Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles.

1 MB·English

by  Xu, Jing

JournalofVisualizedExperiments www.jove.com
VideoArticle
Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer
Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles
Jing Xu,Mansoor Amiji
DepartmentofPharmaceuticalSciences,SchoolofPharmacy,NortheasternUniversity
Correspondenceto:[email protected]
URL:http://www.jove.com/video/3612/
DOI:10.3791/3612
Keywords:Bioengineering,Issue59,GelatinNanoparticle,GeneTherapy,TargetedDelivery,PancreaticCancer,EpidermalGrowthFactor
Receptor,EGFR,
DatePublished:1/4/2012
Citation:Xu, J.,Amiji, M. TherapeuticGeneDeliveryandTransfectioninHumanPancreaticCancerCellsusingEpidermalGrowthFactor
Receptor-targetedGelatinNanoparticles.J.Vis.Exp.(59),e3612,DOI:10.3791/3612(2012).
Abstract
Morethan32,000patientsarediagnosedwithpancreaticcancerintheUnitedStatesperyearandthediseaseisassociatedwithveryhigh
mortality1.Urgentneedexiststodevelopnovelclinically-translatabletherapeuticstrategiesthatcanimproveonthedismalsurvivalstatisticsof
pancreaticcancerpatients.Althoughgenetherapyincancerhasshownatremendouspromise,themajorchallengeisinthedevelopmentofsafe
andeffectivedeliverysystem,whichcanleadtosustainedtransgeneexpression.
Gelatinisoneofthemostversatilenaturalbiopolymer,widelyusedinfoodandpharmaceuticalproducts.Previousstudiesfromourlaboratory
haveshownthattypeBgelatincouldphysicalencapsulateDNA,whichpreservedthesupercoiledstructureoftheplasmidandimproved
transfectionefficiencyuponintracellulardelivery.Bythiolationofgelatin,thesulfhydrylgroupscouldbeintroducedintothepolymerandwould
formdisulfidebondwithinnanoparticles,whichstabilizesthewholecomplexandoncedisulfidebondisbrokenduetothepresenceofglutathione
incytosol,payloadwouldbereleased2-5.Poly(ethyleneglycol)(PEG)-modifiedGENS,whenadministeredintothesystemiccirculation,provides
long-circulationtimesandpreferentiallytargetstothetumormassduetothehyper-permeabilityoftheneovasculaturebytheenhanced
permeabilityandretentioneffect6.Studieshaveshownover-expressionoftheepidermalgrowthfactorreceptor(EGFR)onPanc-1human
pancreaticadenocarcinomacells7.Inordertoactivelytargetpancreaticcancercellline,EGFRspecificpeptidewasconjugatedontheparticle
surfacethroughaPEGspacer.8
Mostanti-tumorgenetherapiesarefocusedonadministrationofthetumorsuppressorgenes,suchaswild-typep53(wt-p53),torestorethe
pro-apoptoticfunctioninthecells9.Thep53mechanismfunctionsasacriticalsignalingpathwayincellgrowth,whichregulatesapoptosis,cell
cyclearrest,metabolismandotherprocesses10.Inpancreaticcancer,mostcellshavemutationsinp53protein,causingthelossofapoptotic
activity.Withtheintroductionofwt-p53,theapoptosiscouldberepairedandfurthertriggerscelldeathincancercells11.
Basedontheaboverationale,wehavedesignedEGFRtargetingpeptide-modifiedthiolatedgelatinnanoparticlesforwt-p53genedeliveryand
evaluateddeliveryefficiencyandtransfectioninPanc-1cells.
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Protocol
1. Preparation of Plasmid DNA Encapsulated EGFR-Targeted Gelatin Nanoparticles
1. Synthesisofthiolatedgelatin
1. Thiolatedgelatinweresynthesizedaspreviousmethod2-5,bycovalentconjugationwith2-iminothiolaneonprimaryaminogroupsof
typeBgelatin.1gramofgelatinwasdissolvedin100mldeionizedwaterandincubatedwith20mg2-iminothiolanehydrochlorideat
roomtemperaturefor15hours.
2. Unreactedreagentwasremovedbydialysisagainst5mMHClsolution,followedby1mMHClsolutionfor3hourseach.Purified
thiolatedgelatinwasfreezedriedandstoredat4°Cforfurtheruse.
2. PreparationofDNA-containingnanoparticles
1. 200mgthiolatedgelatinwasdissolvedinwaterandpHofsolutionwasadjustedto7byadditionof0.2MNaOHsolution.1mgDNA
wasaddedandgentlymixedwithgelatinsolution.
2. Chilledethanolwasaddedslowlyintothemixturewhilestirringsolutionathighspeed.Nanoparticleswereformedwhensolvent
compositionchangedto75%hydro-alcoholicsolution.
3. Nanoparticleswerefurthercrosslinkedbyslowadditionof0.1ml8%(v/v)glyoxalsolution.Unreactedreagentswerequenchedwith0.5
ml0.2Mglycinesolution.
4. Nanoparticleswereultra-centrifugedat16,000rpmfor30minutes.Pelletswerewashedwithdeionizedwatertwiceandpurified
nanoparticleswerefreeze-driedandstoredat4°C.
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3. Surfacemodificationofnanoparticles
1. Nanoparticlesweresuspendedin0.1Mphosphatebuffer(pH7.4)withconcentrationof10mg/mlandincubatedwith2timesweightof
methoxy-PEG-succinimidylcarboxymethylester(mPEG-SCM,MW2,000Da)ormaleimide-PEG-succinimidylcarboxymethylester
(MAL-PEG-SCM,MW2,000Da)for2hoursatroomtemperaturewithslowstirring.
2. PEGylatednanoparticleswerecollectedwithultra-centrifugationat16,000rpmfor30minutes.Pelletswerewashedwithdeionized
watertwiceandpurifiednanoparticleswerefreeze-driedandstoredat4°C.
3. MAL-PEG-SCMmodifiednanoparticlesweresuspendedin0.1Mphosphatebuffer(pH6.5)withconcentrationof10mg/mland
incubatedwith10%weightofEGFRspecificpeptide(Y-H-W-Y-G-Y-T-P-Q-N-V-I-G-G-G-G-C)for6hoursatroomtemperaturewithslow
stirring.
4. Peptidemodifiednanoparticleswerecollectedwithultra-centrifugationat16,000rpmfor30minutes.Pelletswerewashedwith
deionizedwatertwiceandpurifiednanoparticleswerefreeze-driedandstoredat4°C.
2. Characterization of EGFR-Targeted Nanoparticles
1. Particlesizeandzetapotentialmeasurement

