Nanozyme-basedAnticancerNanocompositesforCancerTherapyandinvitroAnalysisUsingElectrochemicalCellChip

Abstract

종양미세환경(Tumormicroenvironment,TME)반응성나노플랫폼을통한표적종양치료는정상세포에는최대한영향을주지않고암세포만을선택적으로죽이기위해사용되는치료전략이다.대표적인항암치료법중하나인화학역학요법(Chemodynamictherapy,CDT)은암세포내부의과산화수소를금속이온의촉매작용하에독성이강한하이드록실라디칼로전환시켜암세포의성장을억제하고결국에는사멸에까지이르게하는새로운전략인데,레이저,전기또는초음파와같은외부에너지의개입이없어도종양미세환경에풍부하게존재하는내인성물질(예:포도당,과산화수소)에의해화학역학요법의촉매반응이시작된다는장점이있다.화학역학요법을위한치료제로서,우수한효소모방활성을가지는나노물질,즉나노자임이다양한나노치료적접근법에널리적용되어왔다.초미립자크기,열적안정성,높은촉매활성,간단한합성과정,우수한내구성이라는장점을지닌나노자임을약물운반체역할을하는나노구조체에포집하여치료제로서의생체적합성을높이는연구또한꾸준히이루어지고있다.그러나화학역학요법에는단점또한존재하는데,불충분한양의암세포내인성과산화수소와강력한항산화제가하이드록실라디칼의생성을억제하여치료효과를감소시킬수있다는것이며,이러한한계를극복하기위해서는화학역학요법을다른치료법과결합시켜해당단점을보완하고단일요법보다높은치료효율과낮은부작용을보이는복합적방식의치료법개발이요구된다. 나아가,나노자임기반나노치료제의생체적합성,치료적효율성및항암제로의응용가능성을확인하기위해서는목적에맞는생체외(invitro)임상적분석이반드시수행되어야한다.하지만,흡광도측정에기반한기존의생체외세포분석법으로는유색의항암나노자임또는나노복합체의효능을검증하기어려우며,이로인해간혹거짓양성/음성과같은분석오류를범할수있다는결정적인단점이있다.따라서나노물질기반항암제의새로운발견을위해서는기존분석법의대체가가능하고유색약물의효능평가가쉽고빠르며간편하게이루어지는분석플랫폼의개발이필요하다. 세포기반칩은살아있는세포를배양하기위한소형화된플랫폼으로,암세포증식을억제하고사멸을유도하는항암제의치료효과를모니터링하는데유용한장치가될수있다.세포칩의장점으로는조작이용이하고신속및정확하며민감한분석이가능하다는점을들수있는데,이를전기화학적센싱기법과접목시켜개발한것이바로전기화학적세포칩이다.분석절차가간단하고특이적인표지가필요없으며,비침습적인방식의세포센싱플랫폼을구현할수있어서항암약물의생체외효능을평가하기위한장치로써널리연구되어오고있다. 본학위논문의2장에서는,전기화학적방식을이용한세포패터닝기법과세포칩의개발및항암약물스크리닝플랫폼으로의응용이소개되었다.불소도핑주석산화물(Fluorine-dopedtinoxide,FTO)전극표면에서발생하는전기장이암세포(Skinmalignantmelanoma,SK-MEL28)내/외부이온불균형을유발하여세포가손상을입고사멸하는방식을통하여,전기적자극을받지않는부분의모양대로세포패턴을구현하였다.전해질,인가전압의세기및시간과같은전기화학적조건의차이를이용하여세포패터닝기법을개발하였고이를다양한종류의세포에적용하였다.또,전기화학적으로세포가패턴된기판을고분자필름기반의미세유체칩과결합시켜항암약물(사포닌)의주입에따른생존세포수변화를정성적/정량적으로분석하는방식으로약물스크리닝을실시하였다. 3장에서는,세포기반의전기화학적바이오센서의개발및이를이용한암세포감지와항암약물의효능평가법이소개되었다.전기화학적으로세포감지가가능한기법/플랫폼을개발하여항암약물(사포닌)이SK-MEL28암세포에미치는성장억제및사멸효과를민감하고빠르게평가하였다.전기촉매적특성과생체적합성향상을위하여금나노입자(Goldnanoparticles,AuNPs)와폴리라이신(Poly-L-lysine,PLL)분자를투명FTO전극위에퇴적및코팅하였다.일정전압을세포가자라는전극에인가하면표면에고정된세포와전극표면간의직접적인전자전달로인해전기적신호가나타나게되고,결국측정전류값이세포수에정비례함을이용하여약물의항암효능이암세포의생존율에미치는영향을전기화학적으로정량화하였다.전기화학적측정에서,암세포의선형감지(검출)범위는장치당2,880–40,000개로나타났으며,해당범위에서세포수와전류세기의사이의선형성을확인하였다("R"^"2"=0.9952).또한,SK-MEL28암세포에대한사포닌의항암효과가20"μM"이상의농도에서명확하게나타났으며,이는기존분석법과매우일치하는결과임을확인하였다.결과적으로,유색약물의분석을할때에측정파장이겹친다는단점이있던비색측정법보다더신속하고(<2분)민감하며(검출한계:2,880세포/장치)비침습적인방식의항암효능평가법을개발하였다. 4장에서는,종양미세환경에반응하는항암나노복합체의개발및항암제로서의치료효능검증에대한연구가소개되었다.과산화효소활성을가지는나노자임(PrussianBluenanoparticle.PBNPs)과포도당산화효소(Glucoseoxidase,GOx),그리고이들을둘러싸는금속-유기골격체(Metal-organicframework,MOF)로구성되는나노구조체(ZIF@GOx@PBNPs)를개발하여이를악성종양의효율적인치료를위한소재로써활용하였다. 나노자임에의한화학역학요법(CDT)과포도당산화효소에의한기아요법(Starvationtherapy,ST)이함께결합된복합적치료요법으로써,ZIF@GOx@PBNPs는연속촉매반응을통한활성산소유도특성을이용해암세포내부의산화적손상을유발하여암성장을현저하게억제하였고생체외/내(invitro/vivo)분석을수행하여높은치료효율성및낮은부작용을검증하였다. 