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Theranostics2015,Vol.
5,Issue4http://www.
thno.
org357TThheerraannoossttiiccss2015;5(4):357-370.
doi:10.
7150/thno.
10657ReviewOfftotheOrganelles-KillingCancerCellswithTargetedGoldNanoparticlesMohamedKodiha1,YiMengWang1,ElizaHutter2,DusicaMaysinger2,UrsulaStochaj11.
DepartmentofPhysiology,McGillUniversity,Montreal,Canada;2.
DepartmentofPharmacology&Therapeutics,McGillUniversity,Montreal,Canada.
Correspondingauthor:UrsulaStochaj,Ph.
D.
DepartmentofPhysiology,McGillUniversity,3655PromenadeSirWilliamOsler,Montreal,QC,Canada,H3G1Y6.
Phone:514-398-2949Fax:514-398-7452E-mail:ursula.
stochaj@mcgill.
ca.
IvyspringInternationalPublisher.
Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsLicense(http://creativecommons.
org/licenses/by-nc-nd/3.
0/).
Reproductionispermittedforpersonal,noncommercialuse,providedthatthearticleisinwhole,unmodified,andproperlycited.
Received:2014.
09.
27;Accepted:2014.
12.
16;Published:2015.
01.
21AbstractGoldnanoparticles(AuNPs)areexcellenttoolsforcancercellimagingandbasicresearch.
However,theyhaveyettoreachtheirfullpotentialintheclinic.
Atpresent,weareonlybeginningtounderstandthemolecularmechanismsthatunderliethebiologicaleffectsofAuNPs,includingthestructuralandfunctionalchangesofcancercells.
Thisknowledgeiscriticalfortwoaspectsofnanomedicine.
First,itwilldefinetheAuNP-inducedeventsatthesubcellularandmolecularlevel,therebypossiblyidentifyingnewtargetsforcancertreatment.
Second,itcouldprovidenewstrategiestoimproveAuNP-dependentcancerdiagnosisandtreatment.
OurreviewsummarizestheimpactofAuNPsonselectedsubcellularorganellesthatarerelevanttocancertherapy.
Wefocusonthenucleus,itssubcompartments,andmitochondria,becausetheyareintimatelylinkedtocancercellsurvival,growth,proliferationanddeath.
Whilenon-targetedAuNPscandamagetumorcells,concentratingAuNPsinparticularsubcellularlocationswilllikelyimprovetumorcellkilling.
Thus,itwillincreasecancercelldamagebyphotothermalablation,mechanicalinjuryorlocalizeddrugdelivery.
Thisconceptispromising,butAuNPshavetoovercomemultiplehurdlestoperformthesetasks.
AuNPsize,morphologyandsurfacemodifi-cationarecriticalparametersfortheirdeliverytoorganelles.
Recentstrategiesexploredallofthesevariables,andsurfacefunctionalizationhasbecomecrucialtoconcentrateAuNPsinsub-cellularcompartments.
Here,wehighlighttheuseofAuNPstodamagecancercellsandtheirorganelles.
WediscusscurrentlimitationsofAuNP-basedcancerresearchandconcludewithfuturedirectionsforAuNP-dependentcancertreatment.
Keywords:Goldnanoparticles,AuNPs,cancercellimagingThisreviewprovidesanupdateonthethera-peuticpotentialofgoldnanoparticles(AuNPs)foroncology.
Tothisend,weintroduceAuNPsasthera-peutictools,summarizethecurrentstrategiesthattargetAuNPstospecificcellcompartmentsanddis-cusshowthistargetingimpactscancercellkilling.
Forsubcellulartargeting,ourfocusisonnucleiandmi-tochondria,sincebothorganellesareintimatelylinkedtocancercellsurvival,growthandprolifera-tionandthereforeprimarytargetsforanti-canceragents[1,2].
Weconcludebyhighlightingunsolvedquestionsandpotentialroadblocksinthefield.
1.
IntroductionNanotechnologyisinthespotlightoftherapeuticinnovation[3],andAuNPsareparticularlypromisingtoolstoimprovecancertreatment[4].
Duetotheiruniqueopticalproperties,non-toxicnature,relativelysimplepreparationandfunctionalization,AuNPsareexcellentcandidatesformanybiologicalapplications,IvyspringInternationalPublisherTheranostics2015,Vol.
5,Issue4http://www.
thno.
org358suchasimaging,drugdeliveryandphotothermaltherapy.
Theseapplicationscommonlytakead-vantageoftheparticles'stronglightscattering,in-tenseabsorption,andelectromagneticfieldenhance-mentthatresultfromlocalizedsurfaceplasmonres-onance[5,6].
AuNPscanbeproducedinlargequantitieswithdefinedshapesandsizes.
Themostcommonap-proachessynthesizeAuNPsinsituthroughchemicalreductionofgoldsaltsandseed-mediatedgrowth[7],whichenlargestheparticlesstepbystep.
ThismethodisidealtocontrolAuNPsizeandshape[8-10]andusedtoproducelargespherical,semi-spherical,rod-like,branchedorotherparticleshapes[7].
AuNPsurfacesareamenabletocovalentandnon-covalentsurfacemodifications;thispropertyiscrucialforcel-lularandsubcellulartargeting.
Asthephysi-co-chemicalcharacterizationofAuNPsandtheirde-tectionhavebeenreviewedbyothers[11-15],itwillnotbediscussedhere.
ThedevelopmentofAuNP-basedstrategiesfortheeradicationofcancercellsisimportant,becauseeffectivetherapiesarefrequentlynotavailableforrapidlyprogressingcancers[16].
Sofar,manyofthestudiesonAuNPssuggestthatcancercellsareespe-ciallyvulnerabletotheseparticles.
Thus,AuNP-basedtreatmentcandestroycancercells,withminimalin-jurytohealthycells[17].
ThetherapeuticvalueofAuNPsisbasedon(i)theirdistinctivephysicalpropertiesand(ii)theirabil-itytointeractwithtumorsanddamagecancercells.
Thus,theenhancedpermeabilityandretention(EPR)characteristicsofmany,butnotall,tumorsfacilitateAuNPinfiltrationintothetumor[18].
Duetothispassivetargeting,AuNPs(~6-200nm)accessthetu-mortissue,wheretheyaccumulateintheextracellularmatrixbeforeenteringthecells[19].
Followingtheirassociationwithtumorcells,AuNPspromoteuniquewaysofkilling(Fig.
1).
Theycandestroycancercellsbyphotothermalablation,asexemplifiedbyAuro-Shell[20,21],throughmechanicaldamage,orasdrugdeliverysystemsforanticanceragents,suchastumornecrosisfactor[21,22]ordoxorubicin[23,24].
WhatarethebenefitsofsubcellularAuNPtargetingWhileAuNPsarerelevantfordifferentclinicalapplications,furtherimprovementsofAuNP-basedstrategiesareexpectedtooptimizethetherapeuticoutcomes.
