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无机化学学报CHINESEJOURNALOFINORGANICCHEMISTRY生长微环境下原位矿化磷酸钙诱导的细胞死亡•1刘朋陈燕王广川王本徐旭荣唐容康*浙江大学理学院化学系,杭州310027摘要本文主要研究了不同带的磷酸钙在生长环境下对骨肉瘤细胞死亡的影响通过在无钙培养基中加入氛化钙原位合成得到所需磷酸钙颗粒,然后用无钙磷培养基按照不同的比例稀释所得磷酸钙颗粒MTT结果表明,24h后的细胞凋亡与培养基中磷酸钙的屋有关当浓度超过240pug-mL-1时,细胞出现大鼠死亡细胞超薄切片结果也表明,当磷酸钙浓度超过240fig-mL1时,可以观察到有大段磷酸钙存在于细胞内;而磷酸钙浓度低于240ag・mLT时,细胞内观察不到磷酸钙的存在这些结果表明240jig-mL-1对于细胞死亡是一个非常重要的转折点,町能超出了细胞的自我修复范围或者承受能力.这些结果对于磷酸钙在生物医学工程,如组织工程、药物输送、基因转染等方面具有一定的指导意义关键词生物矿化;磷酸钙;细胞死亡;纳米颗粒;临界浓度中图分类号
0611.65文献标识码A文章编号1001«
4861201301.
0089.06DOI
10.3969/j.issn.l001-
4861.
2013.
00.032CellDeathInducedbyInSituMineralizedCalciumPhosphateinGrowthMicroenvironmentLIUPengCHENYanWANGGuang-ChuanWANGBenXUXu-RongTANGRui-Kang*DepartmentofChemistryCollegeofScience^ZhejiangUniversity9Hangzhou310027ChinaAbstract Thecorrelationofcalciumphosphatequantityandtheirlethaleffectonhumanosteosarcomacellsingrowthmicroenvironmentwasinvestigated.ThecalciumphosphatenanoparticlesCaP-NPSwereinsitugeneratedbyaddingcalciumsolutiontocellculturemedia.ThemediawithdifferentamountsofCaP-NPSwerepreparedbysuspendingtheobtainedCaP-NPSintocellculturesolutions.TheviabilityofhumanosteosarcomacellsculturedinthemediawasexaminedbyMTFmethod.WefoundthalCaP-NPSinduceddeathofthecoculturedcellsdependingontheiramounts.MTTassayrevealedthatCaP-NPScouldobviouslyreducethecellviabilitybyinducingcelldeathwhentheirquantitywasabovethethreshold240jig•mL~l.ThetransmissionelectronmicroscopyTEMresultsrevealedthatlotsofCaP-NPSwereobservedincytoplasmofcoculturedcellswhentheconcentrationofCaP-NPSwas240jxg・mL・IncontrastnoCaP-NPSwasfoundwithinthecoculturedcellswhentheconcentrationofCaP-NPSwas240ixg^mLLOurresultsimpliedthat240£g-mLlCaP-NPSwasacriticalpointforcellviabilityexceedingwhichwouldcauseseverelycelldeathbecauseitexceededthelimitofcellself-clarifyortolerantability.ThisstudymayprovideaguidefortheCaP-NPSapplicationsinmedicalengineeringsuchastissueengineeringdrugdeliveryandgenetransfection.Keywords biomineralization;calciumphosphate;celldeath;nanoparticles;criticalconcentration收稿日期2012-08-
30.收修改稿日期2012-10-09o国家自然科学基金No.91127003资助项目•通讯联系人E-mail:rtang@zju.edu.cnTel057-87953736;员登记号S0600155i4M°CalciumphosphatenanoparticlesCaP-NPShavereceivedconsiderableinterestinaninterdisciplinarystudyinvolvingchemistrymaterialsbiologyandmedicine1111particularlyinbiomedicalfield.Calciumphosphatethemajorinorganiccompositionofnaturalhardtissuesbonedentinandenamelisextensivelyappliedinbiomedicalapplicationsduetoitsexcellentbiocompatibilitybioactivityandosteo-conductivitycharacteristics
1591.Calciumphosphatebasedcompositenanoparticlesisalsosuggestedasanattractive.candidateforbioimagingandtherapeuticdeliveryapplications10*.UnfortunatelylargeamountsofCaP-NPScanalsoinducecelldeathbutthissideeffecthasnotbeenbroadlyinvestigated.Celldeathincludestwotypicalformsnecrosisandapoptosis.Necrosisisadegenerativephenomenonthatfollowsirreversibleinjuryandapoptosisappearstobeanactiveprocessrequiringproteinsynthesisforitsexecution.Innatureaphenomenonisthatalmostallcelldeathinducedbyphosphateisreliedonthepresenceofcalciumwhichimpliesthatthecelldeathcanbeinducedbytheco-existenceofcalciumandphosphateinthegrowthmicroenvironment
1121.Bothcalciumandphosphatearetheessentialelementsinbiologicalfluidsandgenerallytheirco-existenceisinertoncelldeath.Thereforetheamountcontrolmaybethekeytotheinsecurityofcalciumphosphatematerials.PreviousstudieshavesuggestedthatitistheCaP-NPSratherthanthefreecalciumorphosphateionsinbiologicalsolutionsthatinducethecelldeath1Howeverthesestudiesaboutthelethalinducing-effectarelimitedtotheinfluencesofCaP-NPSsizesorcrystallities113-
