinnanocompositescaffolds(Camargo, Satyanarayanaand Wypych2009) as well asdiagnostictools,suchasDNA microarrays(Caputoet.al 2007) . Lookingatthe examplesof ceramicnanoparticlesapplications,itcanbe seenthatthere are differenttypesof ceramicnanoparticles,rangingfromamorphoussilica(e.g.LUDOX® CLcolloidal silica) to fibroussilicates (e.g.Halloysite).However, thisresearchproject willbe lookingattwospecificceramic nanoparticles:i) CloisiteNa+ montmorillonite,and;ii) Lucentite SWN. i. Cloisite Na+ montmorillonite (MMT) Cloisite Na+ montmorillonite (MMT) isa white-coloured naturally-occurringclaymineralderivedfrom volcanictuffsandash and producedbyhydrothermal activity(Figure1A) (Mindat.org2015). Thisclay mineral belongs tothe large smectite claymineral group.Assuch,its’structure consistsof analuminium oxide/hydroxide layer(Al2(OH)4) sandwichedbetweensilicate layers(Figure 1B) (Uddin2008). Figure 1:A) Cloisite Na+ montmorillonite B)Layered structure of Cloisite Na+ montmorillonite (MMT) Figure 1: (A) Montmorillonitefrom Karnasurt(B) Layered structureof montmorillonite,a member of the smectite group of ceramic nanoparticles (Mis the exchangeable cation which connects the sheets of silicates [in the caseof CloisiteNa+ montmorillonite(MMT), M is Na+]) (Source: de Graaf 2015 and Pusch et. al 2012, respectively) A B
  • 11. DominicThum:N0625034 11 Due to its’layeredstructure,MMT exhibitphysical propertiessuchas:i) absorbingwaterthuscausing swelling;ii) absorborganicliquids(e.g.glycerolandpolyhydricalcohols),and; iii) ahighcapacityto exchange cationsininorganicreactions(EncyclopædiaBritannica2015; Uddin2008; Hackman and Hollaway2006). These propertiesallowMMT nanoparticles tohave manyapplications rangingfrom beingcomponentsof drillingmud(Uddin2008),reinforcementof foodpackaging(Maisanabaet.al 2013) as well aslinersforirrigationditches(due toits’affinitytoswell whenexposedtowater) (Murray 2000). The biomedical applicationsof MTTnanoparticlesincludethe fieldof tissue engineeringas stabilizingagentsintheirscaffolds (due totheirease of availability)(Camargo,Satyanarayanaand Wypych2009) as well asbeingusedasdrug carriersfor anti-cancerdrugs incancer treatments(Lee et.al 2005; Fenget.al 2009). Due to its’use ina diverse range of applications,establishingthe cytotoxicityof MMT nanoparticlesisanimportanttask.However,there doesnotappeartobe a general consensusinregardsto the cytotoxicityof MMT nanoparticles.Some studieshave claimedthatMMT nanoparticlesare notcytotoxicunlessorganicallymodified(Liuet.al 2011; Sharma et.al 2010) or have anti-cancerdrugsattachedto it (Lee et.al 2005) whilstotherstudiessuggestthat regardless of modifications,MMT nanoparticlesare cytotoxic(Lordan,KennedyandHigginbotham2010; Baek,Lee and Choi 2012). ThisdiscrepanciesonMMT cytotoxicitycanbe contributedtothe lack of standardizationinrunningthe cell viability tests(Konget.al 2011). Furthermore,the cell linesusedby the studiesare alsodifferentfromone another.These range from humancells(HepG2humanhepatic carcinoma(Lordan,KennedyandHigginbotham 2010; Maisanabaet.al 2013) andCaco-2 human colon carcinoma(Jorda-Beneytoet.al 2013; Sharma et.al 2010) beingthe mostpopular) tomouse cells(e.g. NIH 3T3 mouse embryonicfibroblasts(Liu et.al 2011)).It ishopedthat thisresearchprojectwouldnot onlydecrease the discrepancyonthe cytotoxicityof MMT butalsoprovide a standardizedapproachto investigatingits’cytotoxicity. ii. Lucentite SWN Lucentite SWN,onthe otherhand,is a white odourlesspowder,composingof lithiummagnesium sodiumsilicate.Asitsharesthe same layeredstructure asMMT itis consideredtobe partof the smectite claymineral group(Co-opChemical Co.Ltd2001) (Figure 2).
  • 12. DominicThum:N0625034 12 Figure 2: Layered Structure of Lucentite SWN However,unlikeMMT,SWN isa syntheticclaythatcan be made viatwoproductionmethods:i) reacting magnesiumsilicatesandalkali cationstogether(Jiang2007) or ii) reactingsmectite claywithorganic compounds(Co-opChemical Co.Ltd2001). The rationale behindthe productionof thissynthetic smectite wasdue tothe fact thatprevioussmectite productsshowedlow transparency, low brightness as well aslowdispersion.These propertiesare essential assmectite claysare usuallyusedasgelling agentsfor beautyproducts(e.g.lipsticks),medicinesandwater-solublepaints(Co-opChemical Co.Ltd 2001; KoboProducts,Inc 2015). Due to the demandfora betteralternatives,the propertiesof Lucentite SWN include hightransparencyandbrightnessaswell asprovide highviscositywhendispersedinwater (Co-opChemical Co.Ltd2001) as well ashavingthe sharedpropertiesof smectite clays(asdescribedin 1.b.i).Beingthatthe primaryapplicationof SWN isin cosmetics(KoboProducts,Inc2015), there would have been excessive cytotoxicstudiesdone todetermineits’appropriatenessof use inthese products. Strangelyenough,there appearstobe noexistingliterature investigatingthe cytotoxicityof SWN. 2) Aims The above sectionhasgivenan insightintothe currentknowledge inregardstothe cytotoxicityof nanoclaysandalsohighlightedthe gapswithinit, specificallyinregardstothe cytotoxicityof Cloisite Na+ montmorillonite (MMT) andlucentite SWN.Therefore,the aimsof thisresearchis: Figure 2: Layered structureof lucentite SWN (The exchangeable cation being lithium(Li+) or magnesium (Mg2+) (Source: Co-op Chemicals Co. Ltd 2001)
  • 13. DominicThum:N0625034 13 i) To investigate andcompare the cytotoxicityof natural smectite Cloisite Na+ montmorillonite (MMT) andsyntheticsmectite lucentite SWN,and; ii) To investigate mechanismof cytotoxicactionof nanoclayson muscle cell growthand differentiation. For thisresearch,C2C12 mouse myoblastcellswereselectedasthe cell model.C2C12cellswere first discoveredbyYaffe and Saxel whentheyserial passagedmyoblastsderivedfrom dystrophicC3Hmouse thighmuscle (Yaffe andSaxel 1977). Beingpluripotentmesenchymal satellitecells,C2C12 cellsare are able to entertwophases:i) self-renewal(wherebytheyrapidlyproliferate),and;ii) differentiation (wherebytheydifferentiatedintomyotubesunderspecificconditions) (YaffeandSaxel 1977; Buratinni et.al 2004). These characteristicscomplimenttheirrole inmuscle growthandrepair.Whenitwas discoveredthatserumconditionswithinthe culture mediums affectedwhichphase thesecellswould enter(C2C12 cellsrapidlyproliferate andgrow inhighserumconditionswhilstlow serumconditions inducesdifferentiation)andthatthe serumconditionscouldbe controlled(LawsonandPurslow 2000), C2C12 cellswere seenasan ideal cell modeltostudyskeletalmuscle growthanddevelopment (KhodabukusandBaar2007). The fact that mice alsoshare similarmuscle physiologytohumansalso contributedtothis (The JacksonLaboratory2014). Furthermore, C2C12 cellshave alsobeenusedin cytotoxicitytestsfornanoparticles,suchasquantumdots(Rahabet.al 2012) and zincoxide nanoparticles(Panduranganet.al 2014).However,littletonocytotoxicitystudiesforclaynanoparticles have usedC2C12 cell as cell modelswhichwasone of the reasonstheywere chosenascell modelsfor thisresearchproject.Furthermore, differentiatedC2C12myotubes have distinctcellularmorphology in comparisontotheirmyoblastcounterparts(Figure 3) whichalsoplayedarole intheirselectionforthis researchproject. Figure 3: C2C12 mouse (A) myoblasts, and; (B) myotubes
  • 14. DominicThum:N0625034 14 MTT formazanassay andneutral red (NR) assaywas usedto measure cell viability.MTTformazanassay isa colorimetricassaythatmeasurescell viabilityinterms of cell metabolicactivity viathe conversionof the MTT dye intopurple formazandye bythe NADPH-dependentoxidoreductase enzymesinthe cells. These can be foundon the mitochondrial membrane orthe cytosol of the cell.The presence of the purple dye once the cellsare introducedtothe MTT dye signifiesthatcellularmetabolicactivityis present,hence the cell isalive (Berridge et.al 2005; Berridge andTan 1993). Neutral redassay,on the otherhand,measurescell viabilityintermsof cellularuptake viathe stainingof lysosomes.Thisstaining representsthe abilityof the cellstotake upthe red dye and indirectly,substancesfromextracellular environment((Repettoet.al2008; Lӧwiket.al 1993). Usingthese twocell viabilityassayswouldprovide a betterperspective onhowthe nanoparticlesaffectedthe cell viabilityof the C2C12 mouse myoblast cells. In the eventthe cell viabilityassaysshowedadecrease incell viabilityof the C2C12 mouse myoblast cellsafterthe introductionof the nanoparticles,the methodbehindthe effectof the nanoparticle on the cell viabilitywasinvestigated. Itwasassumedthattwoof the followingmechanismswouldoccur: a) Necrosis,and; b) Apoptosis Necrosisisdefinedascell deathcausedbyphysical andmechanical damage tothe cells(e.g.shearingor physical puncturing) whichcausesthe cellularmembrane integritytobe lost,causingcell lysisand release of cellularcomponentstothe extracellularenvironment(Proskuryakovet.al 2003). Apoptosis, on the otherhand,is definedascontrolledcell deathwherebyspecificcell signalingcascadesare activatedandregulated whichcausescell death(Albertset.al2008). Some of the different morphological characteristicsof cellsundergoingnecrosisandapoptosisare described inTable 3below: Figure 3: (A) C2C12 myoblasts and (B) C2C12 myotubes. (Source: Gundry et.al 2009)