이러한결과는본연구에서개발된나노자임기반의항암나노구조체가효과적인암치료를위한종양표적치료법으로써기능가능함을입증한다.또한,전기화학적세포칩플랫폼과세포센싱기법은빠르고정확하며민감한방식으로다양한항암약물의효능평가를위해확장하여적용할수있을것으로기대된다. |Targetedtumortherapythroughtumormicroenvironment(TME)-responsivenanoplatformsisanemergingtreatmentstrategyusedtoselectivelykillcancercells.Amongtherepresentativetherapeutics,chemodynamictherapy(CDT)isanovelstrategythatconvertsendogenoushydrogenperoxide(H2O2)incancercellsintohighlytoxichydroxylradicals(∙OH)underthecatalysisofmetalions.Evenwithoutexternalenergyinput,suchaslaser,electricity,orultrasound,thecatalyticreactioninCDTcanbeinitiatedbytheglucoseabundantintheTME,thuspromotingROSgenerationtoinhibitthetumorcellgrowth.AsaCDTagent,nanomaterial-basedartificialenzymes,callednanozymes,havebeenwidelyusedinmanynanotherapeuticapproachesbasedontheirexcellentenzyme-mimickingactivity.Undertheadvantagesofultrafineparticlesize,thermalstability,highcatalyticactivity,easypreparation,andgooddurability,theycanbeincorporatedintovariousnanostructuresthatfunctionasdrugcarrierstoenhancebiocompatibility.InCDT, however,theinsufficientintratumoralH2O2levelandrobustantioxidantsincancercellsmightsuppresstheproductionof∙OH,therebyreducingthetherapeuticeffects.Toovercomethislimitation,itisrequiredtoreinforceCDTbycombiningitwithothertherapeuticmodalities,yieldingthemultimodaltherapieswithhigherefficiencyandlowersystemicsideeffectsthanthemonotherapy. Furthermore,clinicalevaluationofthenanozyme-basednanotherapeuticdrugsforcancertherapiesmustbeconductedtodeterminetheefficiencyandbiocompatibilityfortheirtherapeuticusage.However,traditionalassaysbasedonabsorbancemeasurementshaveacriticaldrawbackthatitisdifficulttoverifytheefficacyofanticancernanocompositehavingitsuniquecolor.Therefore,itisrequiredtodevelopsimpleandaccuratemethodsthatcanbeimplementedeasilyfordiscoveringnewanticanceragents. Acell-basedchipisaminiaturizedplatformforculturinglivingcellsandcanbeausefuldeviceformonitoringthetherapeuticeffectsofanticancerdrugsthatinhibitcancercellproliferation.Duetotheeaseofoperation,rapid,accurate,andsensitiveanalysis,ithasbeenappliedincombinationwithanelectrochemicalsensingtechniqueforinvitrocellcultureanalysis.Theelectrochemicalcellchipshavebeenwidelyusedassimple,label-free,andnon-invasivetechniquesfortheevaluationoftheeffectsofanticancerdrugsoncancercells. Inchapter2ofthisdissertation,asimpleelectrochemicalapproachforarrangingcellsintovariouspatternswasintroducedtoenablerapidandaccuratelocalizationforcellchipformation.Avoltagepulsewasapplieddirectlytothepre-patternedconductingsubstrate,causingcellulardamage,andrapidlyforminglive/deadcellarrays.Thearrayplatform,whichfocusesonassayminiaturization,wasapplicabletocell-baseddrugscreeningbydeterminingtheanticancereffectofdrugsonvarioustypesofcells. Inchapter3,anovelandsimplecell-basedelectrochemicalbiosensor,referredtoasacytosensor,wasproposedtoinvestigatetheelectrochemicalbehaviorofhumanskinmalignantmelanoma(SK-MEL28)cellsandtheanticancereffectofsaponinoncellviability.Toenhancebothelectrocatalyticpropertiesandbiocompatibility,goldnanoparticleswereelectrochemicallydepositedontoaconductivesubstrate,andpoly-L-lysinewasfurtheraddedtotheelectrodesurface.ElectricsignalsfromSK-MEL28cellsontheelectrodeswereobtainedfromcyclicvoltammetryanddifferentialpulsevoltammetry.Thecathodicpeakcurrentwasproportionaltothecellviabilityandshowedadetectionrangeof2,880–40,000cellsperdevicewithanexcellentlinearcellnumber-intensityrelationship(R2=0.9952).Furthermore,theanticancereffectofsaponinonSK-MEL28cellswasclearlyestablishedatconcentrationshigherthan20μM,whichwashighlyconsistentwithconventionalassays.Moreover,thedevelopedelectrochemicalcytosensorforevaluatinganticancereffectsenabledrapid(<2min),sensitive(LOQ:2,880cells/device),andnon-invasivemeasurements,thusprovidinganewavenueforassessingtheanticancerdrugsinvitro. Inchapter4,wedevelopedananosizedzeoliticimidazolateframework-8(ZIF-8)thatsimultaneouslycontainsnaturalglucoseoxidase(GOx)andPrussianbluenanoparticles(PBNPs),whichactasaperoxidase-mimickingnanozyme,toconstructTME-activatablenanocomposites(denotedasZIF@GOx@PBNPs)foreffectivetumorsuppression.InweakacidicTME,GOxeffectivelycatalyzedtheoxidationofintratumoralglucosetoH2O2andgluconicacid.Meanwhile,PBNPsreleasedfromtheZIF-8frameworkdissociatedbyacidicpHconvertthegeneratedH2O2intoharmful∙OHradicals.Inthisway,thecascadecatalyticreactionsofZIF@GOx@PBNPsenhancedreactiveoxygenspeciesproductionandcausedintracellularoxidativedamagetotumorcells,resultinginremarkableinhibitionoftumorgrowth.Giventhesignificantantitumorefficiencybothinvitroandinvivo,ZIF@GOx@PBNPscouldbeappliedasapromisingtherapeuticplatformenablingstarvation/chemodynamicsynergism,hightherapeuticefficiency,andminimalsideeffects. Theseresultsaboveclearlydemonstratethepotentialofnanozyme-integratedMOFnanocompositesastumor-targetedsynergistictherapeuticsforeffectivecancertreatment.Furthermore,thedevelopmentofelectrochemicalcellchipsanditsapplicationforinvitroanalysiscanbeextendedfortheassessmentofotheranticancerdrugsinarapidandsensitiveway.