151.InordertounderstandtherolesofcalciumionphosphateionandCaP-NPSinthecelldeathquantitativelyweinvestigatetheireffectsonMG-63cellsderivedfromosteosarcomaacommonmalignanttumorofboneindifferentculturemedia网.Byaddingcalciumintocalcium-freeculturemediaCaP-NPScanbeinsitusynthesizedintheculturemedia.WerevealthatthecellviabilityisdirectlyrelevanttotheconcentrationofCaP・NPSintheculturemedia.Aninterestingfindingisthereexsitsacriticalpointof240|ig•mL1CaP-NPStoensurethecellviability.LessthanthisvaluetheinfluenceofCaP-NPSoncellcanbeignoredwhereasthecelldeathisinducedremarkablywhentheCaP-NPSamountinmediaexceededthiscriticalpoint.WesuggestthatapossiblereasonmaybetheendocytosisofCaP-NPS.ThefindingcanprovideaguidanceontheappropriateamountofCaP-NPSinmedicalbioengineeringsuchasdrugdeliverybioimagingandgenetransfectionetc.1ExperimentalLIPreparationofcalciumphosphatenanoparticlesThechemicalsareanalysis-gradepurchasedfromSinopharmChemicalReagentCo・,LtdChinawithoutadditionalpurificationpriortotheirusage.Allwaterintheexperimentswastriplydistilled.Thecellculturemediawithoutcalciumorphosphatewerepreparedasdescribedeverywhere[
17.Calciumphosphatenanoparticleswereinsitupreparedintheculturemediabyadding10mmol•L1calciumintothecalciumfreesolution.CharacteristicsTransmissionelectronmicroscopyTEMJEM-200CXJapanandscanningelectronmicroscopySEMS-4800HitachiJapanwereusedtoinvestigateCaP-NPS.X-raydiffractionXRDD/max-2550pcRigakuJapanwasemployedtoidentifythestructure.Theconcentrationsofcalciumandphosphateionsweredeterminedbyultraviolet-visibleexaminationUV・VisT6NewcenturyChinaaccordingtotheliteratures118_2C.CellcultureHumanosteoblast-likeMG-63celllineAmericanTypeCultureCollectionUSAwasused.CellswereseededatadensityofSxlO3perwellin24-wellplates.IncubationwasperformedinDulbeccosmodifiedEaglesmediaDMEMGenomBiomedTechnologyInc.Chinacontaining10%fetalbovineserumFBSHangzhouSijiqingBiologicalEngineeringMaterialsCo.Ltd.China.Whenthecellsinplatereachedmonolayerconfluencetheculturemediawerechangedtocalciumandphosphatefreeculturemediawithdifferentquantityofcalciumphosphate
0.
12.
24.
360.480and6X|ig*inL\respectivelyandtheincubationcontinuedfor24h.MTTassayCellviabilitywasmeasuredusingMTT3・[45・liin*lyhhiazdr-2-yl|-
2.5-liphe!yltetrazoliumbromideassay.TheMTIreagentwasenzymaticallyconvertedbylivingcellsintoaLhitVpiiiplefonnazanproduct.SincetheintensityofcolorproducedwasdirectlyrelatedtotherniinlMTofviablerellsitreflectedcellviabilityinvitro.MTIreagentwasaddedtoeachsampleandincubatedat37for4hina24-wellplate.TheblueibrmazanproductwasdissolvedusingdinirthylsulfoxideandIheliquidofeachsamplewasHMnovrdla96-wellplateforassay.Absorbancewasreadonainicroplatereaderat490nmBio・TekInslnnnrnts.UltrathinsectionobservationHiecellsculturedfor24hweredetachedby
0.25%trypsin-EDTAandfixedwith
2.5%glutaral-lrhvle.AfteralreatmBntwith1%osmiumletroxide.thesamplesurrrdehydratedwithalcoholsandnG11ratedwilhe|Mxyresin.TheresinsampleblockwastrimmrcLihin-sectionedandcollectedonIbrmvar-coaledcoppergrils.TheobtainedultrathinsectionswerestainedusingiranylacetaleandleadcitratefortheTEMexamination.StatisticsDatashownan*ineans±SI.ComparisonsbetweenIwomeanswere[M*rfrinrlusingtheStudent/lest
1131.ResultsCharacteristicsofCaP-NPSTheCaP-NPSformedintheculturemediawereexaminedbyTEMFig.1AandSEMFig.1B.TheinsilubiominrnilizedCaP-N*weregrain-likeandconsistedofnunirrousHake-likenanoparticleswiththediameterofabout5nm.TheformedCaP-NPSwithllu1diameterof2Xnmwerewelllis|ersedintheculturemedia.The\B1patternofCalNPSisshownbyFig.IC;allthediffractionpeakscouldbeassignedusinglhestandardhydroxyapatiteXKDpatternPDFNo.09・
0432.ThereforelheprecipitatednanosolidphasewashydroxyapatitethemostlherinostablephaseofcalciumphosphatephaseunderphysiologicalconditionJ.20/°ATEMimage.BSEMimage.CXRIpatternFig.ICharacterizationsofinsitumineralizedCaP-NPSfonnedinthecuhurt*Cellviabilitywithdifferentquantity^aP-NPSTheeffectofcalciumphosphatequantityontheviabilityofM;・63cellswasalsoevaluatedFig.