OnesuchimprovementisbasedontheconceptthatAuNPtargetingtospecificorganellesmaximizestheimpactontumorcells.
Tothisend,AuNPsarebeingdevelopedthataccumulateinsub-cellularcompartmentswheretheydestroyintrinsiccancercellfunctionsthatareessentialfortumorsur-vival.
Onceintheirproperintracellularlocation,AuNPscanenhancecancercelldestructionbydif-ferentmeans.
Thisincludestheconfineddeliveryofanti-canceragents[25],localizedsubcellularmechan-icaldamage,andimprovedefficiencyofphotothermalablationduetohighlocalAuNPconcentrations[26,27].
SuchcontrolledAuNPactionwillnotonlyin-creasecancercellkilling,butalsodiminishtoxicsideeffects,becauseitreducesthenecessaryamountsofAuNPsanddrug-load.
Candidatecompoundsfornanoparticle-dependentsubcellulardeliveryaredox-orubicin[23],platinum-baseddrugs[28]andpaclitaxel[29].
Theseanticanceragentsinterferewithnuclearandmitochondrialfunctions,respectively[30-33]andhavebeenusedtofunctionalizeAuNPs[23,34-37].
Asidefromdrugs,AuNPscanalsodeliveroligonucleotidestoaltergeneexpressionorsplicing([38]andreferencestherein).
Figure1.
ImpactofAuNPsoncancercells.
Size,morphology,functionalgroupsontheAuNPsurfaceandthecelltypedeterminethesubcellulardistributionofAuNPs.
AuNPscancausetumorcelldeathbyphotothermalablation,me-chanicaldamage,andincreaseinthelocalizeddrugconcentration.
Theseeventscanbecombinedtoenhancetheirkillingefficiency.
WhatarethebottlenecksforAuNPtargetingtospecificsubcellularcompartmentsOnceaccumulatedintumortissue,AuNPshavetoovercomemultipleobstaclesbeforetheycancon-centrateinthedesiredcellcompartment:(i)cellsur-facebinding,(ii)cellularuptake,(iii)escapefromly-sosomes/endosomes,and(iv)associationwithapar-ticularsubcellularlocation,suchasnucleiormito-chondria(Fig.
2;[39]).
ThefirstthreestepsaregeneralfeaturesthatregulatetheintracellulardestinationofallAuNPs.
Thesestepshavebeenreviewedexten-sively[13,39-42];wewillonlybrieflysummarizethemhereandthenprovideamoredetaileddiscus-sionofAuNPtargetingtonuclei,mitochondriaandtheER.
Theranostics2015,Vol.
5,Issue4http://www.
thno.
org359Figure2.
ObstaclesAuNPshavetoovercomeforsuccessfultargetingtointracellularorganellesorcompartments.
OnceAuNPsareintheextracellularmatrixofthetumor(ECM,barrier1),theyhavetobindtothecancercellsurface.
Cellularuptakerequirestranslocationacrosstheplasmamembrane(barrier2),byendocytosisorothermechanisms.
Insidethecell,AuNPshavetoescapefromendosomesorlysosomes(barrier3)tosubsequentlyassociatewiththedesiredorganelleorcellcompartment(barrier4).
Possiblefinaldestinationsarethenucleus(blue)ormitochondria(yellow).
Bindingtothecancercellsurface,internaliza-tionandescapefromendosomes/lysosomesAuNPuptake,subcellulardistributionandtox-icityaredeterminedbyparticlesize,morphologyandsurfacemodification.
AlthoughAuNPsofdifferentshapes(spherical,shells,rods,diamonds)orsizes(1-100nm)accumulateinvariouscancercells,theiruptakekineticsandtoxicitymayvaryprofoundly(Fig.
1,reviewedby[13]).
Besidesshapeandsize,AuNP-basedbio-nanointeractionsarefurthermodu-latedbytheirfunctionalization[11,43,44].
Ingeneral,positivechargesontheAuNPsurfacestimulatecel-lularuptake,possiblyduetoelectrostaticinteractionswiththecellsurface[45].
PositivechargescanalsoimproveAuNPtransporttothenucleus,becausenu-clearlocalizationsequences(NLSs)ofmanyproteinsareenrichedforbasicaminoacidresidues.
ParticleuptakenotonlydependsonAuNPproperties,butalsoreliesonthecelltype.
Whengrowninculture,cancerandnon-tumorigeniccellsdiffersignificantlyinthisrespect.
Notably,tumorcellsareoftenmorevulnerabletoAuNPs[46,47].
Nevertheless,thereisvariabilityevenamongcancercells;forexample,AuNPuptakediffersinhepatocel-lularcarcinoma(HepG2)andcervixcarcinomacells(HeLa)[48].
Despitesuchcell-typespecificdiffer-ences,nanoparticlebindingtocancercellscanbeen-hancedbyexploitingtumor-relatedchangesinplas-mamembranecomposition(AdditionalFile1:TableS1andassociatedreferences[41-91]).
Tothisend,particlesurfaceshavebeenfunctionalizedwithlig-andsthatimprovedockingatthetumorcellmem-brane.
LigandsthatpromotehighaviditybindingtocancercellsincludeEGF(epidermalgrowthfactor;associateswithEGFR)orpeptidescontainingtheRGDmotif(arginine-glycine-asparticacid;recognizedbysomeintegrinfamilymembers)[32,92-94].
Nucle-olin,transferrinorantibodiesagainstHer2andEGFR[55,95-97]canalsoenhancenanoparticlebindingtocancercells.
Onceboundtothecellsurface,AuNPsenterthecell;inmostcasesthisoccursbyanenergy-dependentprocess,forwhichendocytosisisthemainroute(re-viewedin[13,14]).
Followingcellularuptake,AuNPsinitiallylocatetoendosomesand/orlysosomes,wheretheorganellarmembraneintegritydeterminesAuNPretention(reviewedin[14]).
However,properfunctionalizationofAuNPscanstimulatetheirendo-somal/lysosomalescapeorpromoteuptakebynon-endocytoticpathways([13,14]andreferencestherein).
2.
NucleirepresentprimarytargetsforcancertherapyAsnucleimastermindessentialaspectsoftumorcellbiology,theyhavebecomeimportanttargetsincancertherapyingeneral,andAuNP-dependentin-terventionsinparticular.
Thenucleuscontrolscellgrowth,proliferationandapoptosis,andmanyan-ti-cancerdrugsobliteratethesefunctions.
Atthesametime,nuclearhomeostasisisoftenalteredincancercells,whichdisplaychangesinnuclearsize,shape,envelope,laminaandchromatinorganization,nucle-olarfunctionornucleocytoplasmictrafficking[98-104].
Forexample,theconcentrationofnucleartransportcarriersisfrequentlyincreasedintrans-formedcellsortumorsamples,andthiscorrelateswithaugmentedsignal-mediatednuclearimportandexport[105].
Ascomparedtotheirnormalcounter-parts,transformedcellsoftentransportlargerAuNPs,andAuNPnucleartranslocationismoreefficient[106-108].