  • 15. DominicThum:N0625034 15 Table 3: Comparison of morphological characteristics betweencellsundergoingnecrosisand apoptosis Morphological Characteristics Necrosis Apoptosis Swellingof cytoplasmdue toinflux of waterand ions Condensationof the nucleusandshrinkingof cytoplasm Loss of plasmamembrane integrity Plasmamembrane blebbing Complete lysisof vesicles Apoptoticbodies(membrane-boundvesicle) formed Swellinganddisintegrationof internal organelles Increasedpermeabilityof mitochondrial membrane Complete cell lysis Fragmentation of cell intosmallerapoptotic bodies To investigate if necrosisoccurred,cellularimagingwasusedtoidentifythe morphological characteristicsof necroticcells.Toinvestigateif apoptosisoccurred,westernblottingwasusedtodetect the presence of caspase 3. Caspase 3 (cysteinyl-aspartateproteases) are proteasesinvolvedinthe apoptoticcell signalingpathwaysandexistsasinactive pro-caspase 3(Porterand Jänicke 1993). When apoptoticsignals(e.g.release of cytochrome Cfrommitochondriaor deathsignals) are detected,the prodomainof pro-caspase 3 wascleavedintoactive caspase 3 whichisresponsible foractivating apoptosissignalingcascadesincells.Therefore,caspase 3 was a suitable apoptoticmarker. (Source: Baba 2009;University of Colorado Colorado Springs (UCCS) 2015)
  • 16. DominicThum:N0625034 16 3) Materials and Methods a) Materials a. Cell lines Cell line Details Source/manufacturer C2C12 Mouse myoblastcells AmericanType culture collection(ATCC),Manasas, VA (donatedfromDr. John BrameldtoDr Jane Harper) b. Antibodies Antibody Type Description Manufacture and catalogue no. Dilutionfactor Anti-active +proCaspase 3 antibody Primary Polyclonal,Rabbit Abcam(ab47131) 1:1000 (WB) Beta-TubulinLoadingControl Antibody(BT7R) Primary Monoclonal, Mouse ThermoScientific (MAS-16308) 1:2000 (WB) MyosinHeavyChain2 antibody(EPR52801) Primary Monoclonal, Rabbit GeneTex (GTX63258) 1:10000 (WB) Anti-Mouse IgGantibody Secondary Polyclonal, Alkaline Phosphatase conjugated,Goat ThermoScientific (31322) 1:5000 (WB) Anti-RabbitIgGantibody Secondary Polyclonal, Alkaline Phosphatase conjugated, Donkey Santa Cruz Biotechnology, Inc. (sc-2315) 1:5000 (WB)
  • 17. DominicThum:N0625034 17 c. Assay kits Name Details Manufacturer PierceTM BCA Assaykit Contents:BCA ReagentA (sodium carbonate, sodiumbicarbonate pierce BCA detectionreagent,and sodium tartrate in0.1N sodiumhydroxide),BCA ReagentB (4% cupric sulfate) and bovine serumalbumin(BSA) standard (sodiumchloride solutioncontaining sodiumazide) ThermoScientific(23225, 23227) d. Chemicals Name Details Manufacturer Ammoniumpersulphate Sigma-Aldrich(215589) BCIP-T 5-bromo-4chloro-3-indolylphosphate ThermoScientific(R0821) Bovine serumalbumin(BSA) ThermoScientific(B14) Biocleanse Teknon(TK210) Cloisite Na+ montmorillonite (MMT) 0.3% suspension The SouthernClay ProductsCo. USA Dulbecco’s Modified Eagle Medium(DMEM) 1X 4.5 g/L D-glucose,0.11 g/L sodium pyruvate andwithoutL-glutamine Gibco (11995-073) Dulbecco’sModifiedEagle Medium(DMEM) 1X 4.5 g/L D- glucose withoutL-glutamine Lonza (BE12-614F) Dimethyl Sulfoxide(DMSO) FisherChemical (D128-500) EDTA Ethylenediaminetetraaceticacid Sigma-Aldrich(E9884) Ethanol 70% and99.9% EZ-RunProteingel solution (12.5%) Contents:Water,acrylamide, 4- Morpholinepropanesulfonicacid, Tris (hydroxymethyl) aminomethane Fisher-Scientific(BP7712-100; BP7712-30; BP7712-500)
  • 18. DominicThum:N0625034 18 EZ-RunRunningBuffer20X Fisher-Scientific(BP7700-500) Foetal bovine serum(FBS) Heat inactivated Lonza (DE14-830F) L-Glutamine Lonza (17-605) Glycine Fisher-Scientific(BP3811) HEPES buffer(1M) 1M in 0.85% sodiumchloride (NaCl) Lonza (17-737E) Laemmli buffer 5X Laemmli Sample buffer Lucentite SWN 0.3% suspension CoopCorporation,Japan Methanol Laboratory Reagent,≥99.8% FisherChemical (A412-4) Milkpowder Skimmed Marvel Monosodiumphosphate (NaH2PO4) BDH GPR MTT formazan 1-(4,5-Dimethylthiazol-2-yl)-3,5- diphenylformazan Sigma-Aldrich(M2003) NitroBlue Tetrazolium(NBT) ThermoScientific(R0841) Neutral Red 3-Amino-7-dimethylamino-2- methylphenazine hydrochloride Sigma-Aldrich(N7005) Penicillin/Streptomycin Lonza (17-602E) Phosphate buffersaline (PBS) Sterile phosphatebufferforcell culture (0.0067M PO4 withoutcalciumand magnesium) Lonza (BE17-516F) PageRulerTM PlusPrestained ProteinLadder Proteinladder ThermoScientific(26621) Protease InhibitorCocktail Contents:Aprotinin,Bestatin,E-64, Leupeptin,PepstatinA and4-(2- Aminoethyl)benzenesulfonylfluoride hydrochloride (AEBSF ) serineproteases Sigma-Aldrich(P8340) Sodiumdodecylsulphate(SDS) Sigma-Aldrich(L3771) SodiumChloride (NaCl) Melford (S0520) Sodiumorthovanadate (Na3VO4) Sigma-Aldrich(S6508) TEMED Tetramethylethylenediamine Sigma-Aldrich(T9281) Trypan blue Sigma-Aldrich(T8154)
  • 19. DominicThum:N0625034 19 Tris Base Fisher-Scientific(BP154-1) Tris HCl Melford (T1513) TrypsinEDTA (ethylenediaminetetraaceticacid) solution 0.25% trypsin,1mMEDTA Lonza (CC-5012) Tween20 Polyoxyethylenesorbitonmonolaurate Fisher-Scientific(BP337-500) e. Equipment Equipment Details Manufacturer BarnsteadTM nanopureTM watersystem ThermoScientific Cell culture flask andplates 25 cm2 and 175 cm2 (red cap ventilated);6,24 and 96 well culture plates SARSTEDT AG & CO. Centrifudges 15/80 MSE HeraeusMultifuge X3 ThermoScientific Harrier ChemidocTM MP Imagingsystem Bio-Rad Disposable cell scrapers Fisher-Scientific Sterile hood ClassII microbiological safetycabinets Walker FilterPaper Standardgrade Whatman FisherbrandTM WaterBath Fisher-Scientific Haemocytometer NanoEntek Incubator MCO 18AC Panasonic Mini-PROTEAN® Tetracell SDS-PAGEkit Bio-Rad Ministartsingle use filterunit 0.2 µg and 0.4 µg Sartoriusstedimbiotech Microscope Inverted phase contrast microscope- Primovert Carl Zeiss Microscope camera AxiocamERc 5s Carl Zeiss Particle Size analyzer CoulterN4 Plus BeckmanCoulter
  • 20. DominicThum:N0625034 20 Plate Reader(24 and 96 well) UVM340 Asys Pippettes Sterile Pippettes:1,2, 5, 10, 25 ml Micropippette:0.5- 10 µl,2-20 µl,20-200 µl, and 100-1000 µl Automaticpropipetter SARSTEDT AG & Co. FisherScientific FisherScientific pH/ORPmeter Hanna instruments(HI2211) Powerpack PowerPacTM Universal PowerSupply Bio-Rad Rapidsemi-dryblottersystem WSE-4020 HorizeBLOT 2M-R ATTO Flatbedorbitershaker KS 260 Basic shaker IKA Transfermembrane ProteanTM nitrocellulose Whatman Tubes Eppendorf,20 ml and 50 ml SARSTEDT AG & Co. Vortex mixer Lab Dancer IKA OxfordWeighingscale FisherScientific f. Software Software Manufacturer SPSS IBM ZEN 2012 software Carl Zeiss
  • 21. DominicThum:N0625034 21 b) Methods a. Cell culture i. Growth The C2C12 mouse myoblastcellswereprocuredfrom AmericanType culture collection(ATCC), Manasas, VA (donatedfromDr.John BrameldtoDr Jane Harper).These cellswere grownin growth mediumconsistingof highglucose Dulbecco’sModifiedEagle Medium(DMEM) (4.5 g/l D-Glucose,0.11 g/l sodiumpyruvate,withoutL-Glutamine) mixedwith10% foetal bovine serum,1% penicillinand streptomycinand1%glutamine.The growthof the C2C12 cell line wasmaintainedviaincubationat 37o C withan atmosphere of 95% airand 5% carbon dioxide (CO) withamediumchange everythirdday of the week(Burattini et.al 2009; Brownet.al 2012; Fisher-AylorandWilliams2011). ii. Sub-culturing Sub-culturingwasdone whencellsreached80to 90% optical congruence.The growthmediumwas discardedandthe cellswere washedwith20ml phosphate buffersaline (PBS).AsC2C12 were adherent cells,theirdetachmentfromthe surface of the cell culture flaskwas inducedbyincubatingthe cellsin5 ml freshlythawedtrypsinEDTA (0.25%trypsin,1mM EDTA) for 5 minutes.After5 minutesincubation, cellswere observedunderaninverted phasecontrastmicroscope (Carl ZeissPrimovertInvertedphase contrast microscope) todetermine degree of detachmentfromcell flasksurface.10ml of growth mediumwasaddedtostop trypsinreaction.The mediumcontainingthe cellswere thentransferredto centrifuge tubesandcentrifugedat300 rpm for5 minutes(ThermoScientific15/80 MSE centrifuge). Supernatantwasdiscardedand2 ml of growth mediumwasusedtoresuspendthe cellsforcounting. Cellswere counted before beingseeded ataconcentrationof 2 x 103 /25 cm2 cell culture flasks. iii. Viabilityand cell counting Trypan blue wasusedtoassesscell viabilityandthe dye wasmixedwiththe cell suspensionwitha1:1 dilution(0.2ml of cell suspensionand0.2ml of trypan blue stain).Haemocytometer(NanoEntek) were usedand filledwiththisdilution.Phase contrastmicroscope wasusedtoobserve the cells.Cellsthat were stainedblue were considerednon-viablecellsastheyabsorbedthe dye.The numberof viable cells ineach of the four1X1 mm squareswere countedwiththe average countbeingusedtodetermine the numberof viable cellswithinthe cell suspension.
