纳米生物学及其应用 29页

纳米生物学及其应用亓立峰浙江加州国际纳米技术研究院\n前言：纳米微粒概述PartI:纳米药物与靶向设计PartII:肿瘤纳米分子影像与实时治疗PartIII:纳米探针前景展望\nWhatisNano?Quantumdots,magneticNPPolymerNPFig1.AschemeticofnanoscalematerialPhycoerythrin前言\n纳米粒子分类某种物质或元素纳米级别粒子:nanoZinic;SiO2，nanogold,CdSe/ZnS,Fe2O3,PolymerNPDegradablenanospherenanocapsulePolymericmatrixPolymericmembraneOilyoraqueouscore前言\n化学法：(1)沉淀法/透析法把沉淀剂加入到盐溶液中反应后，（将沉淀热处理）得到纳米材料。其特点简单易行，但纯度低，颗粒半径大，聚合体纳米粒子、氧化物。(2)微乳液法/乳液扩散法两种互不相溶的溶剂在表面活性剂的作用下形成乳液，在微泡中经成核、聚结、团聚、（热处理）后得纳米粒子。其特点粒子的单分散和界面性好，聚合体纳米粒子、Ⅱ～Ⅵ族半导体纳米粒子多用此法制备纳米微粒制备方法前言\n纳米粒子表征方法测量方法测量功能适用的尺寸范围使用的主要仪器离心沉降法等效直径>25nm高速离心机，分光光度计或暗场法光学系统气体吸附法（容量法或重量法）比表面积尺寸：约1～10nm比表面积：0.1～1000m2/gBET吸附装置或重量法装置光散射法平均直径>约3nm喇曼光谱仪X射线衍射峰宽法晶粒平均尺寸约<500nm常用于<50nmX射线衍射仪小角度X射线散射线晶粒平均尺寸约<100nmX射线衍射仪电子成像法（TEM）直接观察粒子形貌并测量粒径尺寸约>2nm透射电子显微镜扫描隧道显微镜法（STM）形貌与尺寸宽范围扫描隧道显微镜粒径分析粒径分布粒径分析仪荧光光谱法荧光图谱荧光光谱仪\nThesenewlyformedtumorvesselsareusuallyabnormalinformandarchitecture.Theyarepoorly-aligneddefectiveendothelialcellswithwidefenestrations,lackingasmoothmusclelayer,orinnervationwithawiderlumen,andimpairedfunctionalreceptorsforangiotensinII.Furthermore,tumortissuesusuallylackeffectivelymphaticdrainage.Allthesefactorswillleadtoabnormalmolecularandfluidtransportdynamicsespeciallyformacromoleculardrugs.Namely,thisphenomenonwascoined“enhancedpermeabilityandretention(EPR)-effect”ofmacromoleculesandlipidsinsolidtumors.http://en.wikipedia.org/wiki/Enhanced_Permeability_and_Retention_effectPartI:纳米药物与肿瘤靶向治疗EPReffectsEnhancedpermeabilityandretention(EPR)effect.Long-circulatingdrugcarriers(1),penetratethroughtheleakypathologicalvasculature(2),intothetumorinterstitium(3),anddegradethere,releasingafreedrug(4)andcreatingitshighlocalconcentration.AAPSJournal.2007;9(2):Article15.DOI:10.1208/aapsj0902015\nPartI:纳米药物与肿瘤靶向治疗Nanoparticles:clinicaladministrationofanticancerdrugs.Advantages:ControlledandtargeteddeliveryofthedrugReducedsystemicsideeffectsFacilitatedextravasationintothetumorcells(EPReffect:enhancedpermeabilityandretention)HighcapabilitytocrossvariousphysiologicalbarriersOmicC.Farokhzad,andRobertLanger.ACSNano,2009,3:16\n1.1ChitosanNPforcancertherapyFig.3SizeandzetapotentialofCNPandCNP-Cu50mV96mVCNP-Cu13mVChitosanCNPCNP-CuL.Qi,etal,ColloidsSurfacesA:PhysicochemEngAspects2004;251:183-190;CarbohydrateResearch2004;2693-2700CNP\nPanelofcelllineCelllineCytotoxicity(IC50,μg/mL)ChitosanCNPCNP-CuLiverL-02nananaColoncancerCalo3201200(±27)28(±3)14(±2)GastriccancerBGC8231200(±35)21(±2)8(±1)LivercancerBEL74021200(±22)15(±2)6(±1)Valuesaremeansofthreeexperiments,standarddeviationisgiveninparentheses(na=notactive).Table1.Cytotoxicactivityofchitosan,CNP(meanparticlesize=40nm),andCNP-CuagainstdifferentcelllinesAntitumoractivityofCNPL.Qi,etal,Bioorganic&MedicinalChemistryLetters2005;15:1357-1399\nFig.5CNP-inducedchangesofMMPandzetapotentialofmembrane.A:LossofMMPandzetapotential;Histogramofuntreatedcells(B)andcellstreatedwith100μg/mLCNP(C)CNPcouldneutralizethenegativesurfacechargeofBEL7402cellssoastodamagethecellmembrane.ThestrongdissipationinMMPsuggestsapossibledisruptionofmitochondrialmembraneaftertreatedwithCNP.Lossofzetapotentialandmitochondrialmembranepotential(MMP)inducedbyCNPL.Qi,etal,Eur.J.Cancer2007;43:184-93\nFattyacidControl(μg/mg)CNPgroup(μg/mg)C14:029.93±2.0130.29±2.21C16:0216.5±13.73215.51±5.41C18:0255.61±14.86252.11±10.99C18:1142.62±8.52108.66±5.11aC18:2226.22±8.29195.78±4.54aC20:412.97±1.469.87±0.45aaP<0.01vsControlTable2EffectsofCNPonfattyacidcompositionofmembranephosphoriclipidfromBEL7402cellsFig.6Lipidperoxidationmeasuredasthiobarbituricacidreactivesubstances(TBARs)productionofBEL7402cellsnon-treatedortreatedfor24hwithvariousdosesofCNP.InteractionwithlipidbilayeroftumorcellsIncreasedROS(Reactiveoxygenspecies)productionandLPOdegree,decreasedunsaturatedfattyacidindicatedthatCNPexertedmembranepenetrationactivitybyinductionofLPOL.Qi,etal,Eur.J.Cancer2007;43:184-93\nGroupsBWincrease(g)TW(g)TIR(%)Control1.54±0.562.95±0.46-Chitosan1.78±0.972.24±0.41b2440nmCNP2.40±0.441.13±0.22b6270nmCNP2.38±0.481.21±0.29b59100nmCNP1.96±0.851.92±0.46b35Cisplatin-2.3±0.990.56±0.13b81InvivoantitumoractivityofCNPTable4EffectofCNPwithdifferentparticlesizeonBEL7402tumorcellgrowthinnudemicebyoraladministration(1mg/kg/day)Fig.7EffectsofadministrationroutesonantitumorefficacyofCNPagainstSarcoma-180subcutaneoustumorformationandgrowthinICRmiceL.Qi,etal,,Bioorganic&MedicinalChemistryLetters2006;EuropeanJournalofCancer,2007.Sideeffectsofchemotherapy\nPartII:肿瘤纳米分子影像与实时治疗Imaging:cancerdiagnostics,staging,radiationplanning,andevaluationoftherapy.Standardclinicalimagingmodalities:CT,MRI,ultrasound,limitationindetection(<0.5cm),anddistinguishingbetweenbenignandcanceroustumors.Molecularimaging:Multifunctionalnanoparticles:MNP,QDs,goldNP,drugdelivery(siRNA,DNA,drug),nanothermotherapy,photodynamictherapy.\nWhatisQdotsSize-tunablelightemissionPhotostableSimultaneousexcitationofQDsLargeStokesshiftWuetal.Nat.Biotechnol.2141–6；Qi&Gao,ACSnano,2008\n2.1QuantumdotsfordrugdeliveryandtherapyQiandGao,ExpertOpin.DrugDeliv.2008;5:263-267Figure13.Schematicillustrationofamultifunctionalquantumdotcoatedwithamphiphilicpolymer.\nProtonspongecoatedQDforsiRNAdeliveryandrealtimeimagingYezhelyev,Qi,O’Regan,Nie,Gao.JACS,2008Fig.14.SchematicdiagramshowingthestepsofsiRNA-QDinmembranebinding,cellularentry,endosomalescape,capturingbyRNAbindingproteins,loadingtoRNA-inducedsilencingcomplexes(RISC),andtargetdegradation.