| Nanomaterials have been broadly studied for intracellular delivery of diverse compounds for diagnosis or therapy. The evolution of these nanoparticles(NPs) was followed by their successful “stealth†by modifying the NPs surface using polyethylene glycol(PEG) that can prevent non-specific binding blood components and therefore reduce their rapid uptake and clearance in vivo by the cells of the mononuclear phagocytic system(MPS), leading to prolonged blood circulation. Currently it remains challenging for discovering new biomolecules that can prominently enhance cellular internalization of NPs. Herein we report that a mussel-inspired engineering approach may notably promote cellular uptake of NPs. In this strategy, the catechol moiety is covalently anchored onto biodegradable NPs. Thus fabricated NPs can be more effectively internalized by sensitive and multidrug resistance tumor cells as well as some normal cells, resulting in remarkably potentiated in vitro activity when an antitumor drug is packaged. Moreover, the newly engineered NPs showed increased adhesion at the tumor site and in the mucosal of gastrointestinal tract via local delivery or oral administration, respectively. This biomimetic approach is promising for creating functional nanomaterials for biosensing, molecular imaging, drug delivery, and cell-based therapy.Methods1. Synthesis of carrier materialsThe kinetically controlled acetalization reaction of β-CD was performed in the presence of excess amount of 2-methoxypropene(MP), using pyridinium p-toluene sulfonate(PTS) as a catalyst. Briefly, 20 m L MP(210 mmol) was added into 100 mL anhydrous DMSO containing 5 g β-CD(4.4 mmol), into which 80 mg PTS was added. After 3 h of acetalation under magnetic stirring at room temperature, the reaction was terminated by adding 2 mL triethylamine into the mixture. The acetalated product(Ac-bCD) wasprecipitatedfromwater,collectedbyfiltration,thoroughlywashedwithdeionizedwater,andlyophilizedtoawhitepowder.dopamine(dopa)-conjugated1,2-distearoyl-sn-glycero-3-phosphoethanolamine-n-[carboxy(polyethyleneglycol)-2000](dspe-peg-dopa)wassynthesizedbycouplingreaction.tothisend,100mgofdspe-peg-cooh(35μmol)wasdissolvedinto40mlpbs(ph7.4).then,20mgofn-hydroxysuccinimide(nhs)(176μmol)and67.3mgn-(3-dimethylaminopropyl)-n′-ethylcarbodiimidehydrochloride(edc·hcl)(350μmol)wereadded.after12 hofactivationatroomtemperature,66.4mgofdopa·hcl(350μmol)wasaddedintothereactionmixture,followedbymagneticstirringfor24hundertheprotectionofnitrogenat4oc.theobtainedpolymerwaspurifiedbydialysisfor24husingdialysistubing(withmolecularweightcut-offof1000da)indeionizedwaterandthencollectedbyfreeze-drying.2.materialscharacterizationdspe-peg-dopaandacetalatedβ-cd(ac-bcd)wascharacterizedbyfourier-transforminfraredspectroscopy(ft-ir)andnuclearmagneticresonance(nmr)spectroscopy.3.preparationofnanoparticlesamodifiednanoprecipitation/self-assemblymethodwasemployedtoprepareac-bcdnps.briefly,50mgac-bcdwasdissolvedin2 mlacetonitrile.lecithinanddspe-pegatthemolarratioof7:3weredispersedin0.8mlethanol,andthen19.2mldeionizedwaterwasadded.thusobtainedaqueousdispersionwasheatedto65ocfor1h.then,theac-bcdsolutionwasaddedintothepreheatedaqueoussolutiondropwise(1ml/min)undergentlestirring,followedbyvortexingfor3 min.after2hofincubation,themixturewascooledtoroomtemperature.thesolidifiednpswerecollectedbycentrifugationat16,000rpmfor10 