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1、Cell-specificGeneTransfectionfromGene-functionalizedPoly(D,L-lacticacid)SubstrateFabricatedbyLayer-by-LayerAssemblyTechniqueKaiyongCai*,YanHu,ZhongLuo,TingKong,MinLai,XiaojingSui,YuanliangWang,LiYang,andLinhongDengExperimentalMaterials:ThepSV-β-galactosidasecontrolvecto
2、rcontainingSV40precursor,enhancerandLacZgenewassuppliedbyPromegaCorporation(Madison,WI).pSV-β-galactosidaseplasmidwasamplifiedinEscherichiacoli,andthenisolatedandpurified.Chitosan(fromcrabshells,lowmolecularweight,viscosity:2000cp,Sigma),1-Ethyl-3-(3-dimethylaminopropyl
3、)-carbodiimide(EDC),lactobionicacid(LA),3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide(MTT),N-hydroxylsuccinimide(NHS),tetramethylethylenediamine(TEMED)werepurchasedfromSigmaChemicalCo.(MO,USA).Lipofectamine2000waspurchasedfromInvitrogenCo.(CA,USA).Synthesi
4、sandcharacterizationofLA-NHSester:3.58gramofLAwasdissolvedin100mlofNaHCO3/aceticacidbuffersolution(pH4.7)andthenactivatedwithamixtureofNHS(0.58g)andEDC(3.83g)(molarratioofNHS/EDCis0.25),reactingfor3hwithstirringatroomtemperature.Thereactedsolutionwaspouredintoexcessivea
5、nhydrousalcohol(analyticalgradepurity)tocollectwhitesolidsediment.Subsequently,collectedsamplewassubjecttoberinsedwithanhydrousalcoholfor10times.Samplewasdriedviaavacuumdryingovenandkeptinadissectorforfurthercharacterization.SynthesisandcharacterizationofGC:Galactosylat
6、ionofchitosanwascarriedoutusingEDC[20]andNHSascouplingagentsaccordingtoapreviousreport.Briefly,differentamountsofLAweredissolvedin100mlofNaHCO3/aceticacidbuffersolution(pH4.7)andthenactivatedwithamixtureofNHSandEDC(molarratioofNHS/EDCis0.25),reactingfor3hwithstirringatr
7、oomtemperature.ThemolarrationforNHS/LAis0.5inallstudies.Subsequently,2.2gchitosanwasaddedintoeachsolutiontoobtainvariousLA/chitosanmolarratiosof0.5,0.8,1.0and2.0.Thereactionwasperformedfor3dayswithstirringatroomtemperature.Theresultingproductswerepurifiedusingadialysist
8、ube(8000-14,000MWCO)againstdistilledwaterfor3days,followedbylyophilizationfor3days.PreparedGCswithdifferentLA/