  • 22. DominicThum:N0625034 22 b. Preparation of nanoclay suspensions The Cloisite Na+ montmorillonite (MMT) wasprocuredfrom The SouthernClayProductsCo.USA whilst the lucentite SWN wasprocuredfromCoopCorporation,Japan.The nanoclay suspensionswere preparedat 0.3% concentrationusingdeionizedwaterasa solventandhand-shaken.The suspensions were thenautoclavedat120o C to decrease the riskof biological contamination.Laserlightscattering usinga particle size analyzer(BeckmanCoulterN4Plus) wasusedonthe suspensionbeforeandafter treatingthe cells todetermine changesinparticlesize anddistribution. c. Experimental Design For the initial cell viability experiment,the cellswere grownon24-well cell culture plates(Sarstedt) and were seededattwocell densities:1x 104 cells/cm2 (low celldensity)and5 x 104 cells/cm2 (highcell density).Thiswastodetermineif cell densityaffectedthe effectof the nanoparticle suspensionsonthe viabilityof the C2C12 cells. The cellswere allowedtogrow for 24 hoursto ensure attachmentbefore additionof nanoparticle treatments.The treatmentswere asfollows: 1) 10% FBSDMEM growthmedium (i.e.control) 2) 100 µg/ml of nanoclayin10% FBS DMEM (0.1%) 3) 10 µg/ml of nanoclay in10% FBSDMEM (0.01%) 4) 1 µg/ml of nanoclayin10% FBS DMEM (0.001%) 5) 1% FBS DMEM (differentiationmedium)* The wellswere washedwithPBSgentlybeforethe treatmentswere added.The treatmentswere added intofourwells andwere randomlydistributedamongstthe 24-well plate.Inlaterstagesof the experiment,the cellswere seededat1 X 104 cells/cm2 asitwas believedthatthe highcell densitywas affectingthe growthof the cellsindependentof the treatmentsgiven.The treatedplateswere incubatedoverthe spanof three dayswithplatesbeingtakenoutat specifictime points(Day1,Day 2 and Day 3) to run the cell viability assays. For proteinestimationand extractionexperiment,asimilarexperimental setupasthe cytotoxicitytest was done ( withcellsseededat1 x 104 cells/cm2 ) exceptthe platesusedwere 6-well cellculture plates and the treatmentswere asfollows: *This treatment was used to determine if the cells were ableto differentiate
  • 23. DominicThum:N0625034 23 1) 10% FBSDMEM (control) 2) 1% FBS DMEM (control) 3) 100 µg/ml of nanoclayin10% FBS DMEM (0.1%) 4) 100 µg/ml of nanoclayin1% FBSDMEM (0.1%) 5) 1 µg/ml of nanoclayin10% FBS DMEM (0.001) 6) 1 µg/ml of nanoclayin1% FBS DMEM (0.001%) The treatmentswere organizedasseeninFigure 3below: Figure 3: Organisation of treatments in 6 well plate The plateswere incubatedandharvested ontimepoints day1, 2 and 3 forfurtheranalysis. d. Cytotoxicityassay i. MTT formazan assay The MTT viabilityassayprotocol wasderivedfromImmutec Method51 withsome minordifferences. Thiscolorimetricassaymeasuresviabilitybasedon NADPH-dependentoxidoreductase enzymeactivity inconvertingthe MTT (1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan) (Sigma-Aldrich)dye into purple formazan(Berridge et.al 2005; Berridge andTan 1993). The MTT solutionwaspreparedby 10% FBS in DMEM (Primary control) 1% FBS in DMEM (Secondarycontrol) 100 µg/ml of nanoclay in 10% FBS DMEM (0.1%) 100 µg/ml of nanoclay in 1% FBS DMEM (0.1%) 1 µg/ml of nanoclay in 10% FBS DMEM (0.001%) 1 µg/ml of nanoclay in 1% FBS DMEM (0.001%)
  • 24. DominicThum:N0625034 24 weighingout500 mg MTT and dissolvedin100 ml PBS. The solutionwasthensterile filteredthrough0.2 µM and 0.4 µM before beingdistributedin50ml centrifuge tubes.The tubeswere coveredin aluminiumfoiltopreventthe MTT solution beingexposed tolightandstoredat -20o C. Duringthe differenttime pointsof the treatedcell incubation (Day1,Day 2 andDay 3), MTT solutionat a concentrationof 0.5 mg/ml (i.e.100 µl perwell) wasaddedtoeachwell of the selected platesbefore theywere incubatedforanotherhour.Afterincubation,the wellswere washedgentlywithPBSbefore 500 µl of Dimethyl sulfoxide(DMSO) wasaddedtoeachwell.The plateswere thengentlyshaken ona flatbedplate shaker(IKA KS260 Basic shaker) for2 minutesat80 rpm before aplate reader (Asys UVM340) was usedto readthe absorbance at 570 nm.This wasdone within30 minutesof the addition of DMSO as the formazandye will begintodilute whenexposedtolight. ii. Neutral Red assay The protocol for the neutral redassay wasderivedfromLӧwiket.al (1993) withslightmodifications. This colorimetriccell viabilityassayserves toestimate the abilityof cellstoendocytose nutrientsviastaining lysosomesred.Neutralred(NR) solutionwaspreparedbyadding0.1g of neutral redpowderin100 ml of nanopure waterwhichwasthenfiltersterilizedusing0.2µM and 0.4 µM filters.The NRsolutionwas thenaddedto 1.8% sodiumchloride solutionat1:1 dilutiontoformthe workingsolution.0.2ml of the workingsolutionwasthenaddedtoeachwell of the selectedplatesof differenttime points.These plateswere lefttoincubate foranother1.5 hours.The plateswere thenwashedtwice usingPBSbefore 1 ml of 0.05M NaH2PO4 in50% ethanol wasaddedto the wellstoextractthe dye.A plate reader (Asys UVM340) was thenusedto readthe absorbance at 550 nm, with620 nmbeingthe reference wavelength. e. Cell harvest Cell harvestandproteinextractionmethodswere describedinBrownet.al 2011. The C2C12 cellswere resuspendedintothe mediumusingcell scrapers.Thismediumwasthentransferredintoa20 ml centrifuge tube.Wellsthatwere scrapedwasalsowashedwith 2ml PBSwhichwere alsotransferred intothe centrifuge tube.The cell suspension were thencentrifugedat500g for 5 minutes (Harrier HeraeusMultifuge X3centrifuge).The supernatantwasdiscardedand1ml of PBS wasaddedto the tubes.The tubeswere centrifugedagainat2000g for2 minutes.The supernatantwasagaindiscarded and the tubeswere air-dried.The cell pelletswere thensuspendedin0.2ml of cell lysisbuffer(10mM
  • 25. DominicThum:N0625034 25 Tris Base,5mM EDTA, 150mM NaCl,1mM sodiumorthovandate,1% SDSand 0.2% Protease Inhibitor cocktail).The cell extractswere thenboiledat100o C before beingstoredin -18o C. f. Proteinextraction and estimation The bicinchoninicacidassay(BSA) protocol wasderivedfromthe manufacturer’sinstructionsforthe Pierce® BCA Assaykit (ThermoScientific).The dilutedalbuminstandards(BSA)(2000 µg/ml,1500 µg/ml, 1000 µg/ml,750 µg/ml,500 µg/ml,250 µg/ml,125 µg/ml,25 µg/ml and 0 µg/ml) were prepared followingthe instructionsgiven,usingthe cell lysisbufferasdiluent.Thiswasdue tothe presence of SDS withinthe bufferwhichcouldinterfere withthe BCA reagents.Toensure propermixing,avortex mixer was usedtostir the BCA standardsandthe cell extracts.25 µl of standardsand extractswere pipetted intoa 96-well plate,eachstandardand extractreplicatedtwice.200 µl of workingsolutionconsistingof 50:1 dilutionof ReagentA andReagentBwas thenaddedtoeach well.The 96-well plate wasshakenfor 30 secondsbefore beingincubatedfor30 minutesat37o C. Afterincubation,aplate readerwasusedto readthe absorbance at 565nm. A standardcurve wasdrawn usingthe absorbance readingsof the standardsand the concentrationof the standards.The curve was thenusedto determinethe protein concentrationof the cell extracts. g. SDS-PAGEand WesternBlotting i. SDS-PAGE Proteingelswere assembledbasedoninstructiongivenbymanufacturers(Fisher- Scientific).10ml of 12.5% EZ-Rungel solutionwasmixedwith60µl of ammoniumpersulfate(APS) and6 µl of TEMED. This mixture wasimmediatelypouredbetweenglassplatesof anassembledgel casting stand(insertproduct line here) (Bio-Rad).Combswere thenaddedtothe topof the glass platesbefore the gel wasleftto polymerise.Once polymerized,the gelswereremovedfromthe gel assemblyandplacedintoaprotein gel tank (productline here).800 ml of 20:1 dilution runningbuffer(40ml 20X EZ-RunBufferdilutedin 760 ml of deionizedwater) wasthenaddedtothe gel tank. The selectedcell extractswere thenthawedbefore 10µl of 5X Laemmli buffer(findoutreagentshere) was addedtothem.The mixture wasthenboiledbefore20µl of the mixture waspipettedintoeach well,togetherwith2.5µl of the PageRulerTM PlusPrestainedProteinLadder(Thermo-Scientific).The gel was thenallowedtorunfor1 hour at 180v or until the proteinmarkersreachedthe endof the plate.
  • 26. DominicThum:N0625034 26 ii. Proteintransfer Once the run was complete,the gel wasthenplacedintotransferbuffer(48mMTRIS Base,39mM glycine,0.0375% SDS, 20% methanol).Eightfilterpapersand a nitrocellulose membrane (both Whatman) were cut at 10x8 dimensionsandwere alsosoakedintransferbuffer.A transfersandwich ( fourfilterpapers– nitrocellulose membrane –polyacrylamide gel –fourfilterpapers) was stackedon the anode surface of the rapid semi-dryblottersystem(ATTO WSE-4020 HorizeBLOT2M-R). A glass rod was usedtoremove anyair bubbleswhilstexcesstransferbufferwaswipedaway. The cathode plate of the semi-drytransferapparatuswasthenplacedandthe blotterwasallowedtorunfor 1 hour at 50 mA pergel. The nitrocellulose membrane wasthenremovedwiththe aidof tweezers. h. Immunoprobingof Westernblots The transferrednitrocellulose membrane wasthencut at70 kDa and36 kDa to formthree sections (Figure 4) in accordance to the molecularweightof the primaryantibodiesused withproteinmarkers beingusedasreference pointsforcuttingthe nitrocellulose membrane. Figure 4: Horizontal slices on nitrocellulose membrane The proteinschosentobe detectedwere caspase-3(anindicatorof apoptosis),myosin heavychain(an earlyindicatorof differentiation) andβ-tubulin(acommonhousekeepingproteinexpressedinmouse myoblaststhatservesasthe loadingcontrol).Assuch,these three antibodieswere chosen:
  • 27. DominicThum:N0625034 27  Anti-active +proCaspase 3 antibody (32kDa) (Abcam)  MyosinHeavyChain2 antibody(200kDa) (Genetex)  Beta-TubulinLoadingControl Antibody (51kDa) (Thermo- Scientific) The secondaryantibodiesusedwere:  Goat anti-mouse antibodyconjugatedwithalkalinephosphatase (usedtodetect Beta-Tubulin LoadingControl Antibody) (ThermoScientific),and;  Donkeyanti-rabbitantibodyconjugatedwithalkalinephosphatase (usedtodetectboth Anti- active + pro Caspase 3 antibody andMyosinHeavyChain2 antibody) (SantaCruzBiotechnology, Inc.) The cut nitrocellulose membraneswere thenplacedintoblockingbuffer(5% Marvel milkpowderinTris buffersaline-Tween(TBST) (50mM Tris Base,200 mM NaCl at pH 7.4 and 0.1% Tween-20)) for1 hour at room temperature withcontinuousshaking.The blockednitrocellulose membraneswere then transferredintoprimaryantibodysolutions(dilutedprimaryantibodies(see3.a.b.fordilutions) in5% BSA inTBST) whichwere thenincubatedat4o C overnight. The incubatednitrocellulose membraneswere thenwashedinTBSTfor 50 minutesatroom temperature,TBSTbeingreplacedevery10minutes.The washedmembraneswere thenplacedinto secondaryantibodyblockingsolutioninrespecttothe type of primaryantibodiesusedinitially(1:5000 secondaryantibodydilutionin5%BSA in TBST) and was lefttoincubate for2 hoursin4o C. The membraneswere thenwashedwithTBSTforanother50 minutes,withTBSTbeingreplacedevery10 minutes. The nitrocellulose membrane wasthenrinsedwithdeionizedwaterbefore beingimmersed insubstrate buffer(0.75 mMTris Base pH 9.5, 0.13 mMNitroblue tetrazolium and0.29 mM5-bromo-4chloro-3- indolyl phosphate (BCIP)) whichwaskeptinthe dark.The membrane wasobservedforvisualizationof bandsat 5 minute intervalsbefore the developmentprocesswashaltedbyextensive washingin deionizedwater(Blake et.al 1984). Bio-RadChemidocTM MPImagingsystemwasusedto digitize the WesternBlots.