Nuclearinteractionsofnon-targetedAuNPsEvenintheabsenceofsubcellulartargetingsig-nals,certainAuNPtypesassociatewiththenucleusanditssubcompartments.
Insomebutnotallcasesthisnuclearlocalizationisaccompaniedbyorganelledamage.
Assuch,AuNPs(3.
7nmaveragediameter)weremodifiedwithboth3-mercaptopropionicacidandpolyethyleneglycol(PEG)andconjugatedtoFITC[58].
AlthoughtheparticlesaccumulatedinHeLacellnucleiaftera24-hourincubationperiod,cellTheranostics2015,Vol.
5,Issue4http://www.
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org360viabilitystillamountedto85%ofthecontrolsamples.
Evenafter72hoursandwithupto10MofAuNPs,cellsurvivalwasmaintainedat70%,suggestinglowcytotoxicityinHeLacells[58].
Ontheotherhand,Au55goldclusters(1.
4nm)enteredthecellnucleuswheretheyinteractedwithDNAandelicitedtoxiceffects[109].
Comparisonofhealthyandtumorcelllinesrevealedmaximumtox-icityforthemetastaticmelanomacelllinesMV3andBLM.
TheinteractionbetweenAu55goldclustersandnuclearDNAislikelytheunderlyingcauseofthistoxicity.
OurrecentstudiesexaminedAuNPsofdifferentsizesandmorphologies.
Inbreastcancercells,nuclearmembranes,nuclearlaminaeandnucleolarfunctionswerecompromisedbysmallsphericalAuNPsorgoldnanoflowers,butnotbylargesphericalAuNPs([46],Fig.
3).
Thedamageinflictedbynon-sphericalgoldnanoflowersisparticularlyinteresting;despitetheirlargesize(40-120nm),theyenteredthenucleusanddestroyednuclearhomeostasisinbreastcancercells,butnotinnormalbreastcells.
Figure3.
(A)Goldnanoflowersandlargegoldnanospheres(red)associatewithnucleiofdifferentbreastcells,i.
e.
MCF7andnon-tumorigenichumanmammaryepithelialcells(HuMEC).
NotethatgoldnanoflowerscanbedetectedinthenuclearinteriorofMCF7cells,wheretheydisruptthenuclearlamina(green).
Scalebarsare10m.
(B)Smallgoldnanospheres(15.
6nmdiameter)andgoldnanoflowers(40-120nm),butnotlargegoldnanospheres(60nm),alterthenuclearorganizationinMCF7cells.
Inparticular,nuclearporecomplexes(NPC,red)andthenuclearlamina(LaminA,green)showseverechanges.
Arrowsmarksomeofthenucleiwithalteredmorphology;scalebaris20m.
(C)SmallgoldnanospheresandgoldnanoflowersinhibitdenovoRNAsynthesis(magenta)inthenucleolus.
Scalebaris3m.
Adaptedfrom[46]withpermission.
Theranostics2015,Vol.
5,Issue4http://www.
thno.
org361TargetingAuNPstothenucleusWhichAuNPsizefitsthenucleusTheoptimalAuNPpropertiesfornucleartargetingdependonthedesiredimpactoftheparticle.
IfAuNPsaredestinedforthenuclearinterior,thesizeofthetransportchannelofthenuclearporecomplex(NPC)hastobeconsidered.
Accordingly,sphericalparticlesof9nmorlessindiametermaycrosstheNPCbydiffusion,whereasparticlesupto39nmindiametercanbede-liveredtothenucleusiftheycarryanucleartransportsignal[110].
Thesearenotfixedvalues,asthenucleartransportmachineryisoftenalteredincancercells(seeabove).
Inthecontextofcancertherapy,threedifferentscenarioscanapplytonuclearAuNPs:(i)theyaretransportedintothenuclearinterioror(ii)clogtheNPC,or(iii)releasecytotoxicdrugsinthevicinityofnuclei.
NuclearimportofAuNPs.
Theground-breakingworkbyCarlFeldherrbuiltthefoundationforAuNPtargetingtothenucleus.
HislaboratorycoatedAuNPswithconjugatesthatcontainedserumalbuminandthenuclearlocalizationsequences(NLSs)derivedfromSV40T-antigenornucleoplasmin.
Uponinjec-tionintothecytoplasm,thesefunctionalizedAuNPstranslocatedacrossthenuclearporecomplex(NPC)andaccumulatedinsidethenucleus[111,112].
Feldheim'sgroupwentbeyondthesestudiesanddefinedthedifferentstagesofAuNPnuclearlocaliza-tioninintactculturedcells[39,59].
Thesestudiesshowedthatasidefromtheinitialbinding,cellularuptakeandreleasefromendosomes/lysosomes(seeabove),targetingtothenucleusandpassageacrossthenuclearenvelope(NE)arecriticalstepsthatlimitthedeliverytothenucleus.
WhileAuNPsdestinedforthenucleusshowcell-typespecificdifferencesincel-lularuptake,escapefromendosomes/lysosomesandnucleartargeting[113],theyalsosharecommonfea-tures.
Forexample,uptakeofNLS-modifiedAuNPsistemperature-dependentinculturedcells,consistentwithendocytosis[59,113].
Furthermore,increasingthenumberofNLS-peptidesontheparticlesurfaceenhancesAuNPnuclearlocalizationandtoxicity[59].
Variousstrategieshavebeenemployedtotargetgoldnanoparticlestothenucleus.
Sofar,thereisnoprotocolthathasbeenuniversallysuccessfulfordif-ferenttumorcells.
Therefore,wediscussspecificex-amplesforAuNPsthatwerefunctionalizedwithNLSs,cell-penetratingpeptides,peptidecombina-tions,oligonucleotidesorothermoieties(summarizedinAdditionalFile1:TableS1).
Peptidesequencesaredepictedintheone-lettercode.
ThesuccessofmultiplepeptidemodificationswasdemonstratedforAuNPsmodifiedwithSV40-NLSoranNLSderivedfromadenovirusfiberproteins;theseparticlesdidnotenterthenucleusofHepG2cells.
However,uponfurtheradditionoftheadenovirusreceptor-mediatedendocytosissequence,AuNPslocalizedtonuclei[39].
Notably,whenAuNPscarriedtheNLSandthereceptor-mediatedendocyto-sispeptideasseparateentities,nucleartargetingwasenhancedascomparedtoalongerpeptidethatcom-binesbothsequences.
Ifcombinedintoonepeptide,thetwosequenceelementsmaybelessaccessibletocellularbindingpartners.
Inotherstudies,AuNPsstabilizedwiththenon-naturalaminoacidtiopronin[94]werefunction-alizedwithGRKKRRQRRR,apeptidederivedfromtheHIV-1proteinTat[61].
Aftera1-hourtreatment,Tat-modifiedparticleswerelocatedinthenuclearinteriorofhTERT-BJ1humanfibroblasts.