Nanoparticlesweresuspendedinwaterwithconcentrationof1mg/ml.SuspensionwasanalyzedusingZetasizerNano(MalvernInc).Particle
sizeanalysiswascarriedoutatascatteringangleof90degreesat25°C.Zetapotentialwasmeasuredatdefaultparametersofdielectric
constant,refractiveindexandviscosityofwaterat25°C.
2. ScanningElectronMicroscopy

Lyophilizednanoparticlesweremountedonaluminumsamplemountandsputter-coatedwithpalladiumtoenhanceconductivityandminimize
buildupofcharges.SampleswereobservedforsurfacemorphologyunderaHitachi4800fieldemissionscanningelectronmicroscopeat3
kV.
3. Electronspectroscopeforchemicalanalysis(ESCA)

Freeze-driedformulationofcontrol,PEGylatedandpeptidemodifiednanoparticleswereanalyzedbyESCA.ItwasperformedattheNational
ESCAandSurfaceAnalysisCenterforBiomedicalProblems(NESAC/BIO),UniversityofWashington(Seattle,WA).
1. Sampleswereplacedinultrahighvacuumandexposedtolow-energyx-raybeam,whichinducedanemissionofsecondary
photoelectronsfromthesurface.
2. Byplottingnumberofdetectedelectronsasafunctionofbindingenergy,observedspectrumpeakswereassignedtoeachchemical
components.
3. High-resolutionanalysisofC1sspectrawasperformedtodetermineexactchemicalcompositionfromhydrocarbon(C-CorC-Hat
285mV),ether(C-O)at286.4mV),andcarbonyl(C=Oat288.1mV),andrelativecompositionofeachfunctionalitywasdeterminedby
areaundercurve.
4. Stabilityofencapsulatedplasmid