  • 28. DominicThum:N0625034 28 i. Image analysisand cell imaging Visualisationof cell cultureswasdone usingZeissPrimoVertinverted phase contrastmicroscopewith binocularphototube,ZeissAxioCamERC5S digital camera(Carl Zeiss,Germany) whilstthe processingof the imageswasdone usingZEN 2012 software (Carl Zeiss,Germany). j. Statistical analysis Statistical analysiswasdone usingIBMSPSSStatisticsoftware. Two-wayANOVA wasusedtodetermine if there wasa statistical difference inthe effectof concentrationof nanoclayandtime of exposure to cell viabilitywhilstt-testfordifferenceswasusedtodetermine if therewere differencesinthe effects betweenthe differenttreatments.Pvalues≤0.01 were consideredstatisticallysignificant.Datavalues were presentedasMean± STD (Standarddeviation). 4) Results a) Particle size analysis i. Cloisite Na+ montmorillonite (MMT) Figure 5 belowshowsthe particle size distributionsof pre-autoclaved CloisiteNa+ montmorillonite (MMT nanoparticle suspension andthe autoclaved CloisiteNa+ montmorillonite (MMT) nanoparticle suspension.The particle size distributionswere derivedby comparingthe laserlightintensity(measured in%) to the correspondingparticle size,withnumber% representingthe numberof particlesof aspecific particle size ata specificintensityandvolume% representingthe volume of the particle ataparticular particle size ata specificintensity. InFigure 5A whichrepresentsthe particle sizedistributionsof pre- autoclavedCloisite Na+ montmorillonite(MMT) nanoparticle suspension, three peaks(apeakfor number%andtwopeaksfor the volume%) were observed.Fornumber%,the particle sizerangedfrom 1.00 nm to 215.44 nm. For volume%,the particlesize range forthe firstpeak wasmeasuredtorange from1.00 nmto 146.78 nmwhilstthe particle size range forthe secondpeak rangedfrom215.44 nm to 1000.00 nm.In Figure 5B whichrepresentsthe particle sizedistributionsof the autoclavedCloisiteNa+ montmorillonite (MMT) nanoparticle suspension, threepeaks(apeakfornumber% andtwopeaksfor the volume%) werealsoobserved.Fornumber%,the particlesize rangedfrom1.00nm to 215.44 nm. For volume the particle size range forthe firstpeakwasmeasuredtorange 1.00 nm to 215.44 nm whilst the particle size range forthe secondpeakrangedfrom 215.44 nm to3162.28 nm. Figure 5C providesa comparisonof the particle size distributionsforbothpre-autoclavedandautoclavedCloisite Na+ montmorillonite (MMT) nanoparticle suspensions,respectively.Lookingatboththeir number%,the
  • 29. DominicThum:N0625034 29 peaksremainedinthe same range andat the same intensitylevels.Forvolume%,itcanbe seenthatthe firstpeaksof both pre-autoclavedandautoclaved suspensionsremainedinthe same particle range (albeitthe intensity%forthe autoclavedMMT nanoparticle waslowerthanthatof the pre-autoclaved MMT nanoparticle suspensions).Onthe otherhand,the second peakof the autoclavedMMT nanoparticle suspension showedanelongationanda decrease inintensityincomparisontothatof the pre-autoclavedMMTnanoparticle suspension. Figure 5: Particle size distributionfor A) Pre-autoclavedMMT nanoparticle suspensionand B) AutoclavedMMT nanoparticle suspension;C) Comparisonof particle size distributionbetweenpre- autoclaved and autoclaved MMT nanoparticle suspensions A
  • 31. DominicThum:N0625034 31 Figure 6 belowshowsthe particle size distributionsof pre-autoclavedlucentite SWN nanoparticle suspensionandthe autoclavedlucentite SWN nanoparticle suspension.The particle size distributions were derivedbycomparingthe laserlightintensity(measuredin%) tothe correspondingparticle size, withnumber%representingthe numberof particlesof aspecificparticle sizeata specificintensityand volume%representingthe volume of the particle ata particular particle size ata specificintensity.In Figure 5A whichrepresentsthe particlesize distributionsof pre-autoclavedlucentiteSWN nanoparticle suspension, twopeaks(one peakfornumber% and one peakforthe volume%)were observed. For number%,the particle size rangedfrom1.46 nmto 4.64 nm.For volume%,the peakwasmeasuredto range from 1.47 nm to 4.64 nm. InFigure 6B whichrepresentsthe particlesize distributionsof the autoclaved lucentite SWN nanoparticlesuspension, twopeaks(onepeakfornumber% andone peakfor the volume%) werealsoobserved. Fornumber%,the particlesize rangedfrom2.15nm to 10.00 nm.For volume%,the peakwasmeasuredto range from2.15 nm to46.41 nm. Figure 6C providesacomparison of the particle size distributionsforbothpre-autoclavedandautoclaved lucentiteSWN nanoparticle suspensions,respectively.Lookingatboththeir number% and volume%,itcanbe seenthatthe peaksin autoclavedsuspensions shiftedtothe leftfromthatof the pre-autoclavedsuspensions. Figure 5: Particlesizedistribution for A) pre-autoclaved MMT nanoparticlesuspension,and;B) autoclaved MMT nanoparticlesuspension;C) Comparison of pre-autoclaved and autoclaved MMT nanoparticle suspensions particlesizedistribution
  • 32. DominicThum:N0625034 32 Figure 6: Particle size distributionfor A) Pre-autoclavedSWNnanoparticle suspensionand B) AutoclavedSWNnanoparticle suspension;C) Comparisonof particle size distributionbetweenpre- autoclaved and autoclaved SWNnanoparticle suspensions A
  • 34. DominicThum:N0625034 34 b) Cytotoxicityassay analysis i) MTT analysis i. Initial cell viabilityanalysis on C2C12 treated withCloisite Na+ montmorillonite (MMT) at two differentcell densities:a) 1 x 104 cells,and; b) 5 x 104 cells Figure 7 depictsthe meancell viability scoresobtainedinthe treatmentsusedforthe MTT assay at two differentC2C12 cell densities overincubation time(day).Itcanbe seenthat the treatmentshadan effectonthe cell viabilities of bothcell densitiesincomparisontothe primarycontrol (10% FBS DMEM) withthe cellstreatedwith100 µg/ml (0.1%) MMT showingthe biggestdifference. A one-wayANOVA confirmedthatthe treatmentshada significanteffectoncell viability (F(4)=6.531;p=0.000) whilstthe a post-hocTukey’stestdeterminedthatcellstreatedwith 100 µg/ml (0.1%) MMT showedthe greatest difference incell viabilityrelativetocellsgrowninthe primarycontrol (Meandiff.=0.267750; p=0.000) and cellsgrown indifferentiationmedium(Meandiff.=0.253854; p=0.001). The cellswiththe other MMT treatments(10µg/ml (0.01%) MMT; 1 µg/ml (0.001%) MMT) did notshow any significant difference tocellsgrowninthe primarycontrol andthe differentiationmedium, respectively(see Appendix 1).The resultsalsoshowthatcell densityplayedarole inthe effectof the treatmentsonthe cell viabilityasdemonstratedbythe decline incell viabilityof highercell densitywhentreatedwiththe primarycontrol.Thiswas furtherconfirmedwithatwo-wayANOVA (F(4)=73.384;p=0.000). Incubation time alsoplayedapivotal factorin the effectof the treatmentsonmeancell viabilityasitcan be seen that the cell viabilitiesforlowdensitycellstreatedwith10% FBS DMEM, 1% FBS DMEM, 10 µg/ml (0.01%) MMT and 1 µg/ml (0.001%) MMT show a gradual increase fromDay 1 to Day 3. The exception are the cellstreatedwith100 µg/ml (0.1%) MMT whichshowedaninitial increase fromDay1 to Day 2 but a gradual decrease onDay 3 (Figure 5A).The highdensitycells,onthe otherhand,show anopposite effectwherebycellstreatedwith10% FBS DMEM, 100 µg/ml (0.1%) MMT and 10 µg/ml (0.01%) MMT showedagradual decrease incell viabilityfromDay1 to Day 3. Cellstreatedwith 1µg/ml (0.001%) MMT, on the otherhand,showeda slightincrease incell viabilityfromDay1 to Day 2 before showinga slightdecrease onDay3 whilstcellstreatedwith1% FBS DMEM (differentiationmedium) showedan initial decreasefromDay1 to Day 2 but increasedslightlyonDay3 (Figure 7b).Thisexperimentwas repeatedtwice. Figure 6: Particlesizedistribution for A) pre-autoclaved SWN nanoparticlesuspension,and;B) autoclaved SWN nanoparticlesuspension;C) Comparison of pre-autoclaved and autoclaved SWN nanoparticlesuspensions particlesizedistribution
  • 35. DominicThum:N0625034 35 Figure 7: Mean cell viabilityscore for each treatment at a) Low cell density(1 x 104 cells),and; b) High cell density(5 x 104 cells) B A
  • 36. DominicThum:N0625034 36 Due to the resultsof the statistical analysisaswell asthe initial MTTanalysis,itwas decidedthatthe nextfewMTT cell viabilitytestswere tosee the effectsof MMT treatmentsonlow cell density. Furthermore,differentiationmediumwasremovedasatreatmentasit wasonlyincludedtodetermine if the C2C12 cellswere able todifferentiate. ii. Cell Viabilityanalysison C2C12 cellstreated withCloisite Na+ montmorillonite (MMT) at 1 x 104 cell density Figure 8 showsthe pooledmeancell viabilityscores fromthe treatmentsatlow cell density(1X104 ).It can be seenthatthe MMT treatmentshadaneffectonthe cell viabilityscoresincomparisontothe cells grownin 10% FBS DMEM with100 µg/ml (0.1%) MMT showingthe biggestdifference (asseeninFigure 5A). Similarly,aone-wayANOVA testconfirmedthatthe MMT treatmentshada significanteffecton the cell viabilityscores(F(11)=21.218; p=0.000) witha post-hocTukey’stestconfirming thatcellstreated with100 µg/ml (0.1%) MMT showedthe greatestdifferenceincell viabilityrelativetocellsgrowninthe primarycontrol (Meandiff.=0.224500; p=0.000), followedbycellstreatedin10µg/ml (0.01%) MMT (Meandiff.=0.110819; p=0.002). Onthe otherhand,cellstreatedwith1µg/ml (0.001%) MMT showed no significantdifference withcellstreatedinprimarycontrol (Meandiff. =0.36319; p=0.631). Similarly, incubationtime playedacritical role inthe effectof MMT treatmentsonthe cell viability asitcanbe seenthatcellstreatedwiththatthe cell viabilitiesforlow densitycellstreatedwith10% FBS DMEM, 10 µg/ml (0.01%) MMT and 1 µg/ml (0.001%) MMT show a gradual increase fromDay 1 to Day 3. On the otherhand,cellstreatedwith100 µg/ml (0.1%) MMT whichshowedaninitial increase fromDay1 to Day 2 buta gradual decrease onDay 3 (Figure 6A).Thiswasfurtherconfirmedwithatwo-wayANOVA test(F(6)=20.669; p=0.000) and cell). Thisexperimentwasrepeatedthreetimes. Figure 7: Pooled mean cell viability scores for each treatment againstincubation timeat(A) low cell density (1 x 104 cells) and (B) high cell density (5 x 104 cells)