min,rinsedwithdeionizedwaterforthreetimes.followingsimilarprocedures,ac-bcdnpsbasedondspe-peganddspe-peg-dopaatdifferentmolarratioswerefabricated.similarly,paclitaxel(ptx)-loadedandcy5orcy7.5-labelednpswereproduced.4.characterizationofvariousnanoparticlesmorphologyofvariousnpswascharacterizedbytransmissionelectronmicroscopy(tem).particlesize,sizedistribution,andzetapotentialweremeasuredbydynamiclightscattering(dls).5.invitrohydrolysisofnanoparticlesacertainamountofnpswasdispersedintopbswithph7.4orph5.0.atvarioustimepoints,thehydrolysisofnpswasobservedandthetransmittancewasmeasuredat500nmbyultraviolet–visiblespectroscopy.6.cytotoxicityevaluationcellswereincubatedfor12h,andthentheyweretreatedwiththemediumcontainingvariousconcentrationsofnpsforacertainperiodoftime.thecellviabilitywasquantifiedbythemttassay.thevaluesofhalfmaximalinhibitoryconcentration(ic50)werecalculatedbycurvefittingusingoriginpro7.0.7.intracellularuptakestudybyfluorescencemicroscopymousemelanomacells(b16f10)andhumanbreastcancercells(mda-mb-231)wereculturedfor24h.thentheculturemediumwasreplacedbyfreshmediumcontainingcy5-labelednpsandincubatedfor2,4,8or12 h,respectively.beforeobservation,cellswerestainedwithlysotrackerredand1,1’-dioctadecyl-3,3,3’,3’-tetramethyl--indocarbocyanineperchlorate(dil).afterwashingwithpbs,cellswerecounterstainedwith4’,6-diamidino-2-phenylindole(dapi).cellswereobservedbyconfocallaserscanningmicroscopy(clsm).8.quantificationofcellularuptakebyflowcytometryuptakeefficiencyofdifferentcelllinesfornpswasquantifiedbythefluorescenceactivatedcellsorting(facs)technique.specifically,b16f10,mda-mb-231,mouseleukaemicmonocytemacrophagecells(raw264.7),andmousevascularsmoothmuscle(movas)cellswereplantedin6-wellplatesfor24 hbeforetheexperiments.generally,thecellstreatedwithnpswerewashedthreetimeswithpbs(ph7.4),followedbytrypsinization,centrifugation(1000rpmfor5min),andwashingwithpbs(ph7.4),andthenre-suspendedinfacssolution.thecelluptakeofcy5-labelednpswasquantifiedbyflowcytometry.9.effectsofvarioustreatmentsoncellularuptakeb16f10cellswereincubatedfor24hbeforetheuptakeexperiment.thecellswerepre-incubatedat4ocfor30minorpre-treatedwithvariousinhibitors(nocodazoleat10μg/ml;cytochalasindat5μg/ml)for30min,followedbytheadditionofcy5-labelednps.after4 hofincubation,thecellswerewashedthreetimeswithpbs.thenthecellswerere-suspendedinfacssolution,andanalyzedbyflowcytometry.10.invitroantitumoractivityofptx-loadednanoparticlesallcellswereincubatedunderstandardconditionsfor24hbeforetreatment.cellswerethentreatedwiththemediumcontainingvariousptxformulations(includingfreeptxandptx-loadedac-bcd/dspe-pegnpswithorwithoutdopacoating)atdifferentdoses.after24 hofincubation,thecellviabilitywasquantifiedbythemttmethod,andtheic50 valueswerecalculated.11.studyontheintratumorretentionofnanoparticlesthehumanbreastcancercelllinemcf-7xenograftswereestablishedbysubcutaneousinjectionintherightflankoffemalebalb/cathymicnudemicewithasuspensionofmcf-7cells.whentumorsreachedanaveragevolumeof100-150mm3,tumorbearingmicewererandomlyassignedinto3groups(n=3).then,intratumorinjectionof50μlofaqueoussolutioncontainingcy7.5-labelednps(10mg/ml)wasimplemented.real-timefluorescenceimagingwasperformedandthefluorescenceintensityatvarioustimepointswasdeterminedbyalivingimagingsystem.after72 h,themiceweresacrificed,bothtumorsandmainorganswereresectedforfurtheranalysis.12.studyonthegastrointestinaladhesionofnanoparticlesfour-week-oldmalec57bl/6micewererandomlyassignedinto3groups(n=3)andwerefastedfor24hbeforethetreatment.then,themiceweretreatedwith300μlofaqueoussolutioncontainingcy7.5-labelednps(4mg/ml)throughintragastricadministration.after12or24 