ThestabilityoftheencapsulatedplasmidDNAwasconfirmedbyrunningtheextractedDNAonpre-castgels.Thenanoparticleswere
digestedwith0.2mg/mlproteasecontainingPBS(30minat37°C)and0.2U/mlDNAse(10minatroomtemperature)separately,
simultaneouslyorsequentially.Sampleswerethenloadedonto1.2%agarosegel(GP)(E-Gel,Invitrogen,CA)ataconcentrationof
100ng/wellina18μLvolumeperwell.Nakedplasmidwasloadedascontrolandgelwasrunat75Vfor30minutes.KodakDigitalX-ray
Specimen(DXS)SystemwasusedtovisualizebandswithUVtransluminescence.
5. Determinationofplasmidloading

Plasmidencapsulatednanoparticlesweresuspendedat1mg/mlanddigestedwith0.2mg/mlproteaseat37°Cfor30minutes.Solutionwas
centrifugedat13,000rpmfor10minutesandsupernatantwascollectedandtestedforplasmidconcentrationwithPicogreenassay
(Invitrogen).Encapsulationratiowascalculatedbydividingtheencapsulatedplasmidconcentrationwiththeinitialloading0.5%(w/w).
3. In Vitro Transfection Studies in Panc-1 Pancreatic Cancer Cells
1. Cellcultureconditions