  • 37. DominicThum:N0625034 37 Figure 8: Mean cell viabilityscore for each treatment at lowcell densityVS incubation time iii. Cell Viabilityanalysison C2C12 cellstreated withLucentite SWN at 1 x 104 cell density Figure 7 showsa representative meancell viabilityscoresfromthe treatmentsatlow cell density (1x104 ).It can be seenthatthe SWN treatmentshadan effectonthe cell viabilityscoresincomparison to the cellsgrownin10% FBS DMEM with100 µg/ml (0.1%) SWN showingthe biggestdifference (as seeninFigure 7A).Thiswasfurtherconfirmedwithaone-wayANOVAtest(F(3)=6.596;p=0.000). However,the post-hocTukey’stestshowedthat1 µg/ml (0.001%) SWN showedthe greatestdifference incell viabilityscoresrelativetocellsgrowninthe primarycontrol (Meandiff.=-0.115833; p=0.001). The otherSWN treatments(100 µg/ml (0.1%) SWN and10 µg/ml (0.01%) MMT, respectively) showedno significantdifferencetothatof cellstreatedinthe primarycontrol (10% FBSDMEM) (Appendix 2).Once again, incubationtime playedacritical role inthe effectof SWN treatmentsonthe cell viabilityasitcan be seenthat the lowdensitycellstreatedwith10% FBS DMEM, 100 µg/ml (0.1%) SWN,10 µg/ml (0.01%) SWN showeda gradual increase fromDay1 to Day 3. The exceptionbeingthe cellstreated1 Figure 8: Pooled mean cell viability scores for each treatment againstincubation time
  • 38. DominicThum:N0625034 38 µg/ml (0.001%) SWN showedasharp increase fromDay1 to Day 2 before showing aslightdecrease on Day 3. The effectof incubationtime onSWN treatmenteffectswere confirmedbyatwo-wayANOVA test(F(6)=3.145; p=0.006. Thisexperimentwasrepeatedthree times. Figure 9: Mean cell viabilityscore for each treatment at low cell densityVS incubationtime ii) Neutral Red analysis i. Initial cell viabilityanalysis on C2C12 treated withCloisite Na+ montmorillonite (MMT) at two differentcell densities:a) 1 x 104 cells,and; b) 5 x 104 cells Due to circumstances,initial neutral redanalysisforMMT treatmentsonC2C12 couldonlybe done on Day 1 and Day 3. Figure 10 depictsthe pooled meancell viabilityscoresobtainedinthe treatmentsused for the neutral red(NR) assayat two differentC2C12 cell densitiesoverincubationtime(day). Itcan be seenthatthe treatmentshadan effectonthe cell viabilitiesof bothcell densitiesincomparisontothe primarycontrol (10% FBS DMEM) withthe cellstreatedwith100 µg/ml (0.1%) MMT showingthe biggest difference. A Kruskal- Wallistestconfirmedthatthe treatmentshadsignificanteffectoncell viability (X2(4)=32.536; p=0.000), However,apost-hocpairwise comparisonshowsthatthe cell viabilityscoresof Figure 9: Representative mean cell viability scores for each treatment againstincubation time
  • 39. DominicThum:N0625034 39 cellstreatedwith100 µg/ml (0.1%) MMT were notsignificantlydifferentfromthe cellstreatedinthe primarycontrol (10% FBS DMEM growthmedium) (p=0.062) butrather showedsignificantdifferencesto the cell viabilityscoresof cellstreatedwith1µg/ml (0.001%) MMT and 1% FBS DMEM differentiation medium,respectively(bothp=0.000). The cell viabilityscoresof the cellstreatedinthe otherMMT treatments(10 µg/ml (0.01%) MMT; 1 µg/ml (0.001%) MMT, respectively)alsoshowednosignificant difference tothe cell viabilityscoresof cellstreatedinthe primarycontrol (see Appendix 3). Cell density alsoappearsto playan effectonthe cell viabilityasthe highdensitycellstreatedwithprimarycontrol had highercell viabilityscoresthanthatof the low densitycells. A Kruskal-Wallistestconfirmedthatcell densityhadsignificanteffectoncell viabilityscores(X2 (1)=88.599;p=0.000). Incubationtime alsoplayed a pivotal factorin the effectof the treatmentsonmeancell viabilityasitcan be seenthatthe cell viabilitiesforlowdensitycellstreatedwith10% FBS DMEM, 1% FBS DMEM, 10 µg/ml (0.01%) MMT and 1 µg/ml (0.001%) MMT showa gradual increase fromDay 1 to Day 3. The exceptionforthisiscells treatedwith100 µg/ml (0.1%) MMT as theyshow gradual decrease fromDay1 to Day 3. The high densitycellsfollowsasimilartrendwherebycells treatedwith10% FBSDMEM, 10 µg/ml (0.01%) MMT and 1% FBS DMEM showedanincrease incell viabilityfromDay1 to Day 3. . Cellstreatedwith1µg/ml (0.001%) MMT, on the otherhand, showedaslightdecrease incell viabilityfromDay1 to Day 3 whilst cellstreatedwith1µg/ml (0.001%) MMT showeda sharpdecrease fromDay 1 to Day 3 (Figure 10b). Thisexperimentwasrepeatedtwice.
  • 40. DominicThum:N0625034 40 Figure 10: Mean cell viabilityscore for each treatmentat a) Low cell density(1 x 104 cells),and; b) High cell density(5 x 104 cells) A
  • 41. DominicThum:N0625034 41 Due to the resultsof the statistical analysisaswell asthe initial MTTanalysis,itwasdecidedthatthe nextfewMTT cell viabilitytestswere tosee the effectsof MMT treatmentsonlow cell density. Furthermore,differentiationmediumwasremovedasatreatment asit wasonlyincludedtodetermine if the C2C12 cellswere able todifferentiate. ii. Cell Viabilityanalysison C2C12 cellstreated withCloisite Na+ montmorillonite (MMT) at 1 x 104 cell density Figure 11 showsa representativemeancell viabilityscoresfromthe treatmentsatlow cell density (1x104 ).It can be seenthatthe MMT treatmentshadan effectonthe cell viabilityscoresincomparison to the cellsgrownin10% FBS DMEM with100 µg/ml (0.1%) MMT showingthe biggestdifference (as seeninFigure 9).Thiswas furtherconfirmedwithaone-wayANOVA test(F(3)=5.349;p=0.001) witha post-hocTukey’stestconfirmingthatcellstreatedwith100 µg/ml (0.1%) MMT showedthe greatest difference incell viabilityrelativetocellsgrowninthe primarycontrol (Meandiff.=0.246292; p=0.003). The cellswiththe otherMMT treatments(10 µg/ml (0.01%) MMT; 1 µg/ml (0.001%) MMT) didnot show Figure 10: Pooled mean cell viability scores for each treatment againstincubation timeat (A) low cell density (1 x 104 cells) and (B) high cell density (5 x 104 cells) B
  • 42. DominicThum:N0625034 42 any significantdifference tocellsgrowninthe primarycontrol andthe differentiationmedium, respectively(see Appendix 4).Similarly,incubationtime playedacritical role inthe effectof MMT treatmentsonthe cell viabilityasitcan be seenthatcellstreatedwiththatthe cell viabilitiesforlow densitycellstreatedwith10%FBS DMEM, 10 µg/ml (0.01%) MMT and1% FBSDMEM (differentiation medium) showagradual increase fromDay1 to Day 3. On the other hand,cellstreatedwith100 µg/ml (0.1%) MMT whichshowedaninitial increasefromDay1 to Day 2 but a small decrease onDay3 whilst cellstreatedwith1µg/ml (0.001%) MMT showeda gradual increase fromDay1 to Day 2 buta small decrease onDay 3 (Figure 9) Thiswas furtherconfirmedwithatwo-wayANOVA test(F(6)=3.464; p=0.003). Thisexperimentwasrepeated threetimes. Figure 11: Mean cell viabilityscore for each MMT treatment at low cell densityVS incubationtime Figure 11: Representative mean cell viability scores for each treatment againstincubation time
  • 43. DominicThum:N0625034 43 iii. Cell Viabilityanalysison C2C12 cellstreated withLucentite SWN at 1 x 104 cell density Figure 12 showsa representativemeancell viabilityscoresfromthe treatmentsatlow cell density (1x104 ).It can be seenthatthe SWN treatmentshadan effecton the cell viabilityscoresincomparison to the cellsgrownin10% FBS DMEM with100 µg/ml (0.1%) MMT showingthe biggestdifference (as seeninFigure 9).Thiswas furtherconfirmedwithaone-wayANOVA test(F(3)=9.658;p=0.000) However,the post-hocTukey’stestshowedthatthe SWN treatments(100 µg/ml (0.1%) SWN,10 µg/ml (0.01%) MMT and 1 µg/ml (0.001%) SWN,respectively) showednosignificantdifference tothatof cells treatedinthe primarycontrol (10% FBS DMEM) (see Appendix 5).Similarly,incubationtime playeda role inthe effectof SWN treatmentsonthe cell viabilityasitcan be seenthatcellstreated with10% FBS DMEM and1 µg/ml (0.001%) SWN showedagradual increase fromDay 1 to Day 3. On the otherhand, cellstreatedwith100 µg/ml (0.1%) MMT whichshowedan small decrease fromDay1 to Day 2 buta gradual increase onDay 3 whilstcellstreatedwith10µg/ml (0.01%) SWN showeda sharpincrease from Day 1 to Day 2 buta small decrease onDay3 (Figure 10). However,the two-wayANOVA testshowed that incubationtime didnothave asignificanteffectonthe effectof the SWN treatmentsoncell viabilityscores((F(6)=2.347;p=0.033). Thisexperimentwasrepeatedthree times.