2.TheMTIresultsshowedthatlheco-culturedcellviabilitywasgraduallydecreasedwiththegrowinganunintofCaP-NPSadded.Notablymorethan90%cellswerestillviablewhentheconcentrationofCaP-Eig.2ViabilityofM»-63cellsinthecultureiiK^liawithdifferentconcentrationsofcalciumphcisphatflccr*aselrapid!hhentheconc*nlralioiwaslargerthan240jig*inLLTheseresultsindicatedthal240|ig•mL1CaP-NIAwasacriticalpointforcellvicibililv.ConcentrationofcalciumandphosphateToconfinnthatthedeathofo-ciilhirelcellswascausedbytheCaP・NPS.theconcentrationsoffreecalciumandfreephosphateionsincellco-culturedinkliawithdi伍Tentamountsofcalciutnphosphalf*phaseat37Cfor24hwen*examinrdbyI\is.AsshowninEig.
3.theconcentrationsofcahiuinionandphosphateionwerekepiasthkconslantftof
1.2**
1.5mmol*I/1and12**
1.6mmo•Lr*sptively.TheincreaseofCaP・NPSquantitylilnotobviouslychangeth*concrnlralionsofcalciumand|hospluitrionsindicatingthatthedecreaseofcellviabilitywasnotcausedbythecalciumionorCalciumphosphateconcentration/pgmlphosphateiongeneratesIbythemineraldissolution.Moreimportantlytheconcentrationsofcalciumandphosphateionsinthe:aP・NPScontainingmediaweresimilarwiththoseinthenormal*ulturemediaimplyingthattheinfluenceofcalciumionandphosphateiononcellviabilitycanberxclulrl.UltrathinsectionTodeterminetheinfluenceofCaP-NPSoncellinnerstnicturrsandIhrpresenceofCaP-NISintheinnercelltheullra-thinsectionalsampleswerepreparedandrxaininrdbyTEM.UnderbiologicalTEM.nocalciumphosphateparticleuasobsenrdinsidecellswhenCaP-NI^Sconcentraliotwaslessthan240|ig*tnL1Fig.4A・howeverlotsofCaP-NISwereobservedinthecytoplasmofM;・63cellswhentheconcrntrationexceeded240|xg•inL1Eig.4B.Moreiin|xi*tantlythemoiphologyandsizeofCaP-NISinlhecytoplasmwereconsistrnlwithIheinsifumitieraliztdparticlesinuhiin*mediaFig.4C.indicatingthallhecelldeathinighliinluctMlbytheetidiwylicHoweversuchainduceddea山wasonlyobservedwhenCaP-NPSwasmorethan240|ig*inL1inlhfcculhin*nuMlia.Underaconditionof240pig-nil/1wesuggestlluiltheparticlescoulddissolvedandclearedbycellssothatIheirpresencescouldnotledeleeledallargescale.Discussion\Xeinvestigatedlhecelldeathinducing-rffeclofCaP-NPSdependingontheirquantitywiththeinfluenceofCaP-NPSsizeandcrystalliteexcluded.ThestableCaP-NPSinsitupreparedinabiocompatible/biologicallyfriendlywayprovidedanopportunitytoinvestigatetherelationshipbetweenCaP-NPSandtheirbiologicaleffects.Previousreportsdemonstratedthatthephysicalcharacteristicsincludingsizemorphologyandcrystallinityoftheexsitupreparedcanaffectcellbehaviorssignlicantly^
22241.HereauniformCaP-NPSwiththesamesizeandcrystallitywasinsitupreparedincellculturemediasothatthequantityeffectofCaP-NPScanbeexactlyexamined.WeobservedthatthecelltoxicitiesofCaP-NPSonMG~63cellswereinaccordancewithpreviousreportsonothercellssuchaslivercolonandstomachcancercells125-261andtheeffectisstrengthenedwithincreasingparticlesconcentration.Whenthevalueishigherthan240pig-mL1CaP-NPSincellculturemicroenvironmentcansignificantlyimpaircellviabilityofMG-