Panc-1andCapan-1pancreaticadenocarcinomacelllines,SKOV3ovarianadenocarcinomacelllineandNIH-3T3murinefibroblastcellline
wereobtainedfromATCC.Panc-1andNIH-3T3weregrownwithDMEMsuppliedwithL-glutamine,Pen-strepand10%fetalbovineserumat
37°Cand5%CO2,whileCapan-1requiredDMEMsupplied20%fetalbovineserum.SKOV3wasgrowninRPMI-1640suppliedwith10%
fetalbovineserum.
2. WesternblotanalysisforEGFRexpression
1. Celllysateswerecollectedfrom2millioncellsandanalyzedfortotalproteinconcentrationusingBCAassay(Pierce).NIH-3T3was
usedasnegativecontrolandSKOV3wasusedaspositivecontrolforEGFRexpression.
2. 10μgoftotalproteinextractwasrunonpre-castsodiumdodecylsulfate-polyacrylamidegelelectrophoresis(SDS-PAGE)systemat
135Vfor90minutes.
3. Subsequently,gelwastransferredontoPVDFmembranebyiBlotDryBlottingSystem(Invitrogen).
4. Membranewasblockedwith5%non-fatmilkinTween-containingTrisbuffersaline(TBS-t)for1houratroomtemperature.
5. Membranewascutandincubatedwith1:1,000dilutionofprimaryrabbitbeta-actinantibodiesand1:1,000dilutionofprimaryrabbit
EGFRantibodiesseparatelyovernightat4°C.
6. MembranewasthenwashedtwicewithTBS-tandincubatedwith1:2,000dilutionsofsecondaryanti-rabbithorse-radish
peroxidase-conjugatedIgGinTBS-tfor1houratroomtemperature.
7. AfterrinsingexcessantibodieswithTBS-tandwater,4mlECLsubstrate(Pierce,Rockford,IL,USA)wasaddedandincubatedwith
membranesfor5minutes.
8. ChemiluminescentbandswerethenvisualizedusingKodakDigitalX-raySpecimen(DXS)System.
3. Cellviabilitystudieswithdifferentformulations
1. Panc-1cellsweregrownin96-wellplatesat10,000cellsperwellin200μLofsupplementedDMEMovernight.
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2. Growthmediumwasreplacedwithserumfreemediacontainingdifferentconcentrationsofnanoparticleswith0,0.5,1,2,4,6mg/ml.
1mg/mlPEI,aknowncytotoxiccationicpolymer,wasusedaspositivecontrol.
3. Cellsweretreatedwith200μLnanoparticlesfor6hoursandthenreplacedwith20μLMTSreagentand100μLcompleteculturemedia.
4. Afterpostincubationfor3hoursat37°Cin5%CO2,theabsorbanceofformazanproductwasmeasuredat490nmwithBiotek
SynergyHTplatereader(Winooski,VT).
5. Thepercentviabilityofcellswasexpressedastheratioofabsorbanceofpolymertreatedcellsrelativetonegativecontrol(0mg/ml)
multipliedby100andplottedasfunctionofpolymerconcentrations.
4. Celltraffickingstudies
1. RhodamineBisothiocyanate(RBITC)wasusedtoconjugateonthiolatedgelatinbyreactionwithaminegroup.Afterdialysisand
lyophilization,RBITClabeledthiolatedgelatinwasusedfornanoparticlespreparation.
2. Beforedesolvation,25μLPicoGreenwasmixedwith1mgplasmidfor1minuteandlabeledplasmidswereaddedtogelatinsolution.
Differentformulationsweremadefollowingthepreviousmethod.
3. Panc-1cellsweregrownin6-wellplatescontainingglasscover-slipswith200,000cellsperwell.Afterovernightgrowth,2mloflabeled
nanoparticlesweretreatedintoeachwellwithconcentrationof1mg/mlinserumfreemedium.
4. Afterdifferenttimepoints,from15minutesto6hours,mediumwasreplacedwithculturemediumcontaining1μg/mlofHoest33342
(Invitrogen)for15minutesincubationatroomtemperature.2mlof4%paraformaldehydesolutionwasreplacedintoeachwelltofix
cells.CellswerethenwashedwithPBStwice.
5. Coversilpsweremountedontoglassslides.Laserscanningconfocalfluorescencemicroscopywasusedtotakeimagesoffixedcells.
5. QualitativedeterminationoftransfectionefficiencybyfluorescencemicroscopywithpEGFP-N1encapsulatednanoparticles
1. pEGFP-N1plasmidswereencapsulatedintonanoparticlesandsuspendedinserumfreemediawithconcentrationequivalentto10μg
plasmidspermlforfurthertreatments.
2. Panc-1cellsweregrownovernightin6-wellplatescontainingglasscover-slipswith200,000cellsperwell.2mlofpEGFP-N1plasmids
encapsulatednanoparticlesweretreatedintoeachwell.20μgofplasmidsweremixedwith20μlLipofectin,cationiclipidtransfection
reagent,anditwasusedaspositivecontrol,whileuntreatedcellswereusedasnegativecontrol.
3. Cellswereincubatedwithdifferentformulationsfor6hours.
4. Mediumwasreplacedwithculturemediumandcellswerepost-transfectedfor24,48,72and96hours.
5. Afterpost-transfection,mediumwasreplacedwithculturemediumcontaining1μg/mlofHoest33342(Invitrogen)andincubatedwith
cellsfor15minutesatroomtemperature.
6. CoverslipsweremountedontoglassslidesandexpressionofGFPinthecellswasobservedbyfluorescencemicroscope.Differential
interferencecontrast(DIC)andfluorescenceimagesacquiredusingOlympusBX61microscopeanddigitalimageswereprocessed
withImageJsoftware.
6. QuantitativedeterminationoftransfectionefficiencybyELISAwithpEGFP-N1encapsulatednanoparticles
1. pEGFP-N1plasmidswereencapsulatedintonanoparticlesandsuspendedinserumfreemediawithconcentrationequivalentto10μg
plasmidspermlforfurthertreatments.
2. Panc-1cellsweregrownovernightin6-wellplateswith200,000cellsperwell.2mlofpEGFP-N1plasmidsencapsulatednanoparticles
weretreatedintoeachwell.20μgofplasmidsweremixedwith20μlLipofectin,cationiclipidtransfectionreagent,whichwasusedas
positivecontrolanduntreatedcellswereusedasnegativecontrol.
3. Cellswereincubatedwithdifferentformulationsfor6hours.