Bycontrast,AuNPscarryingtioproninaloneaccumulatedincy-toplasmicvacuolesoratthemitochondrialperiphery[61].
Upona24-hourincubationperiodwithupto10MAuNPs(coresize2.
8nm),toxicitywas≤20%forbothtypesofparticles.
Toimprovecellularuptakeandnuclearaccu-mulationof30nmAuNPs,particlesweredecoratedwithpeptidesthatcontainedthesequenceCALNN.
AdditionofmixedpeptidescontainingCALNNandCALNNpluseightarginineresidues(CALNNR8)promotedAuNPaccumulationinnucleiofHeLacells[52].
Afterincubationwith0.
32nMAuNPsfor24hours,celldeathcouldreach95%.
Infurtherexperiments,16or14nmPEGylatedAuNPsweremodifiedwithdifferentCALLN-derivedpeptides.
FunctionalizationwithCALLN-fusionscontainingthecellpenetratingpeptideofTAT(CALLN-AGRKKRRQRRR)orPntn(CALLN-GRQIKIWFQNRRMKWKK)stimulatedcellentry,whereasanNLS(CALLN-GGFSTSLRARKA)wasaddedfornucleartargeting[66,114].
Inthissce-nario,AuNPsmodifiedsimultaneouslywithNLS,TATandPntn-containingpeptidesledtohigherin-tracellulargoldcontentwhencomparedtoAuNPscarryingonepeptidespeciesonly.
Furthermore,sim-ultaneouscoatingwithNLS,TATandPntnpromotedAuNPnuclearlocalization.
Surprisingly,ifAuNPscarryapositivesurfacecharge,nucleartargetingcanevenbeaccomplishedwithpeptidesthathavenosequencesimilaritytoabasicNLS([64],Fig.
4).
Nucleolinisamultifunctionalproteinthatre-sidesinthenucleolusandothercellcompartments,suchastheplasmamembrane.
Forsomecancercellsnucleolinabundanceishighintheplasmamembrane,wheretheproteincanserveasadockingsiteforAuNPs.
TheaptamerAS1411bindsnucleolinandwastestedinphaseIandIIclinicaltrialsforadvancedsolidtumorsandacutemyeloidleukemia[21].
WhenTheranostics2015,Vol.
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Thisuptakerequiredbothnucleolinonthecellsurfaceandtheaptamer.
AS1411-goldnanostarlocalizationclosetothenucleuscorrelatedwithchangesinnuclearmorphology,suggestingmechan-icaldamagetothenuclearenvelope.
Irradiationlib-eratedtheaptamerfromAuNPs,whichinturnacti-vatedcaspases3and7andincreasedcelldeath.
Col-lectively,thestudysupportsthemodelthattheap-tamerreleaseclosetothenucleusenhanceddamageandtheensuingcelldeath.
Figure4.
Detectionof13nmAuNPsmodifiedwithCIPGNVG-PEG-NH3+in1BR3Gcells(transformedhumanskinfibroblasts).
Cellswereincubatedfor3hourswithfunctionalizedAuNPs,andparticleswerevisualizedbytransmissionelectronmicroscopy.
SomeoftheAuNPswerepresentinthenuclearinterior,asindicatedbytheredarrows.
PanelsAandBdepicttwodifferentnuclei.
Scalebarsare2m.
AdaptedfromOjea-Jiménezetal.
[64]withpermission.
Theexamplesabovesuggestthattargetingtothenucleuscanamplifyorganelle-specificinsults.
ThisideaisvalidatedbyNLS-modifiedAuNPsthatin-creasedDNAdamageandinterferedwithcelldivi-sion,inparticularcytokinesis[56].
Specifically,30nmPEGylatedAuNPsdecoratedwithRGD-andNLS-peptidesaccumulatedinnucleiandtriggeredapoptosisin20%ofthecancercells[56].
Asidefromtargetingtothenuclearinterior,AuNPscanalsobeusedtoobstructtheNPC.
Thus,NLS-modifiedAuNPs(~39or32nminsize)blockednucleartransportinHeLacellsandinducedau-tophagiccelldeath[115].
Interestingly,thiswascelltypespecific,becauseitwasnotobservedinSiHacells,anothercervixcarcinomacellline.
Deliveryofnucleicacids.
OneoftheapplicationsofnuclearAuNPsisthemodulationofgeneexpression;thiscanbeachievedbyknockdownorchangesinsplicing[60,116-119].
Anexampleofthisapproachistheuseof13nmAuNPsthatcarriedoligonucleotidestoadjustthealternativesplicingofmRNAsforpro-survivalfactorsindifferentexperimentalsystems[119].
Assuch,theparticlesweredetectedinnucleiofculturedcellsandledtotumorshrinkageinaxeno-graftmodelbasedonLoVocells(humancoloncarci-noma).
InMCF7breastcancercells,sphericalultrasmall(6and2nm)tiopronin-coatedAuNPsresideinthecytoplasmandenterthenucleusaftera24-hourin-cubationperiod[60].
Whenconjugatedtoafluores-centFITC-tag,only2nmparticlesweredetectedinMCF7nuclei.
FITC-labelingincreasedthesizeof6nmAuNPsto10nm,whichmayhavelimitedthepassagethroughNPCs.
Toinducecelldeath,anoligonucleo-tidewasaddedto2nmtiopronin-AuNPs.
Thisoligo-nucleotidegeneratedtriplexstructureswiththeP2promoterofc-MYC.
Asaresult,expressionoftheproto-oncogenewasdownregulatedandcellviabilitywasreducedto70%.
Whencomparedtothetri-plex-formingoligonucleotidealone,AuNP-attachedoligonucleotidesincreasedc-MYCsilencingandMCF7celldeath[60].
Interestingly,AuNPsfunctionalizedwithdexa-methasonealsodeliveredplasmidDNAtonuclei[76],suggestingthatsteroidhormonescanbeusedforAuNPnucleartargeting.
Thesestudieswereper-formedinculturedcellsandexperimentalanimals;theyarediscussedinsection5.
Deliveryofanti-cancerdrugs.
AuNPsthatlocatetothenuclearperipherycanprovidetransportvehiclesforanti-cancerdrugs.
Onesuchdeliverysystemcar-rieddoxorubicinandwastestedinHeLa(cervixcar-cinoma),A549(lungcarcinoma)andNIH3T3-L1cells(fibroblastswithpre-adiposecharacteristics).
Tothisend,25nmPEGylatedsphericalAuNPswerefunc-tionalizedwithcellpenetratingpeptides,suchasTAT[24].
TAT-AuNPsweretakenupinallcellstested,whereasotherpeptidesdisplayedcelltype-specificdifferences.
Doxorubicin-loadedAuNPswereespe-ciallycytotoxictoHeLaandA549cells,butNIH3T3-L1cellswerelessaffected.
Celldeathwasattributedtothereleaseofdoxorubicinclosetothenucleus,whereAuNPsconcentrated.