63.ItfollowsaquantityeffectoftheinteractionofCaP-NPSandlivingcellswhichprovidesaguideforapplicationofcompositehybridmaterialforbiomedicalgoals.Asthedeath-inducingeffectofcalciumorphosphateionsoncellshasbeenreportedweexaminedwhethertheconcentrationofcalciumionorphosphateioncontributedtothisdeathinducement.ThefinalconcentrationofcalciumorphosphateioninculturemediawasmeasuredandtheirinfluencecouldbeexcludedbecausetheconcentrationofcalciumionorphosphateioninculturemediacontainingCaP-NPSwassimilarwiththatinnormalculturemedia
1161.Inadditiontheultra-thinTEMresultsrevealedthatlotsofCaP-NPSwereobservedincytoplasmofcoculturedcellswhenmediaCaP-NPSwas240pig*mLt.HowevernocalciumphosphatenanoparticlewasfoundincytoplasmwhenmediaCaP-NPSbelowthecriticalpoint.ThemechanismofcelldeathinducedbyCaP-NPSmaybeexplainedbytheincreasedintracellularcalciumconcentration.TheendocyticCaP-NPSarerapidlydegradedinlysosomesandsubsequentacidificationleadstothereleaseofcalciumionsintotheceilandthereleasedcalciumisinitiallysequesteredbycalciumstoresorpumpedoutofthecelLHoweverathighCaP-NPSconcentrationthelargeamountofendocyticCaP-NPSresultinasevereincreasingofcalciumionsincytoplasmwhichexceedstheself-handlingabilityofcellandcausesthelossoffunctionofmembranepumps.1121IncontrastatlowconcentrationtheimpairmentfromtheendocyticCaP-NPScanbeeliminatedbycellselfhandling.Thebiomineralphaseisdegradedinlysosomesandtheacidificationleadstothereleaseofcalciumionswhicharesubsequentlysequesteredbycalciumstoresorpumpedoutofthecell.WenoticethatintheclinicapplicationsofCaP-NPSespeciallygenetransfectionanddrugdeliveryCaP-NPSareexpectedtoenterintocellstoimprovetheefficienciesofgenetransfectionanddrugdeliverybutexceededCaP-NPScanalsoseverelyinducethedeathofcells.ThereforethequantitycontrolofCaP-NPSshouldbeconsideredasavitalfactorbeforeciinicapplicationsarecarriedout.Hereinweclearlydemonstratethat240|xg•mL1isacriticalpointfortheinsitumineralizedcalciumphosphateinbiologicalgrowthmicroenvironment.4ConclusionsInthisstudytheCaP-NPSareinsitusynthesizedincalciumandphosphatefreeculturemedia.ThecorrelationoftheirquantityandlethaleffectonMG-63cellsisevaluated.OurresultsdemonstratethatcelldeathinductioneffectisrelatedtotheconcentrationofCaP-NPSand240isacriticallimitationtokeepcellviability.Thisworkprovidesaninstructionfortheapplicationsofcalciumphosphatenanoparticlestoensurebiosecurityinbiomedicalapplications.References:DorozhkinS.Materials20092⑵:399»498TaoJHJiangWGPanHHetal.J.Cryst.Growth.20073081:151-158KoutsopoulosS.J.Biomed.Mater.Res・2002624:600-612LemosARebeloARochaJetal.KeyEng.Maier.2005284-28617:67-702£l-8Zl:(l)oczooz㈣•岫%rp4s2ucio9u^ax口即394S60ri60l:(£)6r800Z7汕wrwTY•即WT.伊P,AUBHMSflT【9服8[穴]n£g-ifr£(6J)9iti0JW伽a如叫沏uiqznzoriS^iw宓Wug孙$泣[3“湖jbp丫粉Mfc)unS-u!AVW(告王爰)新$咔IV(即军川叫OVD[tZ]869M)69fr:(tt)iriOOZFgWRW・f-p衿、An门y口叽,H0nH[£ZJL8££・08L£(9£)LI匕00・也神砰V7F0跄UM•An门为k^lt\9Vl£-0£l£:(Ll)ItZOQZVPS蚀Iuag^a8uy•印研ddgSu!^MzaioallZ]PL・69⑴Z0€,66【初1°UV爬V•也J9HBW*1puouiui(ua
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