  • 44. DominicThum:N0625034 44 Figure 12: Mean cell viabilityscore for each SWNtreatment at low cell densityVS incubationtime iii) The visual phenotype of C2C12 cellstreatedwith: i. Cloisite Na+ montmorillonite (MMT) Figure 11 depictstypical picturesof C2C12 cellswhenin10% FBSDMEM growthmedium(control),100 µg/ml (0.1%) MMT in 10% FBS DMEM growth medium and 1 µg/ml (0.001%) MMT in 10% FBS DMEM growth medium. Figure 12: Representative mean cell viability scores for each treatment againstincubation time
  • 45. DominicThum:N0625034 45 Figure 13: C2C12 cellsin10% FBS DMEM growth medium(control),100 µg/ml (0.1%) MMT in 10% FBS DMEM growth medium and 1 µg/ml (0.001%) MMT in 10% FBS DMEM growth medium. Figure 14 depictstypical picturesof C2C12 cellswhenin1% FBS DMEM differentiationmedium (control),100 µg/ml (0.1%) MMT in 10% FBS DMEM differentiationmediumand1 µg/ml (0.001%) MMT in1% FBSDMEM differentiationmediumovertime. Figure 13: Cellular imaging of effect of 100 µg/ml (0.1%) MMT and 1 µg/ml (0.001%) MMT treatments on cells over time (Note the gaps between the cells treated with 100 µg/ml (0.1%) MMT on Day 3 in comparison to the control).
  • 46. DominicThum:N0625034 46 Figure 15: C2C12 cellsin1% FBS DMEM differentiation medium(control), 100 µg/ml (0.1%) MMT in 1% FBS DMEM differentiation medium and 1 µg/ml (0.001%) MMT in 1% FBS DMEM differentiation medium. ii. Lucentite SWN Figure 16 depictstypical picturesof C2C12 cellswhenin10% FBSDMEM growthmedium(control),100 µg/ml (0.1%) SWN in10% FBS DMEM growth medium, 1µg/ml (0.001%) SWN in10% FBSDMEM growth medium. Figure 15: Cellular imaging of effect of 100 µg/ml (0.1%) MMT and 1 µg/ml (0.001%) MMT treatments on cells over time.
  • 47. DominicThum:N0625034 47 Figure 16: C2C12 cellsin 10% FBS DMEM growth medium (control),100 µg/ml (0.1%) SWNin 10% FBS DMEM growth medium,1 µg/ml (0.001%) SWN in10% FBS DMEM growth medium. Figure 17 depictstypical picturesof C2C12 cellswhenin1% FBS DMEM differentiationmedium (control),100 µg/ml (0.1%) SWN in 1% FBS DMEM differentiationmedium, 1µg/ml (0.001%) SWN in 1% FBS DMEM differentiationmedium. Figure 16: Cellular imaging of effect of 100 µg/ml (0.1%) SWN and 1 µg/ml (0.001%) SWN treatments on cells over time.
  • 48. DominicThum:N0625034 48 Figure 17: C2C12 cellsin 1% FBS DMEM differentiation medium(control), 100 µg/ml (0.1%) SWN in1% FBS DMEM differentiationmedium,1µg/ml (0.001%) SWNin 1% FBS DMEM differentiation medium. iv) WesternBlot analysis i. Detectionof Caspase-3expressionfollowingMMTtreatment Day 1 Westernblotsampleswere lost,leavingonlyDay2 andDay 3 C2C12 WesternBlots. Western blottinganalysisfailedtodetect the expressionof active caspase-3inanyof the Day 2 andDay 3 C2C12 cell extracts.However,there were bandspresentabove the 36 kDa proteinmarker,signifyingpresence of inactive procaspase-3(Figure 18).Unfortunately,the bandswere notsharpenoughforfurther analysis. Figure 17: Cellular imaging of effect of 100 µg/ml (0.1%) SWN and 1 µg/ml (0.001%) SWN treatments on cells over time.
  • 49. DominicThum:N0625034 49 Figure 18: WesternBlot for caspase-3 expressioncomparedwithhousekeepingprotein β-tubulin (loadingcontrol) on (A) Day 2, and; (B) Day 3 A B Figure 18: Western Blot analysisfor β-tubulin (control) and Caspase-3 on (A) Day 2, and (B) Day 3 (Bands from left to right: Protein Marker, 10% FBS DMEM C2C12 cell extract, 1% FBS DMEM C2C12 cell extract, 100 µg/ml (0.1%) MMT in 10% FBS DMEM cell extract, 100 µg/ml (0.1%) MMT in 1% FBS DMEM C2C12 cel l extract; 1 µg/ml (0.001%) MMT in 10% FBS DMEM C2C12 cell extract; 1 µg/ml (0.001%) MMT in 1% FBS DMEM C2C12 cell extract; protein marker)
  • 50. DominicThum:N0625034 50 ii. Detectionof MyosinHeavy Chain expressionfollowingMMTtreatment Day 1 Westernblotsampleswere lost,leavingonlyDay2 andDay 3 C2C12 WesternBlots. Western blottinganalysis failedtodetectthe expressionof myosinheavychain(MHC) inanyof the Day 2 and Day 3 C2C12 cell extracts(Figure 16).Due to absence of bands,furtheranalysiswasnotpossible. Figure 19: WesternBlot for myosinheavy chain expressioncomparedwith housekeepingprotein β- tubulin(loadingcontrol) on (A) Day 2, and; (B) Day 3 A B Figure 19: Western Blot analysis for β-tubulin (control) and Caspase-3 on (A) Day 2, and (B) Day 3 (Bands from left to right: Protein Marker, 10% FBS DMEM C2C12 cell extract, 1% FBS DMEM C2C12 cell extract, 100 µg/ml (0.1%) MMT in 10% FBS DMEM cell extract, 100 µg/ml (0.1%) MMT in 1% FBS DMEM C2C12 cell extract; 1 µg/ml (0.001%) MMT in 10% FBS DMEM C2C12 cell extract; 1 µg/ml (0.001%) MMT in 1% FBS DMEM C2C12 cell extract; protein marker)
  • 51. DominicThum:N0625034 51 Unfortunately,due toprojecttime constraints,awesternblotanalysis forlucentite SWN cytotoxicity couldnot be done. 5) Discussion a. Cloisite Na+ montmorillonite (MMT) showslow cytotoxicitytowards C2C12 cellsat higherconcentrations As seenfromthe MTT formazanassayand neutral redassayresultsforMMT, the treatmentwiththe highestconcentrationof MMT (0.1%) showssignificanteffectonthe cell viability of the C2C12 cellsin comparisontothe othertreatmentswithdecreasingMMT concentrations(0.01% and0.001%, respectively). Thisisfurtherconfirmedlookingatthe Figure 11 whichshowsthe C2C12 cellsinin10% FBS DMEM growth medium(control),100 µg/ml (0.1%) MMT in10% FBSDMEM growthmediumand1 µg/ml (0.001%) MMT in10% FBS DMEM growthmedium.Whencomparedwiththe C2C12 cellsgrown inthe control,itcan be seenthatthe higherconcentrationof MMT affectedthe phenotypesof the C2C12 cellsgreatlyashuge gaps are observedbetweenthe cellsaswell asthe presence of roundedcells (brightspotsinthe picture).Thisisevenmore apparentwhenthe treatedcellswereincubatedtill Day3. Thisobservationappearstobe a commonobservation asLee et.al (2005) has shownthat high concentrationsof MMT had a greatereffectsoncellulargrowthandviability,statingthat Saccharomyces cerevisaetreatedwiththe highestMMT concentration(5.0g/L MMT) hadthe lowest growthrate and cell viabilityincomparison toS.cerevisaenot treatedwithMMT. Theirresultsalso agree withphenomenonabsorbedinthisresearchthatthe yeast S.cerevisaetreatedlower concentrationsof MMT (0.1 -0.5 g/l MMT) had lessof an effectoncellulargrowthandviabilityin comparisontothat of S.cerevisae treatedwith highMMT concentration(1.0-5.0 g/L). (Figure 20).
  • 52. DominicThum:N0625034 52 Figure 20: (A) The Saccharomycescerevisae growth in differentMMTconcentration (B) Growth rate of S.cerevisae in various MMT concentration However,Lee et.al’sexperimentmeasuredthe S.cerevisaecell viabilityandgrowthusing standardplate countingandprobit analysis(astatistical model thatsuggestthatthe relationshipbetween individual’s tolerance totoxicmaterialsina givenpopulationisalog-normal distribution). Furthermore,Lee et.al doesnotgo intothe cellularmechanismthatcausesthe repressionof S.cerevisaegrowthandviability. Lee et.al 2005 alsoexploresthe effectof intravenousinjectionsof highMMT doses(1.42.9 mg per Kg rat bodyweightin1 ml PBS) intorats before obtaininghistopathological sectionof the heart,lung,kidney and spleen.However,the highMMTdose didnot have any effectsonthe cell morphology. Anotherphenomenonobservedwas thataftertwo daysof incubation,the MTTanalysisshowed that C2C12 cellstreatedwithhighconcentratedMMT showedaninitial increasebeforeagradual decrease on the thirdday whereasthe otherMMT treatmentsshowedagradual increase incell viability(albeit lessthancellstreatedin10% FBS DMEM growthmedium). Thisshowsthatthe nanoparticleisnotvery cytotoxiceveninhighconcentrationsalthoughitisable toimpede the cellularmetabolicactivityof Figure 20: (A) S. cerevisae growth curves in different MMT concentration (B) Growth rate of S. cerevisar in various MMT concentration. (Source: Lee et.al 2005)
  • 53. DominicThum:N0625034 53 C2C12 cells(basedonthe principle workings of MTT formazanassays).MTT formazanassaysare colorimetricassaysthatmeasures NADPH-dependentoxidoreductase enzymes activityin actively convertingthe MTT (1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan)(Sigma-Aldrich) dye intopurple formazan(Berridge et.al 2005; Berridge andTan 1993). Therefore,anincrease inthe intensity of the purple dye wouldeithersuggest thatthere is highmetabolicactivityorthere are a largernumber of NADPH-dependentoxidoreductaseenzymes withinthe cell. Thisappearstoagree withpreviousMTT formazanassaysdone on MMT cytotoxicity.Maisanabaet.al (2013) useda variationof MTT formazan assayknownas MTS assay(whereby3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulfophenyl)-2H-tetrazolium(MTS) dye isutilizedinstead) todetermine if MMT wascytotoxicto HepG2 humanhepaticcells.TheirresultssuggestthatMMT doesnotcause cytotoxicityevenwhenthe HepG2 cellswere incubatedwithMMTnanoparticle suspensions from24 to 48 hours.However,the highest concentrationMaisanabe usedwas62.5 µg/ml MMT insteadof the 100 µg/ml usedinthisstudy as their rationale wasthatthe higherconcentrationwouldcause complicationwiththe measurementsystem theyused.Furthermore,Maisanabe seeminglydoesnotfurtherexpandonthisknowledge,instead focusingonMMT’s organicallymodifiedcounterpart,Cloisite® 30Bmontmorillonite (MMT). Thisis unlike Lordanet.al (2011) studieswhocomparedthe cytotoxiceffectsof Cloisite Na+montmorillonite (MMT) and Cloisite 93® montmorillonite toHepG2humanhepaticcells.Althoughhisresults suggests that MMT significantlydecreasescell viability,Lordanet.al furtherinvestigatedthe reasonforthis phenomena,statingthatMMT was causingthe necrosisof the HepG2 via productionof intracellular reactive oxidative specieswhichledtocellularmembrane damage (Lordanet.al 2011).However,the concentrationof MMT usedby Lordan et. al was significantlyhigherthanthatof the one usedinthis research,specifically500µg/ml. Previousstudieshave alsoshownthatMMT causescellularmembrane damage inP388D macrophages(Gormley et.al 1978) and N1E-115 neuroblastomacells(Murphyet.al 1993). Gormleyet.al suggeststhatthe membrane damage isdue tothe clay mineral reducingthe membrane potentialof the macrophages,leadingtocell lysis.However,inalaterstudy,Gormleyand Addison(1983) explainsthatthe variable levelsof ancillaryminerals (e.g.quartz) withinthe MMT clay mineral isresponsibleforthe mineral’scelllysisability.Thisisunderstandable consideringthatCloisite Na+ montmorillonite (MMT) isa naturallyoccurringmineral (Section1i) derivedfromvolcanicash.As such,not onlyisit not pure but(dependingonthe locationitismined) the levelsof extraneousminerals minedwithitalsovaries.Another potential contributingfactortoits cell lysisability,couldbe MMT’s affinitytoswell whenincontactwithwater(Murray 2000). Thiswouldexplain the reasonbehindthe