4. Mediumwasreplacedwithculturemediumandcellswerepost-incubatedfor24,48,72and96hours.
5. Afterpost-transfection,celllysateswerecollectedfromeachwellandanalyzedfortotalproteinconcentrationusingBCAassay
(Pierce).
6. wellplatewascoatedwith100μlof1:1000dilutionsofmonoclonalanti-GFPantibodiesineachwell.After2hoursincubation,platewas
washedwithPBS-0.5%(w/v)Tween-80for4times.
7. 300μlTBSblockingbufferswereaddedintoeachwellandincubatedfor2hours.ThenplatewasthenwashedwithPBS-0.5%(w/v)
Tween-80for4times.
8. 30μgofproteinsofeachgroupwereaddedintotheplateandincubatedovernightat4°.ThenplatewasthenwashedwithPBS-0.5%
(w/v)Tween-80for4times.
9. 100μlof1:2400dilutionsofsecondaryanti-GFPantibodiesrelativetoalkalinephosphatasewereaddedtoeachwellandincubatedfor
1hour.ThenplatewasthenwashedwithPBS-0.5%(w/v)Tween-80for4times.
10. 100μlalkalinephosphatasesubstrateswereaddedtoeachwellandincubatedfor30minutesto1hour.Platewasmeasuredwith
BioTekSynergyHTplatereaderforabsorbanceat405nm.
11. ExpressedGFPconcentrationwasreportedasnanogramspermilligramsoftotalprotein.
7. QualitativedeterminationoftransfectionefficiencybyRT-PCRwithwt-p53plasmidencapsulatednanoparticles
1. Wt-p53plasmidswereencapsulatedintonanoparticlesandsuspendedinserumfreemediawithconcentrationequivalentto10μg
plasmidpermlforfurthertreatments.
2. Panc-1cellsweregrownovernightin6-wellplateswith200,000cellsperwell.2mlofwt-p53plasmidsencapsulatednanoparticleswere
treatedintoeachwell.20μgofplasmidsweremixedwith20μlLipofectin,cationiclipidtransfectionreagent,whichwasusedaspositive
controlanduntreatedcellswereusedasnegativecontrol.
3. Cellswereincubatedwithdifferentformulationsfor6hours.
4. Mediumwasreplacedwithculturemediumandcellswerepost-incubatedfor48hours.
5. mRNAwereextractedfromeachwellbyusingHighPureRNAisolationkit(Roche,Indianapolis,IN)andmeasuredwithNanodrop
2000(ThermoScientific,Wilminton,DE).
6. RT-PCRwasdonebyusingQIAGENOne-stepRT-PCRkit(QIAGEN,Valencia,CA).Primersforp53,Bax,Bcl-2,Beta-actin,DR5,
Apaf-1,PUMA,SurvivinweresynthesizedbyEurofinsMWGOperon(Huntsville,AL).
7. PCRproductswereevaluatedwithgelelectrophoresisandthepixelsofcDNAbandswereanalyzedwithImageJsoftware.
8. Quantitativedeterminationoftherapeuticefficiencywithwt-p53palsmidencapsulatednanoparticles
1. Wt-p53plasmidwasencapsulatedintonanoparticlesandsuspendedinserumfreemediawithconcentrationequivalentto10μg
plasmidspermlforfurthertreatment.
2. Panc-1cellsweregrownovernightin6-wellplateswith200,000cellsperwell.2mlofwt-p53plasmidsencapsulatednanoparticleswere
treatedintoeachwell.20μgofplasmidsweremixedwith20μlLipofectin,cationiclipidtransfectionreagent,whichwasusedaspositive
controlanduntreatedcellswereusedasnegativecontrol.
3. Cellswereincubatedwithdifferentformulationsfor6hours.
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4. Mediumwasreplacedwithculturemediumandcellswerepost-incubatedfor24,48,72and96hours.
5. ChromatinCondensation/MembranePermeability/DeadCellApoptosisKit(Invitrogen,Carlsbad,CA)wasusedtolabelapoptoticcells,
necroticcellsandlivecellswithdifferentdyes.
6. iCysResearchImagingCytometerfromCompuCyte(Westwood,MA)wasusedtoanalyzeandcompareapoptosislevelsafter
treatment.Basedonthefluorescencemicroscopicimages,theintensitiesofallcolorswasrecordedandplottedversuscountsandthe
percentagesfordifferentpopulationswerecalculated.
7. Comparedtothenegativecontrol,whichmeanstherewasnotreatmentforthecells,theapoptoticcellsfoldchangeswerecalculated
outandlistedinthegraph.
8. 3.8.8Apo-ONEHomogeneousCaspase-3/7Assaykit(Promega,Madison,WI)wasusedtoexaminethepro-apoptoticactivityafter
transfectionofwt-p53plasmid.1mg/mlPEIwasusedasanegativecontroltoeliminatealltheapoptoticactivity.Afterpost-transfection,
cellsweretreatedwithrhodamine110,bis-(N-CBZL-aspartyl-L-glutamyl-L-valyl-L-asparticacidamide;Z-DEVD-R110),whichisa
substrateofcaspase3/7,forupto18hours.
9. PlatewasmeasuredwithBioTekSynergyHTplatereaderforfluorescenceat490/520nm.Basedontheintensityofgreen
fluorescence,pro-apoptoticactivitycouldbeevaluated.
4. Representative Results
1.SynthesisandChatacterizationofEGFRTargetedNanoparticles
EGFRtargetingpeptide-modifiednanoparticlesweresynthesizedastheschemeshowedinfigure1.Thenanoparticlespreparedbydesolvation
werecharacterizedforparticlesizeandzetapotential.Theaveragesizeandsurfacechargeoftheparticlespreparedfromthiolatedgelatinswith
differentdegreesofthiolationarelistedinTable1.Themeanparticlediametersofdifferentnanoparticleswerebetween150-250nm.Thiolated
nanoparticleshavesmallersizecomparedtogelatinnanoparticles,mightduetothedisulfidebridgeformationinsideparticles.Withdifferent
surfacemodifications,sizesofnanoparticleshaveincreased.Thezetapotentialsofdifferentformulationswerearound-20mV.WithSEM
analysis,thesizes,surfacemorphologyandsphericalshapeofnanoparticleswereobservedandcorrespondingtoZetasizerresult.DNAloading
efficienciesingelatinnanoparticlesandthiolatedgelatinnanoparticleswerehigherthan95%(Table1).