\nQuantumDot-AmphipolNanocomplexforsiRNAdeliveryFig.16.SchematicdrawingofthehybridstructureofQDandamphipolforsiRNAdeliveryandreal-timeimaginginlivecells.QiandGao,ACSnano,2008,2(7),1403–1410,2008\nGelloadingefficiencyandprotectionagainstnucleaseFig.17QDloadingcapacityandprotectionofsiRNAmoleculesagainstnucleasedegradationdeterminedbygelelectrophoresis.QiandGao,ACSnano,2008,2(7),1403–1410\nSiliencingefficiencyandcytotoxicityFig.18GenesilencingefficiencyofsiRNAtargetingHer-2usingQDPMALcomparedwiththeclassictransfectionagents,LipofectamineandPEI.QiandGao,ACSnano,2008,2(7),1403–1410\n纳米分子造影剂产品1）3TP的癌症诊断技术，基于MRI的诊断乳腺癌、前列腺癌，区分恶性/良性，〉5mm瘤块。2）主要以钆/氧化铁为组分核磁纳米造影剂3）Palatin技术公司2007年获批准新型造影剂neutrospec，neutrospec含有一个以锝为标记的抗cd15单克隆抗体，可以选择性的结合于参与免疫应答的嗜中性粒细胞。neutrospec被注射入血后，可与感染部位存在的嗜中性粒细胞相结合，使这些细胞被放射性示踪剂所标记。\nPartIII:纳米探针Fig.22ModulatingsignalingpathwaysandprobingbiologicalinteractionswithnanotopographyOligo\n3.1InteractionofNanotopographywithCells**†*†****Fig.25.CytotoxicityofCNCdeterminedbydetectionofLDHreleasefromBJ-5acellscoculturedwithextractsofCNCfor72h.Fig.26ActinlocalizationinspreadingcellswithtimeControlCNCChitosan3h3d11dL.Qi,etal,J.Biomed.Mater.Res.A.2007Dec.18\n3.2纳米纤维转染DNAFig.27.Humanembryonickidneycellline(HEK293T)culturedonSiNWs(c)withand(d)withoutPEItreatmentpriortoGFPplasmiddeposition.Yanget.al.J.AM.CHEM.SOC.2007,129,7228-7229\n3.3靶分子监测纳米探针Fig.28.Well-dispersedantibody-nanoshellconjugatesintheabsenceofanalytepossessawell-definedextinctionpeakinthenear-IR.Inthepresenceofthecomplementaryanalyte,multiplenanoshellsbindtotheanalyte,causingagglutinationandacorrespondingreductionintheextinctionpeak.MethodsinMolecularBiology™•303NANOBIOTECHNOLOGYPROTOCOLS\nFig.29.Signaldetectionofoligonucleotide-derivatizedNBCshybridizedwithcomplementaryCy5oligonucleotide.3.3纳米金荧光检测核苷酸序列MethodsinMolecularBiology™•303NANOBIOTECHNOLOGYPROTOCOLS\n3.4硅纳米纤维用于DNA监测Fig.30.Flowchambersetupforhybridizationandvisualizationoffluorescencesignalsonthenanoarray.Opticalmicroscopeimageofanopticalfiber-bundlebasednanoarraycontainingindividual300nmdiameterfibers;(ii)magnifiedSEMimageofaregionofthearraycontainingnanobeads;(iii)fluorescentnanobeadsinthenanoarray.BiosensorsandBioelectronics24(2009)2488–2493\nFig.31.Schematicofhybridizationassaysonthefibernanoarrays.BiosensorsandBioelectronics24(2009)2488–24933.4硅纳米纤维用于DNA监测\n展望：纳米技术在肿瘤诊断和治疗方面应用Earlydiagnosisforlow-stagecancer1)Identificationofeffectivetumormarkersbygenomicandproteinomics2)Insituquantitativeprofilingofmultiplemarkersbymulticolorquantumdotsdopednanobeads(nanobarcode)Multimodalimaging:NIROpticalimaging+MRI1)Developmentofnovelimagingprobewithnon-toxicelements2)Imagingsinglecellsandsinglemolecules,probethemechanismoftumormetastasisTargetedtherapeutics1)Synthesisofmultifunctionalnanoparticles(self-trackingandtargeteddrug/siRNAdelivery),aptamers,peptide2)Rationallydesignbiodegradablenanoparticlesfortargetedtherapeutics