3.
TargetingcancercellmitochondriawithAuNPsMitochondriaarethemajorsitesofcellularen-ergyproduction,andtheirdysfunctionisassociatedwithawiderangeofdiseasesandpathophysiologies,includingcancer[120].
Changesinbioenergeticsareahallmarkofmanytumors,butmitochondriaarealsoTheranostics2015,Vol.
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org363keyregulatorsofapoptoticcelldeath.
Theseproper-tiesmakemitochondriaprimetargetsforcancertreatment[121,122].
AuNPsoftenimpairmitochon-drialfunctionsandtherebyinducecelldeath.
How-ever,thisdoesnotalwaysinvolvestablephysicalcontactbetweenAuNPsandtheorganelle.
Here,wefocusonexamplesthatreportAuNPassociationwithmitochondria.
TheemphasisisonstudiesthatweredesignedtodeliverAuNPsspecificallytomitochon-dria.
WhichAuNPsizefitsmitochondriaTheconcep-tualdifferencesforAuNPtargetingtonucleiandmi-tochondriaarebasedonthedistinctorganelleborderstothecytoplasm.
Unlikethenucleus,mitochondriadonotcontainlargeporesthatprovideeasyaccessforAuNPs.
BoththeouterandtheinnermitochondrialmembranespresentbarrierstoAuNPsthataredes-tinedforthemitochondrialmatrix.
Inheartcells,3nmparticles,butnot6nmAuNPs,translocatedacrosstheoutermitochondrialmembrane[86].
Intheinnermi-tochondrialmembrane,proteinimportchannelspro-videopeningsof<2nm([123],reviewedin[124]).
ThisrestrictsAuNPentryintothematrixofintactmito-chondria.
Accordingly,mitochondrialtargetingse-quences,whichinteractwiththemitochondrialpro-teinimportapparatus,haveonlylimitedvalueforAuNPdelivery.
However,AuNPtranslocationacrossthemitochondrialmembranesisnotobligatorytodamagetheorganelle,andotherstrategieshavebeensuccessful.
CommoneffectsofAuNPsonmitochon-driaaremorphologicalchanges,lossofmembranepotentialandproductionofreactiveoxygenspecies.
MitochondrialdeliveryofAuNPs.
Analternativetopeptide-derivedAuNPtargetingwasliposomesthatfusewithmitochondrialmembranes[87].
AuNPs(8-12nm)weredeliveredbyencapsulationinMi-to-porter;thisenvelopecontainsfusogeniclipidsandocta-argininesurfacemodifications.
Octa-arginineservedmultiplefunctions;itstimulatedparticleup-takebymicropinocytosis,escapeintothecytosol,andbindingtomitochondria.
Oncetheparticlesboundtomitochondria,Mito-porterlipidsmediatedmembranefusion,andAuNPswerereleasedintomitochondria.
AnotherapproachwasbasedonAuNPfunc-tionalizationwithcetyltrimethylammoniumbromide(CTAB)[88].
Thiscompounddamageslipidbilayersandcanfacilitatethepermeationofmembranes,leadingtothemitochondrialassociationofgoldna-norods.
CTABstimulatedlysosomalescapeinsomecelllines,likelybecauseitcompromisedlysosomalmembraneintegrity.
Amonglungcarcinoma(A549),normalbronchialepithelial(16HBE)andprimaryadultstemcells,thenanoparticlesaffectedA549cellstothegreatestextent[47].
SuchCTAB-goldnanorodsaccumulatedinmitochondria(Fig.
5)andcausedcelldeaththroughacollapseofthemitochondrialmem-branepotentialandthegenerationofoxidativestress.
Figure5.
A549cellswereincubatedfor24hourswithgoldnanorods.
Swellingandroundingwasobservedforafractionofthemitochondria.
Moreover,somecristaewerelostandvacuolesappearedinmitochondria.
Goldnanorodsassociatedasaggregateswithmitochondria(M),asindicatedbythearrows.
Thetransmissionelectronmicrographwasadaptedfrom[47]withpermission.
ThebiologicalpropertiesofmitochondriacanbeexploitedforAuNPtargetingwithtri-phenylphosphonium.
Drivenbythemitochondrialmembranepotential,thislipophiliccationcanin-creasetheconcentrationofvariousagentsinthemi-tochondrialmatrix(reviewedin[125]).
Tri-phenylphosphoniumtargetedgoldnanoclusterstoHeLacellmitochondria[89].
However,undertheconditionstested,thecytotoxicityofthesegoldnanoclusterswaslow,as≥80%ofthecellsremainedviable.
Pre-photoactivationseemedtoenhancethedel-eteriouseffectsofAuNPsoncancercells[90].
Thus,AuNPsweremoretoxic,ifphotoexcitedbeforeaddi-tiontopancreaticcancercells(1.
4E7cells).
Theirheightenedtoxicitycorrelatedwithincreasedmito-chondrialdamage,suchasswellingandlossofmito-chondrialmembranepotential.
Pre-photocativationalsopromotedAuNPlocationtomitochondriaandtheproductionofreactiveoxygenspecies.
Apro-apoptoticpeptidecontainingthesequence(KLAKLAK)2targeted13.
5nmAuNPstoHeLacellmitochondria[33].
Thesepeptide-functionalizedAuNPsassociatedwithmitochondriaandinducedtheirswelling,lossofmembranepotentialandulti-matelycelldeath.
Peptide-modifiedAuNPsweremoretoxicthantheirnon-functionalizedcounterpartsorthepro-apoptoticpeptidealone.
Takentogether,nucleiandmitochondriahavebeensuccessfullytargetedbyAuNPs.
ThegeneralprinciplesthatrouteAuNPstothenucleusormito-chondriahavebeenidentified(Fig.
6),butmechanisticdetailsarefrequentlyunknown.
Theranostics2015,Vol.
5,Issue4http://www.
thno.
org364Figure6.
UptakeandsubcellulartargetingofAuNPs.
Non-targeted(left)andtargeted(right)AuNPsbindtotheplasmamembrane;thismayinvolvere-ceptorsonthecellsurface.
Uponinternalization,AuNPsinitiallyconcentrateinendosomesorlysosomes.
Afterescapefromthesemembrane-boundcom-partments,AuNPsassociatewithnucleiormitochondria,wheretheycancauseirreversibledamagethatculminatesincancercelldeath.
CellularinjuryisenhancedifAuNPsaretargetedtonucleiormitochondria(right);thissubcel-lulartargetingincreasescancercellkilling.
4.
TargetingAuNPstotheERUnlikeAuNPdeliverytonucleiormitochondria,strategiesforERtargetingarelesswelldeveloped.
However,giventheimportanceoftheERformanytumortypesandthelinksbetweenmitochondriaandtheER[126],AuNPslocatedintheERcouldplayasignificantroleincancertherapy.
AuNPshavebeendetectedinthismembranesystem;forexample,non-functionalized13nmsphericalAuNPscolocal-izedwiththeERandGolgiapparatusinB16F10melanomacells[127].