Figure1.ChemicalReactionScheme,illustratingsurfacemodificationofthiolatedgelatinnanoparticleswithepidermalgrowthfactorreceptor
(EGFR)bindingpeptidethroughapoly(ethyleneglycol)(PEG)spacer.Pleaseclickheretoseealargerversionofthisfigure.
CharacterizationofNanoparticles
Formulation NanoparticleDiameter(nm) ZetaPotential(mV) PlasmidDNALoadingEfficiency(%)
GelNP 151.4±23.5 -17.1±5.23 95.6±2.2
SH-GelNP 132.6±17.9 -24.6±5.16 97.0±3.8
SH-Gel-PEG 179.0±30.9 -22.3±9.50 95.8±6.5
SH-GelPEGPeptide 230.8±41.5 -18.1±4.02 94.8±5.1
Table1.Particlesize,surfacecharge,andplasmidDNAencapsulationefficienciesofcontrolandEGFR-targetedgelatinandthiolatedgelatin
nanoparticles.
High-resolutionC1Sscansofelectronspectroscopyforchemicalanalysis(ESCA)wasusedtoanalyzesurfacecomponentofthiolatedgelatin
(SH-GelNP),PEG-modifiedthiolatedgelatin(SH-GelPEG)andEGFRtargetingpeptide-modifiedthiolatedgelatinnanoparticles(SH-GelPEG
Peptide).TheresultsinTable2showedpeakintensitiesoftheC-H(hydrocarbon),C-O(ether),andC=O(carbonyl)groupsat285.0,286.3,and
288.1eV,respectively.TheetherC-OsignalhasincreasedafterPEGmodificationanddecreasedafterpeptideconjugation.Whilenitrogen
compositionhasdecreasedafterPEGmodificationandincreasedafterpeptidemodification,whichconfirmedthepresenceofEGFR-targeting
peptideonthenanoparticles.ESCAanalysishasfurtherconfirmedPEGandpeptidesurfacemodification.