  • 54. DominicThum:N0625034 54 mineral’sabilitytocause cell lysisasthe MMT nanoparticle wouldabsorbthe watercontentwithinthe cell,resultinginthe membrane potential reducing.However, thisisonlyaspeculation. The neutral redresultsforCloisite Na+ montmorillonite (MMT) cell viabilitytestsfollowsasimilartrend to that of the MTT formazanassayresultsin that C2C12 cellstreatedwithhighconcentrated MMT showedaninitial increase before agradual decrease onthe thirddaywhereasthe otherMMT treatmentsshowedagradual increase incell viability(albeitlessthancellstreatedin10% FBS DMEM growthmedium). This,once again,suggeststhatthe MMT nanoparticle isaffectingthe cell’sability to take up substances(basedonthe principlesof neutral redassay). However,lookingatneutral red resultsatDay 1, itcan be seenthatcell viabilityscoresforthe cellstreatedwith MMT were higherthan that of the cellstreatedinthe control (10% FBS DMEM) (withcellstreatedwith0.1% MMT havingthe highestmeancell viabilityscores).Thiscouldbe attributedtothe principle of neutral redassayswhich workby dyinglysosomesred(Repettoet.al2008; Lӧwiket.al 1993). As lysosomesare activelyprocessto allowendocytosisof nutrientsorsubstances,the rationale follows thatthe cell isalive andanincrease in the red dye suggestsmore lysosomesare beingproduced totake upthe reddye.Withthisrationale,the reasonbehindthe relativelyhighmeanviabilityscores forthe MMT treatedcellscanbe understoodthat a largeramount of lysosomeswere producedbythe cell totake up boththe neutral reddye and MMT nanoparticlesinthe growthmedium. Althoughthere isnodefinite answertohow the MMT nanoparticlesaffectedC2C12cellularuptake,Gormleyet.al(1978) providesapotential explanation. Usingmacrophages,they suggested thatthe cellulardamage beganonce the MMT mineralshadbeen phagocytosedbythe cell.However,due tothe mineralsabilitytoreduce membrane potential,the phagolysosomesrupture andreleasethe MMT mineralsintothe cytosol wherebytheywill continue reducingthe membrane potential untilthe cell lyses.Furthermore,Gormley shows thatthe cellsare still viable butare ‘leaky’ due tothe membrane beingdamagedbyMMT. Therefore,notonlyare the cells unable tocontrol the cellularintake butalsohave issuesretainingcomponentsof the cell membrane (e.g.enzymesandproteins) whichfurtherreducesthe membrane potentialandleadingtocell lysis. These suggestionscoincide withthe researchresults andcouldactas a potential explanationtoMMT’s effectoncellularuptake systems.Unfortunately, neutral redassaysare rarelyusedinthese MMT cytotoxicitystudies,withMTTassaysbeingthe preferreddue toits’reliability. Maisanabaet.al (2013) alsoutilizedneutral redassaytodetermineMMT cytotoxicityonHepG2but similarlytothe MTS assay, didnot furtherexpandonit.
  • 55. DominicThum:N0625034 55 Lookingat Figure 12, it can be observedthatthe cellstreatedwith100 µg/ml (0.1%) MMT in 1% FBS DMEM differentiationmediumshowedthe greatesteffectonthe C2C12 phenotype incomparisonto the cellstreatedwiththe differentiationmedium(1% FBSDMEM) and 1 µg/ml (0.001%) MMT in 1% FBS DMEM differentiationmedium,therebydepictingthatMMT had an effectoncell differentiation. However, MMT concentrationisnotthe onlyfactorcontributingtothe treatedcells’phenotype.Asthe cellsare beinggrowninlowserumconditions,the cellsare inducedtodifferentiate insteadof growing. Notall the cellswoulddifferentiate andthe low serumconditionscancause themto die.Assuch,this lowserumconditioncanfurtheraggravate the effectsof highMMT concentrationoncell growth,a phenomenonconfirmedbyLordanand Higginbotham(2013) wherebylow concentrationof MMT (1 mg/ml) significantlyinhibitedthe cell growthof humanmonocyte U937 growninlow serumconditions (Lordanand Higginbotham2013). Thisisfurtherconfirmedfromthe westernblotdetectionformyosin heavychain.Asstated,myosinheavychainisa proteinexpressedduringthe earlystagesof muscle differentiation.Withthe absenceof MHC proteinbandsonthe westernblotsfromboththe Day 2 and Day 3 cell extracts,itcan be assumedthatnone of the C2C12 cellswere differentiatingatthe time of the treatments. b. Lucentite SWNdecreases cellularmetabolicactivity but has drastic effectoncellular uptake system Looking at Figure 7, it can be seen that lucentite SWN does not have much effect on the cellular metabolic activity of the C2C12 cells as the mean cell viabilities for each of the SWN treatments gradually increase over time (albeit at a slower rate than the cells treated in 10% FBS DMEM). This can also be further confirmed in Figure 13 which showsthe C2C12 cellsinin10% FBS DMEM growthmedium(control), 100µg/ml (0.1%) SWN in 10% FBS DMEM growthmediumand1 µg/ml (0.001%) SWN in10% FBS DMEM growthmedium.Whencomparedwiththe C2C12 cellsgrowninthe control, there appearstobe nodifference betweenthe cellstreatedin0.1% SWN and 0.001% SWN, respectivelyevenamongstthe differenttimepoints. Itisspeculatedthatdue toits’syntheticnature,the SWN nanoparticle ismore pure andhasa lowerriskof carryingtoxicchemical particles.Unfortunately, thisisonlya speculationasnopreviousstudiesonlucentite SWN cytotoxicityexists. The neutral redanalysis,onthe otherhand,showsthat at 0.1% concentration,lucentiteSWN wasable to haltcellularuptake fromDay1 to Day 2 before increasingagaininDay3. Anotherphenomenon observedwasthe sharpincrease inmeancell viabilityscoresof cellstreatedin0.01% SWN from Day 1
  • 56. DominicThum:N0625034 56 to Day 2 before asmall decrease inDay3. Cellstreatedwith0.1% SWN showedgradual increase asseen inthe MTT analysis.The phenomenonseenat0.1% SWN and 0.01% SWN showsthat lucentite SWN has an effectonthe cellularuptake system.Thiscouldbe attributedtothe nanoparticlesabilitytoincrease the viscosityof the solutionthe nanoparticle issuspendedin.Thisrationale couldexplainthe haltin lysosomal uptake of the neutral reddye incellstreatedwith0.1% SWN as the large amountof nanoparticleswouldcause the growthmediumto become more viscous,makingitmore difficultforthe cellstoendocytose the reddye.However,thisisonlyaspeculation. Unfortunately,due totime constraints,awesternblotanalysiscouldnotbe done forlucentite SWN, therefore the mechanismbehindits’effectoncellularuptake couldnotbe analyzed. c. ComparisonbetweenMMT and SWNshows that SWNis lesscytotoxic than MMT from a metabolicactivity standpointand MMT is lesscytotoxicto SWNfrom a cellular uptake standpoint Lookingat the cell viabilityresults,itcanbe seenthat whilstboth Cloisite Na+ montmorillonite (MMT) and lucentite SWN nanoparticlesimpedecellularmetabolicactivityandcellularuptake atmiddle orlow concentrations,the effectsof highconcentrationof the nanoparticlesdiffersoncell viability. Itcanbe seenthathighconcentrationof MMT has a greatereffectoncellularmetabolicactivitywhilsthigh concentrationof SWN has a greatereffectoncellularuptake system.Withthese results,the taskof determiningwhichismore nanoparticleismore cytotoxicisnotas simple asthe questionboilsdownto whichaspectof cell viabilitytoconsider.If cellularmetabolicactivityisconsidered,then CloisiteNa+ montmorillonite (MMT) wouldbe consideredmore cytotoxicandvice versaif cellularuptake is consideredinstead.Tofurtheraddto the confusion,the definitionof cell viabilityisratherambiguous and there existsothercell viabilityassaysthatmeasure otheraspects (e.g.lactate dehydrogenase leakage assay(LDH),(acolorimetricassayaimedatmeasuringcellularmetabolicactivityof lactate dehydrogenaseinconvertingNAD+ toNADH) (Deckerand Lohmann-Matthes1988); . Assuch, previous studiesdone oncytotoxicityof nanoparticles,have alwaysusedatleasttwocell viabilityassaysto give a betterperspectiveoncell viability. d. Conclusionand recommendationfor future research The current researchshowsthatCloisite Na+ montmorillonite (MMT) showsrelativelylow cytotoxicity towardsC2C12 cellsat highconcentrationasit hasan effecton NADPH-dependentoxidoreductase enzymes metabolicactivitywhilsthighconcentrationof lucentiteSWN hasan effectonthe cellular
  • 57. DominicThum:N0625034 57 uptake systemof the C2C12 cells.However, due tothe limitedknowledge onCloisiteNa+ montmorillonite (MMT) cytotoxicityonC2C12 and SWN cytotoxicityingeneral,itisstill tooearlyto conclude if these canbe consideredfact as there were areasthatwere notexploredinthis research projectdue to time constraints. An example of anareato explore in future researchcould be the effectof layerexfoliationon Cloisite Na+ montmorillonite (MMT) andlucentite SWN cytotoxicity. Asseenin3.b., bothCloisite Na+ montmorillonite (MMT) andlucentite SWN nanoparticles were suspended indeionizedwaterviahand- shakinginsteadof the more conventionalmethodof using ultrasonicvibrations. Thiscouldaffectthe nanoparticles’effectoncellsas nanoparticles have atendencytoaggregate withone anotherviaVan derWaals forces overa periodof time due theirlarge surface area(Othmanet.al 2012). Thisalso affectstheirdispersionwithinthe aqueoussolution. Therefore,ultrasonicvibrations are usedto prevent the nanoparticlesfromaggregatingandformingclumps,andensuringthattheymore dispersed within the aqueoussolutiontheyare in (Changet.al 2004; Satoet. al 2008). The appearance of three peaks insteadof twofor the particle size distributionsof Cloisite Na+ montmorillonite(MMT) nanoparticle suspensions beforeandafterautoclaving(Figure 5) confirmsthisaggregationphenomenon.The appearance of a lone peakfornumber%suggeststhat,numerically,mostof the nanoparticleswithinthe suspensionare atthat size range.Therefore,logicallythereshouldbe onlyone peakforthe volume%as mostof the nanoparticlesshouldbe the same size andvolume.However, asFigure 5has shown, volume%hastwopeaksat differentparticle size ranges.Asthe firstvolume% peakcoincideswiththe lone number%peak, itissuggestedthatthe sharedparticle range corresponds tothe particle size distributionof one MMT nanoparticle.The appearance of asecondvolume% peakatadifferentparticle size range suggestthe appearance of aggregatedparticlesand corresponds tothe particle size distributionof aggregated Cloisite Na+ montmorillonite(MMT) nanoparticles. LordanandHigginbotham (2013) alsoaddressesthe implicationsof nanoparticle dispersiononcytotoxicity astheystate thatinlow serumconditions,claynanoparticlesare more evenlydispersedwhereashighserumconditionscauses the nanoparticlestoaggregate intolarge bundles,therebyreducingits’cytotoxicability. Furthermore, the effectof autoclavingonthe nanoparticle size anddispersionshouldbe investigatedthoroughly as Figure 5 and 6 have shownthatautoclavinghadsome effectof the aggregated Cloisite Na+ montmorillonite (MMT) nanoparticle size andthe lucentite SWN nanoparticle size(asevidencedinthe shiftinboththe number%andvolume% peaksof the autoclavedSWN nanoparticlesuspension, suggestinganincrease inparticle size).