InHeLacells,18nmAuNPsassociatedwithcytoplasmicvesiclesthatwerepre-sumablypartoftheER[128].
InKupffercellsoftheliver,13nmPEGylatedAuNPslocatedtolysosomesandthesmoothER[129],andAuNPsderivatizedwithCALNNR8peptidesweretrappedintheERofHeLacells[52].
SimultaneousassociationwithnucleiandtheERhasalsobeenreportedforK562cells(human,chronicmyelogenousleukemia)[130].
Notably,theseparticlesinducedERstress,whichlikelycontributedtotheirtoxicity.
5.
TargetingAuNPstosubcellularorga-nellesinvivoAscomparedtoculturedcellsorspheroids,AuNPtargetingtocellorganellesinvivofacesextrahurdles.
Theseadditionalchallengesincludeaccu-mulationinhealthyorgans,tissue-specificbarriers,clearancebythereticuloendothelialsystemandin-sufficientconcentrationatthetumorsite.
Onceac-cumulatedinthetumor,AuNPswillfollowthesamestepsdepictedinFig.
1.
Assuch,aftercellularuptake,particleshavetoescapelysosomes/endosomesandlocatetotheirfinalsubcellulardestination.
Toovercometheseobstacles,severalvariablescanbechanged,includingsize,surfacemodification,dosageandrouteofentry.
Accumulationatthetumorsitesinvivoisaprerequisiteforsubcellulartargeting,andthistopichasbeendiscussedinpreviousreviews[16,18].
Atpresent,onlyalimitednumberofstudieshaveattemptedtooptimizeAuNPstoreachasub-cellulardestinationinvivo.
Here,wediscussseveralstudiesthatshoworganelletargetingwithAuNPsbothinculturedcellsandinexperimentalanimals.
Targetingnucleiinvitroandinvivo.
Huangetal.
[85]comparedtheperformanceofAuNPsinthreedifferentmodelsystems.
Sphericaltiopronin-coatedAuNPsof2,6or15nmsizewereanalyzedinMCF7cells,growninmonolayersorspheroids.
Theseparti-cleswerealsoexaminedinxenograftsofBalb/cnudemice.
Inallofthesemodelsystems,cellularuptakewassize-dependentandmoreefficientforsmallerAuNPs.
Moreover,particlesizedeterminedthesub-cellularAuNPdistribution(seeAdditionalFile1:Ta-bleS1).
SmallAuNPsof2and6nmlocatedtothenu-cleusandcytoplasm,whereas15nmparticleswererestrictedtothecytoplasm.
Followingintravenousinjection,allAuNPswereclearedfromtheblood;thiswasmostefficientfor15nmparticles.
Bycontrast,tumorsaccumulatedpreferentially2nmAuNPs,but2nmparticlesalsoconcentratedinthekidneyandlung.
AuNPsof15nmsizeassociatedpredominantlywithnon-tumortissue,withpreferencefortheliverandspleen.
Collectively,theseresultssuggestthatthesubcellulardistributionoftiopronin-coatedAuNPsissimilarinMCF7cellmonolayers,spheroidsandxen-ografts.
Chenetal.
[76]examinedAuNPnucleartarget-inginvitroinHep3B(hepatocellularcarcinoma)and293Tcells(humanembryonickidneycellscontainingSV40T-antigen);theexperimentswereextendedtoHep3Bcell-derivedxenografts.
TodeliverDNAeffi-cientlytothecellnucleus,AuNPswerecoatedwithdifferentmoleculesinasandwich-likefashion.
ThesandwichincludedplasmidDNA,polyethylenimine(PEI)anddexamethasone,asyntheticagonistoftheglucocorticoidreceptor.
PEIenhancedendosomalescape,whiledexamethasoneservedseveralpurpos-es.
First,itimproveduptake,whichreached82.
5%inculturedHepB3cells.
Second,dexamethasonestimu-latedAuNPnucleartargetingthroughitsassociationwiththeglucocorticoidreceptor.
Specifically,func-tionalizedAuNPs(carryingdexamethasone,DNAandPEI)formcomplexeswithglucocorticoidreceptorinthecytoplasmandthentranslocatetothenucleus.
Withthisapproach,culturedcellsweretransfectedeffectively,whilecytotoxicitywaslow.
Buildingontheseinvitrostudies,functionalizedAuNPswerealsousedforgenedeliveryinvivoTheranostics2015,Vol.
5,Issue4http://www.
thno.
org365(Balb/cnudemicebearingHepB3-derivedtumors).
DNAencodingtumornecrosisfactor-relatedapopto-sis-inducingligand(TRAIL)wasintroducedintotheanimalsusingAuNPsof~55nmsize.
Tumorspro-ducedthehighestlevelsofTRAILwhentheDNAwasdeliveredbyanAuNPsandwichthatcontainedbothPEIanddexamethasone.
ConcomitantwithefficientDNAdelivery,tumorgrowthwasinhibited.
Thus,functionalizedAuNPsinducedTRAILsynthesisinvivoandtherebyinterferedwithtumorgrowth[76],whileTRAILexpressionwaslowinnon-tumortis-sues.
ThisstudydemonstratedAuNPnucleartarget-inginculturedcells.
Althoughnotshownexperi-mentallyinvivo,resultssuggestthatnucleartargetingalsooccurredinexperimentalanimals.
Thus,AuNPfunctionalizationwithdexamethasonecanbeusefultotargettumorsthatsynthesizeglucocorticoidre-ceptor.
Targetingmitochondriainvitroandinvivo.
ThepreferredrouteofATPproductioninmanyformsofcancerisanaerobicglycolysis;thispathwayreliesontheenzymehexokinasewhichsynthesizesglu-cose-6-phosphate.
Hexokinase2isespeciallyim-portantamongthefourenzymeisoforms,becauseitishighlyabundantinaggressivetumorcellsandpre-dominantlyassociatedwiththeoutermitochondrialmembrane.
Sincehexokinase2interactionwiththeoutermitochondrialmembranestimulatesglycolyticenergyproductionandreducesapoptosis,theenzymehasbecomeatherapeutictargetforcancertherapy.
Hexokinase2isinhibitedby3-bromopyruvate,atoxiccompoundwithoff-targeteffects.
Toimprove3-bromopyruvatespecificityanddiminishtoxicity,AuNPsweredevelopedformitochondrialdelivery[91].
PEGylatedsphericalAuNPs(2.
9–4.
3nm)weremodifiedwithTPP,therebygeneratingT-AuNPswhicharecharacterizedbypositivelychargedandlipophilicmoietiesonthesurface.
ThesepropertiesimprovedT-AuNPuptakeandmitochondrialassoci-ationinPC3cells.
ResultsweresimilarwhenT-AuNPswerefurthermodifiedwith3-bromopyruvate(T-3-BP-AuNP).
After4hours,T-3-BP-AuNPsboundtotheoutermitochondrialmembranewherehexokinase2resides.