ElectronSpectroscopyforChemicalAnalysisofNanoparticlesSurfacecomposition
Formulation C1s(%) O1s(%) N1s(%)
SH-GelNP 59.3±0.8 22.9±0.5 12.9±0.1
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SH-Gel-PEG 58.2±0.6 28.0±1.2 9.5±0.7
SH-GelPEGPeptide 56.7±0.8 25.9±0.7 12.3±0.6
Formulation C-C(%) C-O,N(%) C=O(%)
SH-GelNP 51.5 26.6 21.9
SH-Gel-PEG 17.1 63.1 19.8
SH-GelPEGPeptide 33.1 42.8 24.1
Table2.C1Shigh-resolutionscansofelectronspectroscopyforchemicalanalysis(ESCA)
Inordertoexaminethestabilityofencapsulatedplasmid,nanoparticlesweretreatedwithproteaseorDNAseseparately,simuntaneouslyor
sequentially.Afterelectrophoresis,theresultsinFigure2haveshownthatplasmidDNAencapsulatedinallthenanoparticlesareprotectedby
nanoparticlesandstable,comparabletonakedplasmidDNA.Thesestudiedhaveshownthatallthesenanoparticlescouldencapsulateand
preservetheplasmidstructureafterencapsulation.

Figure2.StabilityofplasmidDNAencapsulatedinthiolatedgelatin,PEG-modifiedthiolatedgelatin,andEGFRpeptide-modifiedthiolatedgelatin
nanoparticlesbyagarosegelelectrophoresis.Thenanoparticlesweretreatedwith0.2mg/mlofproteasetoproveplasmidDNAencapsulation
withinthenanoparticlematrix
2.BaselineEGFRExpressioninPancreaticCancerCells
Twohumanpancreaticadenocarcinomacelllines(Panc-1andCapan-1)wereanalyzedbywesternblotforEGFRexpression.Humanovarian
adenocarcinoma(SKOV3)andmurinefibroblast((NIH-3T3)cellswerechosenaspositiveandnegativecontrols,respectively.Beta-actinwas
analyzedasproteinloadingcontrol.Panc-1cellshaveshownhigherEGFRexpressioncomparedtoCapan-1andthiscelllinewasthenusedfor
thefollowinginvitrostudies
3.CytotoxicityofControlandSurface-ModifiedThiolatedGelatinNanoparticles
Inordertoevaluatethecellularinteractionofnanoparticles,cytotoxicityassayswerecarriedoutaftertreatmentwithnanoparticles.Basedonthe
resultsinFigure3,boththecontrolandthesurface-modifiednanoparticleswererelativelysafeandbiocompatibleinPanc-1cellsevenathigh
concentrations,withcomparisontoPEI.Thefollowingstudieswerecarriedoutwith1mg/mlnanoparticles.

Figure3.PercentcellviabilityasafunctionofnanoparticleformulationconcentrationsinPanc-1cellsasevaluatedbytetrazoliumdye(MTS)
assay
4.ReceptorMediatedCellUptakeinPanc-1Cells
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ToconfirmsurfaceaccessibilityofEGFR-targetingpeptideandreceptor-mediatedendocytoticuptakeofnanoparticles,asystemwasdesignedby
labelingeachcomponentwithdifferentfluorescenceforvisualizationofnanoparticlesuptakeandtraffickingincells.Withthislabelingsystem,
plasmidDNA,nanoparticlesandcellnucleuscouldbeidentified.Laserscanningconfocalfluorescencemicroscopywasusedtotakeimagesat
differenttimepoints,from15minutesto6hours.Bycomparingtheimagesofdifferentformulations,peptideconjugatedgelatinnanoparticles
showedthefastuptakeandplasmidreleasewithin30minutes.ThisresultfurtherprovedthatEGFRpeptide-conjugatednanoparticlesunderwent
facilitatedendocytosiswithquickinteractionbetweentheEGFRspecificpeptideandEGFRreceptorsoncellsurface,whichwasmuchfaster,
comparedtoothernanoparticles,whichunderwentnon-specificendocytosis.
CellTraffickingStudy

Figure4.ConfocalfluorescencemicroscopyanalysisofDNA-encapsulatednanoparticleuptakeandtraffickinginPanc-1cells.
(red=rhodamine-labelednanoparticles,green=PicoGreen-labeledplasmidDNA,andblue=DAPI-labelednucleus).Thelaserpowerwas7times
lessinlastfourfiguresoflowerpanel.
5.QualitativeandQuantitativeInVitroTransfectionwithEnhancedGreenFluorescentProtein
ELISAinFigure5andfluorescencemicroscopicanalysisinFigure6wereusedtomeasurequalitativeandquantitativeGFPtranfectionefficiency
inPanc-1cellsuponadministrationofunmodified,PEG-modifiedandEGFRpeptide-modifiedthiolatedgelatinnanoparticles.Plasmidsdelivered
byEGFR-targetednanoparticlesresultedinthehighestlevelofGFPexpressionafter48hoursrelativetoothercontrols,including
Lipofectin-complexedDNA.

Figure5.GFPexpressionanalyzedbyELISAplottedasafunctionoftimepost-administrationofplasmidDNAincontrolandEGFR-targeted
nanoparticles.
FluoresenceMicroscopicAnalysisforGFPtransfection
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Figure6.QualitativeanalysisofgreenfluorescentproteinexpressioninPanc-1cellsbyepifluoresencemicroscopyafter24,48,72and96hours
post-transfectionwithEGFP-N1.Lipofectin-DNAcomplexwasusedasapositivecontrol.
6.InVitroTranfectionwithWild-Typep53PlasmidinPanc-1Cells
Wild-typep53plasmidspORF-hp53,withEF-1α/HTLVhybridpromoterwereextractedfromE.coliandencapsulatedintonanoparticlestostudy
theapoptotictherapeuticeffect.Panc-1cellsweretreatedwithparticlesfor6hoursandpost-transfectedforadditional24,48,72,and96hours.
Sincep53couldinduceapoptosisincellsandinordertoaccomplishthisfunction,manydownstreamtranscriptionfactorswouldbeinvolvedand
directlyregulatedbyexpressionofwt-p53.Amongthem,Bax,caspase-3,caspase-9,DR5,PUMAandApaf-1wouldbeup-regulatedby
expressionofp53andwhileBcl-2,survivinwouldbedown-regulated.Inordertoexaminethelevelsofthesetranscriptionfactors,mRNAwas
extractedfromPanc-1cellsafter48hourspost-transfectionandusedforRT-PCR.Theproductswereevaluatedwithgelelectrophoresisand
bandswereanalyzedwithImageJ.BasedontheresultsshowedinFigure7,survivindecreasedsignificantlywiththetreatmentofEGFRtargeted
thiolatedgelatinnanoparticlescomparedtoothertreatments,noobviouschangewasseeninBcl-2,Baxandexpressionofcaspase-3,
caspase-9,DR5,PUMAandApaf-1increasedwithtargetednanoparticlestreatment.
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Figure7.ThemRNAlevelsofdownstreamfactorsofwt-p53expressionwerecomparedbyRT-PCRafter48hourspost-transfection.

Afterwt-p53transfection,ChromatinCondensation/MembranePermeability/DeadCellApoptosiskitwasusedtodifferentiateapoptoticcells,
necroticcellsandlivecellswithdifferentdyes.iCysResearchImagingCytometerfromCompuCyte(Westwood,MA)wasusedtoanalyzeand
compareapoptosislevelsaftertreatment.Comparedtothenegativecontrol,apoptoticcellsfoldchangeswerecalculatedoutandlistedinFigure
8.EGFRtargetedthiolatedgelatinnanopaticleshaveshowedthehighestapoptoticcellpopulationafterpost-transfection.Analysisofcaspase3/7
activityalsoshowedthatEGFR-targetednanoparticleshadrapidinternalizationandhighestlevelofapoptoticactivityinPanc-1cells.

Figure8.Cytometricanalysisofpro-apoptoticactivityincontrolwt-p53transfectedPanc-1cellsusingiCys°ImagingCytometer
Discussion
ControlandEGFRtargetedthiolatedgelatinnanoparticleswerepreparedwithefficientDNAencapsulationandstability.Theparticlesizesofallof
thesesystemswereintherangeof150-250nmindiameter.Zetapotentialhasprovedthatthissystemisaslightlynegativesystem.WithSEM
analysis,thesizesofnanoparticleswerethesamewithZetasizerresult.ESCAanalysiscouldconfirmPEGandpeptidesurfacemodification.
WesternblotanalysisshowedthatPanc-1cellshadahighEGFRexpressionlevelsandthiscelllinewasusedfortheinvitrostudies.Boththe
controlandthesurface-modifiednanoparticleswererelativelylesscytotoxicinPanc-1cellsascomparedtoPEI.
CelltraffickingstudiesshowedrapiduptakeandplasmidreleaseofEGFR-targetednanoparticlesinPanc-1cells.Deliveryofreporterplasmids
DNAexpressingwithEGFR-targetednanoparticlesresultedinhighestlevelsofGFPexpressionrelativetoothercontrols,including
Lipofectin-complexedDNA.Withthesamesystem,transfectionwithwt-p53plasmidtriggeredthedownstreamapoptoticpathwayandinduced
rapidapoptosisinPanc-1cells.
ThesepreliminaryresultssuggestthattheEGFR-targetedthiolatedgelatinnanoparticlescanserveasasafeandefficientDNAdeliverysystem
forgenetherapyasatreatmentforpancreaticcancer.
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Disclosures
Noconflictsofinterestdeclared.
Acknowledgements
ThisstudywassupportedbytheNationalCancerInstitute'sAllianceinNanotechnologyforCancer'sCenterforCancerNanotechnology
Excellence(CCNE)grantU54-CA151881.
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