多糖修饰的巨噬细胞靶向超氧化物歧化酶及其体外降低辐射诱导的炎性细胞因子表达作用

多糖修饰的巨噬细胞靶向超氧化物歧化酶及其体外降低辐射诱导的炎性细胞因子表达作用刘金峰a,b，滕莉a，刘纯慧a,b，胡立宽c，王永刚c，刘宏c，王凤山a,b（a.山东大学药学院生化与生物技术药物研究所；b.山东大学国家糖工程技术研究中心；c.山东大学齐鲁医院肿瘤防治研究中心山东济南250012）Targeteddeliveryofsuperoxidedismutasetomacrophagesbychemicalmodificationwithpolysaccharideandattenuationeffectsonradiation-inducedinflammatorycytokineexpressioninvitroJinfengLiua,b,LiTenga,ChunhuiLiua,b,LikuanHuc,YonggangWangc,HongLiuc,FengshanWanga,ba.InstituteofBiochemicalandBiotechnologicalDrug,SchoolofPharmaceuticalScience;b.NationalGlycoengineeringResearchCenter,ShandongUniversity;c.CancerCenter,QiluHospital,ShandongUniversity,Jinan250012,China.Fax:[phone];E-mail:[e-mail]ilityofsuperoxidedismutase(SOD)tosuppressreactiveoxygenAbbreviations:(SOD)Cu,Zn-superoxidedismutase;(ROS)reactiveoxygenspecies;(TMC)N,N,N-trimethylchitosanchloride*Towhomcorrespondenceshouldbeaddressed.Abbreviations:(SOD)Cu,Zn-superoxidedismutase;(ROS)reactiveoxygenspecies;(TMC)N,N,N-trimethylchitosanchloride*Towhomcorrespondenceshouldbeaddressed.species(ROS)-mediatedinjury,chemicallymodifiedderivativesofSODwithN,N,N-trimethylchitosanchloride(TMC)andheparin,cationizedSOD(TMC-SOD)andanionizedSOD(heparin-SOD),weredesignedandprepared.Inthisstudy,theinhibitoryeffectofTMC-SODandheparin-SODonsuperoxideanionreleasefrommacrophageswasstudiedinvitro.BothTMC-SODandheparin-SODexhibitedexcellentinhibitoryeffectsonsuperoxideanionreleasefrommacrophages,andtheeffectsofTMC-SODsurpassedthoseofnativeSODandheparin-SOD.TheeffectsofTMC-SODandheparin-SODoninflammatorycytokineexpressioninvitrowerealsoevaluated.TheresultsshowedthatbothTMC-SODandheparin-SODcouldsignificantlylowerthelevelsoftransforminggrowthfactor-1(TGF-1)andinterleukine-1(IL-1)expressedbyirradiated3T3fibroblasts.TheseresultsdemonstratedthatcationicpolysaccharideoranionicpolysaccharideSODderivativesmightbeusefulinthepreventionandtreatmentofROS-mediatedinflammatorydiseases.ThisstudyalsodemonstratedthatchemicalmodificationofSOD,especiallycationization,greatlyenhancedSODsintracellulardelivery,andasaconsequence,producedasignificantprotectiveeffectagainstROS-mediatedinjury.Keywords:Superoxidedismutase;chitosan;heparin;chemicalmodification;cationicpolysaccharide;anionicpolysaccharide1.IntroductionPhagocytes,suchasmacrophagesandneutrophils,sometimesproduceanexcessamountofreactiveoxygenspecies(ROS)thatcauseoxidativedamage,asobservedinvariousinflammatorydiseases.Thesefreeradicalsareveryunstableandreactrapidlywithothersubstancesorgroupsinthebody,leadingtocellortissueinjury.Thismaybeillustratedbyoneofthemanymechanismswhichoxidativestresscancausedamagebystimulatingthefreeradicalchainreaction.ROSproducedbymacrophagesplayacentralroleincausinginflammatorydiseases,suchasacuteliverfailure,arthritis,andsepsis[1-3].ROSactivatetransforminggrowthfactor-β1(TGF-β1),oneofthemostimportantgrowthfactorsinthepathogenesisoffibroticdiseases,whichpromotesepithelialcellapoptosis[4,5].Superoxidedismutase(SOD)isanenzymethatscavengesROSandhasthepotentialtotreatinflammatorydiseasesviasuppressingROSproductionbymacrophages[6-8].Howeveritspoorhalf-lifeandlowaffinitytomacrophageshavelimiteditseffectiveness.DirectmodificationofSODisapromisingapproachtoimprovethedeliverypropertiesoftheenzyme[9].VariouskindsofCu,Zn-SODderivativeshavebeendevelopedviachemicalmodificationforthetreatmentofliverorkidneyischemia/reperfusioninjuriesinvivo[10-12],includingglycosylatedandPEGylatedSODs.Inourlaboratory,Cu,Zn-superoxidedismutase(Cu,Zn-SOD)hadbeenchemicallymodifiedwithlowmolecularweightheparin(LMWH),andourpreviousstudyhadprovedthatLMWH-SODconjugate,whichhadalowerimmunogenicityandhigheranti-inflammatoryactivity,alsogainedhigherstabilitiesthanCu,Zn-SODtowardsacid,alkali,heatandtrypsintreatment[10,13].Ourearlystudyalsorevealedthatheparin-SODcouldattenuatebleomycin(BLM)-inducedlungfibrosis[14].Inthisstudyheparin-SODwasalsopreparedusingthesamestrategy.AnotherSODchemicalmodificationstrategyisdirectcationization.Cationizationofproteinsisknowntoimprovetheircellularuptakevianon-specificelectrostaticinteractionswiththenegativechargeofthecellsurface,ashasbeendemonstratedwithSOD[15-18].Mitsuruetal.hadreportedthatdirectcationizationofSODgreatlyenhanceditscellularuptakeandsubsequentlyimproveditsinhibitoryeffectonsuperoxideanionreleasefrommacrophages[19].Chitosanisapositivelychargedpolysaccharidecomposedof2-amino-2-deoxy-(1-4)-d-glucopyranose,whichhasreactiveaminoandhydroxylgroups.Chitosanhasattractedmuchattentionasabiomedicalmaterial,owingtoitsuniquebiologicalactivities,suchasanti-tumor,antibacterial,andimmunostimulatoryactivities[20].Recently,theantioxidantactivityandradioprotectiveeffectofchitosanalsostimulatescientists'interests[21,22].ItwasprovedthatlowmolecularweightchitosanhadahighradicalscavengingactivityforOHandlowradicalscavengingabilityfor2O[23,24].N,N,N-trimethylchitosanchloride(TMC),protonatedchitosan,possesmorepositivechargethanchitosan.WespeculatethatTMC-SODpossiblyhashighaffinitytosomecells,suchasmacrophageswithnegativelychargedsurface.Inthisstudy,theinh