TheseAuNPsaccumulatedinthemitochondrialmatrixat12hours.
T-3-BP-AuNPsreducedPC3andDU145(prostatecarcinoma)cellviability,buthadlittleeffectonhu-manmesenchymalstemcells.
Theimpactoncancercellswasattributedtothelossofmitochondrialfunc-tionsandglycolysis.
Overall,AuNPsmodifiedwith3-BPweremoretoxictocancerthannormalcells.
Atthesametime,AuNPstargetedtomitochondria(T-AuNPs)weremoreharmfulthantheirnon-targetedcounterparts(NT-AuNPs).
ThetoxicityofAuNPswasfurtherenhancedbya1minlaserirra-diationat660nm.
Toevaluateinvivoeffects,thebio-distributionandpharmacokineticsofT-AuNPandNT-AuNPwereassessedinmaleSpragueDawleyrats.
ClearancefromtheplasmawasfasterforT-AuNPsthanNT-AuNPs.
Bothtypesofparticlesaccumulatedintheliverandspleen,buttheirsubcel-lularlocalizationwasnotdetermined[91].
6.
Perspectives:LimitationsandfuturedirectionsThepromiseofAuNP-dependentcancertherapyisemphasizedbynumerouspublicationsandclinicaltrials[21].
Atpresent,severalhurdleslimittherapidimprovementofAuNP-basedtreatments.
Forexam-ple,theapproachesforAuNPtargetingtosubcellularlocationsdifferwidely.
SinceAuNPsize,morphology,functionalization,concentrationandthecelltypesanalyzedvarysignificantly,itisdifficulttocompareresultsfromdifferentlaboratories.
Moreover,alt-houghessentialtoimproveAuNP-basedintervention,thebiologicalmechanismsunderlyingAuNP-inducedcellkillingareoftennotdefined.
Nevertheless,somegeneralconclusionscanbedrawnforthediversestrategiesthatleadtoAuNPassociationwithsubcel-lularorganelles(Table1).
OurreviewdiscussesthepotentialbenefitsoftargetingAuNPstospecificcellorganelles,andwepresentthebarriersAuNPshavetoovercometoreachtheirintracellulardestination.
Asidefromthere-strictionsthesebarriersimpose,AuNP-mediatedcancercellkillingcouldbelimitedbyexportviaexo-cytosis(reviewedin[131]).
However,suchparticlelosscanbereducedwithappropriatesurfacefunc-tionalization[132].
Attheorganismallevel,itwillfurtherbeimportanttodesignstrategiesthatcontrolAuNPclearance.
Clearanceismostlyachievedthroughthehepatobiliarysystemandthekidney,anditismodulatedbyAuNPsurfacemodification[133].
Astheimmunesystemcontributestonanoparticleclearance[134],clearancerateslikelydifferamongpatients.
Althoughthesevariationschallengetheuniversalapplicationforcancertherapy,theymayneverthelessoffertheopportunitytoproduceAuNPsforpersonalizedmedicine.
Celltypedependentdifferencesinuptakeandsubsequentintracellulartargetingcomplicatetheop-timizationofAuNPsforcancertherapy.
Ontheotherhand,ifsystematicallyexamined,thesedifferencescouldbeexploredtodevelopAuNPsthatareselectiveforspecifictypesofcancer.
Moreover,AuNPtarget-ingtotumorcellorganellescouldbeenhancedbyexploitingthedifferencesbetweennormalandcancercells.
Forexample,nucleartransportismoreefficientinproliferatingcancercellsascomparedtotheirnon-tumorigeniccounterparts(seeabove).
Thisis-inTheranostics2015,Vol.
5,Issue4http://www.
thno.
org366part-causedbytheoverexpressionofimportin-αandimportin-βfamilymembersandothersolublefactorsornucleoporinsthatsupportsignal-mediatednuclearimport[135-137].
Todate,AuNPnucleartargetingreliespredominantlyonNLSsthatbindimportin-α(SV40T-antigen,nucleoplasmin)orimportin-β1(TAT,[138]).
Inthefuture,nucleardeliverycouldbeimprovedthroughAuNPfunctionalizationthatstim-ulatesNPCbinding.
Somenucleoporinsaremoreabundantincancercells[139],andthereforeprovidepotentialAuNPdockingsitesatthenuclearpore.
Withrespecttomitochondria,functionalizingsmallsphericalorrod-shapedAuNPswithmito-chondrialtargetingsequencesmaybesuitabletodamageorganellarfunctionsorevenclogmitochon-drialproteinimportsites,analogoustowhathasbeenobservedforNPCs.
Whiletargetingtosubcellularorganellesisofparticularinteresttonanomedicine,themajorbottle-neckaftercellularuptakeisAuNPescapefromen-dosomes/lysosomes.
Recentstudiessuggestaddi-tionalstrategiestoenhancethisescape.
Besideslowenergylaserirradiation[140],particlesurfacemodi-ficationsthatincreasethe"protonspongeeffect"couldpromoteendosomeswellingandAuNPrelease[141].
Moreover,AuNPsthatundergopH-dependentaggregationorenhancepH-dependentlipidrupturecouldbeusefulfortheliberationfromendosomes.
ReleasefromendosomesiscriticalforAuNPstoreachnuclei;however,itmaynotbemandatoryformito-chondria.
Forinstance,fusogeniclipidsasdescribedfortheMito-portercouldcircumventtheneedforendosomalescape.
Ontheotherhand,itisconceiva-blethatthedirectdeliveryofmaterialfromendo-somestomitochondria[142]willbeexploitedtobringAuNPstomitochondria.
Sofar,cellculturemodelshavedominatedthecharacterizationofAuNPbio-nanointeractions.
Morerecently,AuNP-inducedcelldamageanddeathhavebeenassessedintumorcellspheroids[85,143],amodelsystemthatmimicsmultipleaspectsoftumortissuesinvivo.
Forexample,AuNPpenetrationacrossmultiplecelllayerswasstudiedinspheroidsderivedfromU87glioblastomacells[143].
TheprogressinspheroidproductionsetsthestagetoexplorethismodelfurtherandexamineAuNPtargetingtonucleiandmitochondria.
Todate,thereareonlyfewstudiesthatexaminethesubcellulardistributionofAuNPsinvivo.
WhileinvitroanalyseslocatedAuNPsinnucleiormitochondria,itisinmostcasesnotclearwhethertheseAuNPsaretargetedtothesametumorcellor-ganellesinexperimentalanimals.
Clearly,thisinfor-mationhastobeprovidedinthefuturetofullyassessthevalueofAuNPsubcellulartargetinginvivo.
Table1.
AdvantagesandlimitationsofcurrentapproachesforAuNPdeliverytosubcellularorganelles.
