Research Project Dissertation_Final_270815_DT

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 .......................................................

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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.

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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

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7) Appendices 65

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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

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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.

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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

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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)

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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

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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+
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

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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).

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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)

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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

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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)

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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)

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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)

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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)

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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)

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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
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
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
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%)
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
1) 10% FBSDMEM (control)
2) 1% FBS DMEM (control)
4) 100 µg/ml of nanoclayin1% FBSDMEM (0.1%)
5) 1 µg/ml of nanoclayin10% 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
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
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
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
 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
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+
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
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

30
ii. Lucentite SWN
B
C

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
Figure 6: Particle size distributionfor A) Pre-autoclavedSWNnanoparticle suspensionand B)
AutoclavedSWNnanoparticle suspension;C) Comparisonof particle size distributionbetweenpre-
autoclaved and autoclaved SWNnanoparticle suspensions
A

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
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
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
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
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
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
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
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
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

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
Figure 12: Mean cell viabilityscore for each SWNtreatment at low cell densityVS incubationtime
iii) The visual phenotype of C2C12 cellstreatedwith:
i. Cloisite Na+
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.

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
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
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
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
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
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
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
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
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
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
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
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+
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
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
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.

59
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6) Appendices
a. Appendix1: Post-hoc Tukeytests results for initial MTT formazan assay analysisfor
Cloisite Na+
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
.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
7) Appendix2: Post-hocTukey testsresults for MTT formazan assay analysison Lucentite SWN
Tukey HSD
Mean
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
.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
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

67
8) Appendix3: Post-hoc pairwise comparisontests resultsfor neutral red assay analysis for
Cloisite Na+

68
9) Appendix4: Post-hoc Tukeytests results for neutral red assay analysis on Cloisite Na+
Tukey HSD
Mean
10% FBS DMEM (0
microgram/ml MMT)
.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
.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

69
10) Appendix5: Post-hoc Tukeytests results for neutral red assay analysis on Lucentite SWN
Tukey HSD
Mean Difference
(I-J) Std. Error Sig.
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
.105845 .000 -.80358 -.13559
10 microgram/ml
SWN
10% FBS DMEM (0
microgram/ml SWN)
.302021 .105845 .025 -.03198 .63602
.105845 .000 .13200 .80000
1 microgram/ml SWN -.003583 .105845 1.000 -.33758 .33041
microgram/ml SWN)
.305604 .105845 .022 -.02839 .63960
.105845 .000 .13559 .80358
10 microgram/ml SWN .003583 .105845 1.000 -.33041 .33758

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