  • 58. DominicThum:N0625034 58 FollowingGormleyandAddison(1983) suggestion,the mineral compositionof MMT and SWN should alsobe analysed beforeacytotoxicity testtodetermine the levelsof ancillarymineralswithinthe clay. Thiscan be done usinglaser-lightscatteringandparticle distribution,andprovidesfuture researchersa bettercomparisonbetweenthe cytotoxiceffectsof Cloisite Na+ montmorillonite(MMT) withvarying levelsof ancillaryminerals.Anotherareaof researchcouldbe to compare the cytotoxicityof purified Cloisite Na+ montmorillonite (MMT) andnatural Cloisite Na+ montmorillonite(MMT). Anotherthingtohighlightinthisresearchwasthat the cell viabilitieswere measuredusingtwodifferent cell viabilityassayswhoworkondifferentprinciples andthere are othercell viability assaysthatworkon differentprinciplesinmeasuringcellviability.Thisisbecause of the ambiguityof whatisconsidereda measure of cell viability.Therefore,atleasttwodifferentcell viabilityassaysare usedtomeasure cell viabilityasitprovidesabetterperspective oncell. Althoughneutral redassay andMTT assayare both consideredthe more sensitive of the optionsavailable(FotakisandTimbrell2006), future researchcould use a biggercombinationof cell viabilityassaystoprovide abetterunderstandingof Cloisite Na+ montmorillonite (MMT) andlucentite SWN cytotoxicityasmostof the previousstudiesdone showan inclinationtowardsMTTformazanassays. As thisresearchisthe firstinvestigation(tothe author’sknowledge) onlucentiteSWN cytotoxicity, there are still areasyetto be explored. Due totime constraints,the mechanismbehindthe effectof lucentite SWN oncellulargrowthandcellularuptake systemwasnotfullyexplored.This,togetherwith the fact that there existsnopreviousresearchon lucentite SWN cytotoxicity,providesanopportunityto investigatethisareaof research. As there appearstobe no literature inregardsto Cloisite Na+ montmorillonite (MMT) andlucentite SWN cytotoxicitytowardsC2C12 mouse myoblastcells,acomparisonstudycouldalsobe done comparingthe effectsof MMT and SWN on C2C12 cellsandotherwell-documentedcellmodels(Caco-2andHepG2 to name a few) toinvestigate if the phenomenaobservedinthisresearchisexclusivetoC2C12 cellsor these observationsare sharedinothercell models.
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  • 65. DominicThum:N0625034 65 6) Appendices a. Appendix1: Post-hoc Tukeytests results for initial MTT formazan assay analysisfor Cloisite Na+ montmorillonite (MMT) Multiple Comparisons DependentVariable: Absorbance Tukey HSD (I) Treatment (J) Treatment Mean Difference (I-J) Std. Error Sig. 99% Confidence Interval Lower Bound Upper Bound 10% FBS DMEM 100 microgram/ml MMT .267750* .064720 .000 .05466 .48084 10 microgram/ml MMT .131333 .064720 .255 -.08176 .34443 1 microgram/ml MMT .001875 .064720 1.000 -.21122 .21497 1% FBS DMEM .013896 .064720 1.000 -.19920 .22699 100 microgram/ml MMT 10% FBS DMEM -.267750* .064720 .000 -.48084 -.05466 10 microgram/ml MMT -.136417 .064720 .220 -.34951 .07668 1 microgram/ml MMT -.265875* .064720 .001 -.47897 -.05278 1% FBS DMEM -.253854* .064720 .001 -.46695 -.04076 10 microgram/ml MMT 10% FBS DMEM -.131333 .064720 .255 -.34443 .08176 100 microgram/ml MMT .136417 .064720 .220 -.07668 .34951 1 microgram/ml MMT -.129458 .064720 .269 -.34255 .08364 1% FBS DMEM -.117438 .064720 .368 -.33053 .09566 1 microgram/ml MMT 10% FBS DMEM -.001875 .064720 1.000 -.21497 .21122 100 microgram/ml MMT .265875* .064720 .001 .05278 .47897 10 microgram/ml MMT .129458 .064720 .269 -.08364 .34255 1% FBS DMEM .012021 .064720 1.000 -.20107 .22511 1% FBS DMEM 10% FBS DMEM -.013896 .064720 1.000 -.22699 .19920 100 microgram/ml MMT .253854* .064720 .001 .04076 .46695 10 microgram/ml MMT .117438 .064720 .368 -.09566 .33053 1 microgram/ml MMT -.012021 .064720 1.000 -.22511 .20107 *. The mean difference is significantatthe 0.01 level.
  • 66. DominicThum:N0625034 66 7) Appendix2: Post-hocTukey testsresults for MTT formazan assay analysison Lucentite SWN Multiple Comparisons DependentVariable: Absorbance Tukey HSD (I) Treatment (J) Treatment Mean Difference (I-J) Std. Error Sig. 99% Confidence Interval Lower Bound Upper Bound 10% FBS DMEM (0 microgram/ml SWN) 100 microgram/ml SWN -.007896 .029628 .993 -.10139 .08560 10 microgram/ml SWN -.061521 .029628 .165 -.15501 .03197 1 microgram/ml SWN -.115833* .029628 .001 -.20933 -.02234 100 microgram/ml SWN 10% FBS DMEM (0 microgram/ml SWN) .007896 .029628 .993 -.08560 .10139 10 microgram/ml SWN -.053625 .029628 .272 -.14712 .03987 1 microgram/ml SWN -.107938* .029628 .002 -.20143 -.01445 10 microgram/ml SWN 10% FBS DMEM (0 microgram/ml SWN) .061521 .029628 .165 -.03197 .15501 100 microgram/ml SWN .053625 .029628 .272 -.03987 .14712 1 microgram/ml SWN -.054313 .029628 .261 -.14780 .03918 1 microgram/ml SWN 10% FBS DMEM (0 microgram/ml SWN) .115833* .029628 .001 .02234 .20933 100 microgram/ml SWN .107938* .029628 .002 .01445 .20143 10 microgram/ml SWN .054313 .029628 .261 -.03918 .14780 *. The mean difference is significantatthe 0.01 level.
  • 67. DominicThum:N0625034 67 8) Appendix3: Post-hoc pairwise comparisontests resultsfor neutral red assay analysis for Cloisite Na+ montmorillonite (MMT)
  • 68. DominicThum:N0625034 68 9) Appendix4: Post-hoc Tukeytests results for neutral red assay analysis on Cloisite Na+ montmorillonite (MMT) Multiple Comparisons DependentVariable: Absorbance Tukey HSD (I) Treatment (J) Treatment Mean Difference (I-J) Std. Error Sig. 99% Confidence Interval Lower Bound Upper Bound 10% FBS DMEM (0 microgram/ml MMT) 100 microgram/ml MMT .246292* .069124 .003 .02817 .46442 10 microgram/ml MMT .020896 .069124 .990 -.19723 .23902 1 microgram/ml MMT .051917 .069124 .876 -.16621 .27004 100 microgram/ml MMT 10% FBS DMEM (0 microgram/ml MMT) -.246292* .069124 .003 -.46442 -.02817 10 microgram/ml MMT -.225396* .069124 .007 -.44352 -.00727 1 microgram/ml MMT -.194375 .069124 .028 -.41250 .02375 10 microgram/ml MMT 10% FBS DMEM (0 microgram/ml MMT) -.020896 .069124 .990 -.23902 .19723 100 microgram/ml MMT .225396* .069124 .007 .00727 .44352 1 microgram/ml MMT .031021 .069124 .970 -.18710 .24914 1 microgram/ml MMT 10% FBS DMEM (0 microgram/ml MMT) -.051917 .069124 .876 -.27004 .16621 100 microgram/ml MMT .194375 .069124 .028 -.02375 .41250 10 microgram/ml MMT -.031021 .069124 .970 -.24914 .18710 *. The mean difference is significantatthe 0.01 level.
  • 69. DominicThum:N0625034 69 10) Appendix5: Post-hoc Tukeytests results for neutral red assay analysis on Lucentite SWN Multiple Comparisons DependentVariable: Absorbance Tukey HSD (I) Treatment (J) Treatment Mean Difference (I-J) Std. Error Sig. 99% Confidence Interval Lower Bound Upper Bound 10% FBS DMEM (0 microgram/ml SWN) 100 microgram/ml SWN .163979 .105845 .410 -.17002 .49798 10 microgram/ml SWN -.302021 .105845 .025 -.63602 .03198 1 microgram/ml SWN -.305604 .105845 .022 -.63960 .02839 100 microgram/ml SWN 10% FBS DMEM (0 microgram/ml SWN) -.163979 .105845 .410 -.49798 .17002 10 microgram/ml SWN -.466000* .105845 .000 -.80000 -.13200 1 microgram/ml SWN -.469583* .105845 .000 -.80358 -.13559 10 microgram/ml SWN 10% FBS DMEM (0 microgram/ml SWN) .302021 .105845 .025 -.03198 .63602 100 microgram/ml SWN .466000* .105845 .000 .13200 .80000 1 microgram/ml SWN -.003583 .105845 1.000 -.33758 .33041 1 microgram/ml SWN 10% FBS DMEM (0 microgram/ml SWN) .305604 .105845 .022 -.02839 .63960 100 microgram/ml SWN .469583* .105845 .000 .13559 .80358 10 microgram/ml SWN .003583 .105845 1.000 -.33041 .33758 *. The mean difference is significantatthe 0.01 level.