ApproachSubcellularorganelleAdvantagesLimitationsExploitationofAuNPphysicalprop-ertiesfororganellardeliverywithoutspecifictargetingmoieties:size(e.
g.
smallorlargeparticles,nanoclusters),shape(spheres,rods,flowers,urchins),chargenonspecificsubcellulardistribution,oftenconcentratedinendo-somes/lysosomes,particlesmayescapeendosomes/lysosomesandassociatewithnucleiand/ormito-chondriaeasytoprepare,lowcost,fastclearancemaylimittoxicity,PEGylatedsmallAuNPsoftendamagecancercells,positivechargesfrequentlyenhanceup-takeuptakenotspecifictocancercells,fastclearancemaylimitaccumulationintumor,effectofpositivechargescell-typede-pendent,lowendosomal/lysosomalescape,lowconcentrationinsubcellularorganelles,hightoxicity,thusdamagetonon-tumortissuesTargetingtocellsurface:RGDtrans-ferrin,EGF,antibodiesoraptamersthatbindcellsurfacecomponentsnonspecificsubcellulardistribution,oftenconcentratedinendo-somes/lysosomes,particlesmayescapeendosomes/lysosomesandassociatewithnucleiand/ormito-chondriaenhancedtargetingtocancercells,therebyimproveduptakebytumorcellsendosomalescapeandsubcellulardeliverymayrequireadditionalmodifications;cellsurfacereceptorsignaturesforefficienttumortargetingnotavailableforallcancertypesImprovedcellularuptake:cellpene-trating(CPP)andotherpeptides;e.
g.
CALNN,CALNNR8,TAT,Pntn,lysosomalsortingpeptidesnucleusandothersubcellularcom-partmentsmayenhancenucleartargetingthroughincreaseofcellularuptake;someCPPsalsofunctionasnuclearlocali-zationsignalsomepeptidesinefficientforendoso-mal/lysosomalescapeandnucleartarget-ing;nuclearlocalizationcandependoncelltypeNuclearlocalizationsignals(NLSs):biologicalandsyntheticsignals;linearandcyclicpeptides:SV40-NLS,adenoviralNLS,cyclic[KW]5enrichedinnucleuspositivechargesofNLSenhancecellularuptake;specificandefficientnucleartargeting;frequentlylowtoxicityinvitro;usefulfordrugdeliverytonucleusmayneedadditionalmodificationstoimprovetumortargetinginvivoandtofacilitateendosomal/lysosomalescapeCombinationofpeptideswithdiffer-entfunctions:e.
g.
CPP+NLS,RGD+NLSenrichedinnucleusimprovedtumortargetingandcellularuptake;usefultoexploitcellsurfacereceptorsfunctionalizationwithmultiplepeptides;specificratioofpeptidesmayberequiredOthermolecules:CTAB,tiopronin,cysteamine,thioglucose,dexamethasonecanleadtoenrichmentinnucleusmaystimulatecellularuptake,endoso-mal/lysosomalescapeorboth;thiscanenhancenuclearassociationsomemodificationshighlytoxic(e.
g.
CTAB);exceptfordexamethasone,nucleartarget-ingnotefficient;targetingmaybecelltypespecific(e.
g.
nuclearaccumulationbydexamethasonereliesonglucocorticoidreceptor)Differenttypesoffunctionalization:octa-arginine,CTAB,TPP,chitosan,polyvinylpyrrolidoneenrichedinmitochondriacanstimulatecellularuptakeand/orendosomal/lysosomalescape;thiscanenhancemitochondrialassociationfrequentlytoxic(e.
g.
TPP);mayrequirepermeabilizationofplasmamembrane(e.
g.
polyvinylpyrrolidone)Theranostics2015,Vol.
5,Issue4http://www.
thno.
org367AsthemodelsystemstoevaluateAuNPper-formancearebecomingmorediverse,soareAuNPs.
Assuch,AuNPscanassembleintochains,plasmonicvesicles[144,145]orotherstructuresthatprovidemultiplefunctionsatthesametime,includingdrugdelivery,enhancedphotothermalablationandparti-cletracking.
LargecomplexAuNPsthatareabletodisassembleintosmallerAuNPunits[145,146]couldhelpovercomethedifferentroadblocksthatlimitAuNPsubcellulartargeting(Fig.
2).
SuchcomplexAuNPs,whendisassembled,mayhavetheadditionalbenefitofrapidrenalorhepatobiliaryclearance.
Takentogether,AuNPsofferuniqueopportuni-tiestotranslatetheinsightsofbasicresearchintoclinicalapplications.
GiventhesuccessofAuNPsforphotothermalablation,mechanicalinjuryandtar-geteddrugdelivery,futurestrategiesthatcombinetheseeffectsforAuNP-dependentcancercellkillingareparticularlypromising.
7.
SummaryandhighlightsRecentstudieshavebeguntorevealhowAuNPsimpingeonthestructural/functionalorganizationofcancerandnormalcells.
Thisknowledgeiscriticalfortwoaspectsofnanomedicine.
First,itwillhelpdefinetheAuNP-inducedeventsatthesubcellularlevel.
Thiswillsetthestagefortheidentificationofnewmoleculartargetsforcancertherapy.
Second,itwilldirectthedesignofAuNPswithphysico-chemicalpropertiesthatovercomethecurrentlimitationstheseparticlesfaceinbasicresearch,diagnosisandtherapy.
Thus,optimizationofAuNPsurfacesforcellandor-ganelle-specificdeliveryisanticipatedtoenhancetheefficiencyofcancercellkilling,whileminimizingtheimpactonnon-tumortissues.
Basedontheiressentialroleforcancercellsurvival,nucleiandmitochondriaareprimetargetsforthisapproach.
AbbreviationsAuNP:goldnanoparticleCALNN:cys-ala-leu-asp-aspCTAB:cetyltrimethylammoniumbromideEGF:epidermalgrowthfactorEGFR:EGFreceptorEPR:enhancedpermeabilityandretentionLSP:localizedsurfaceplasmonresonanceMTS:mitochondrialtargetingsequenceNIR:nearinfraredradiationNLS:nuclearlocalizationsequencePEG:polyethyleneglycol,surfacecoatingofAuNPsthatreducesparticleaggregation,non-specificinteractionswithbiomoleculesanduptakebythere-ticuloendothelialsystemPEI:polyethyleniminePntn:Penetratin,peptidesequencederivedfromDrosophilaproteinAntennapediathatpromotesentryintothecellTAT:HIV-1-trans-activatingproteinTPP:triphenylphosphoniumTRAIL:tumornecrosisfactor-relatedapopto-sis-inducingligandSupplementaryMaterialAdditionalFile1:TableS1:ImpactofAuNPsize,morphologyandfunctionalizationoncellularuptake,subcellularlo-calizationandcellsurvival.
http://www.
thno.
org/v05p0357s1.
pdfAuthorContributionsThemanuscriptwaswrittenthroughcontribu-tionsofallauthors.
Allauthorshavegivenapprovaltothefinalversionofthemanuscript.
CompetingInterestsTheauthorshavedeclaredthatnocompetinginterestexists.
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