h,themiceweresacrificed.stomach,intestine,andothermainorganswereresectedandimagedbyalivingimagingsystem.results1.characterizationbyft-irand1hnmrspectroscopyshowedthesuccessfulconjugationofdspe-peg-dopaandac-bcds.accordingtothe1 hnmrspectrum,themolarratiooflinearacetaltocyclicacetalwas1.65forthesynthesizedac-bcd.2.npscanbepreparedthroughamodifiednanoprecipitation/self-assemblymethodwithwell-definedsphericalshape,uniformparticlesizedistributionandgooddispersion.thesphericalnpswithaverageparticlesizeof200 nm.thehydrolysisrateofnpsinpbs(ph5)washigherthanthatinpbs(ph7.4).3.comparedwiththecontrolnpswithoutdopa,500μg/mlofac-bcdnpswithdifferentcontentsofdopadidnotdramaticallydecreasethecellviabilityofbothb16f10andraw264.7murinemacrophagecells.afterincubationwithb16f10cellsfor24 h,ac-bcdnpsat0%dopaand80%dopadisplayedthesimilardose-dependentcellviabilityprofiles.therewasnosignificantdifferentbetweentheic50.4.clsmobservationshowedthattheintensityofgreenfluorescencecellsweredramaticallyincreasedaftercellswereco-incubatedwithnpsfor2,4,8,and12 h.themaximalfluorescenceintensitywasobservedat8h,whichwasdecreasedat12 h.atdifferenttimepoints,cellsincubatedwithnpsmodifiedbydopashowedhighgreenfluorescentintensity.whenthecontentofdopawas60%,thehighestfluorescenceintensitywasobserved.flowcytometrywasusedforthequantitativeanalysisoftheresultsandfurtherconfirmedthenpsmodifiedbydopashowedincreasedendocytosis.themaximaluptakeefficiencywasobservedfornpswith60%dopa.5.asthecellswerepretreatedat4°c,theuptakeefficiencyofnpswithdifferentcontentsofdopadecreasedsignificantly.similarly,endocytosiswasinhibitedafterthecellswerepretreatedwithcytochalasindornocodazole.6.observationbytemindicatedthatptx-loadednpswith0%dopaand60%dopaexhibitedwell-definedsphericalshape,uniformparticlesizedistribution,andgooddispersion.nocrystallizationofptxwasobserved.theaverageparticlesizeof0%dopanpswas170nmandzetapotentialof0%dopanpswasabout-33 mv.ptx-loadednpswith60%dopashowedanaverageparticlesizeof184nmandzetapotentialof-32 mv.thedrugloadingcontentofnpswith0%and60%dopawas9.8%and9.7%,respectively.forb16f10,hepg2,mcf-7,andmda-mb-231 cells,pharmacologicalactivityofptx-loadednpswasbetterthanthatoffreeptx.therewassignificantdifferencebetweenptx/ac-bcdnpsat0%dopaandptx/ac-bcdnpswith60%dopa.theobtainedic50valuesofptxnpsat60%dopawerelowerthanthoseofnpswith0%dopaandfreeptx.7.cy7.5-labelednpswereinjectedintothetumor.fornpswithoutdopa,thefluorescencewasmainlylocatedaroundthetumorat24and48h(particularlyat48h).bycontrast,fluorescencewashomogeneouslydistributedinthetumorinjectedwithnpswith60%dopa.moreover,relativelyhighintensitycouldbedetectedfornpswith60%dopa,ascomparedwiththosewithoutdopa,andtherewassignificantdifferenceat48 h.inlinewiththisresult,exvivoimagingandquantificationoftheresectedtumortissuesrevealedthatthefluorescenceintensityat60%dopawasprominentlyhigherthanthatat0%dopa.8.theresultsofexvivoimagingandquantificationatpredeterminedtimepointsshowedthatthefluorescenceintensityinthestomachandintestinaltissueof60%dopanpsgroupwashigherthanthatof0%dopanpsgroupat12h.at24 h,thefluorescenceintensityof60%dopanpsgroupinthestomachwasweakerthanthatof0%dopanpsgroup.ontheotherhand,thefluorescenceintensityinintestinaltissueof60%dopanpsgroupwashigherthanthatof0%dopanpsgroup.also,thefluorescenceintensityintheliverof60%dopanpsgroupwasstrongerthanthatof0%dopanpsgroup.conclusions1.ac-bcdwithgoodph-sensitivitywassynthesizedbykineticallycontrolledacetalationreaction.dspe-peg-dopawassynthesizedwithamidationreaction.2.bothcontrolanddopa-coatednpswerefabricatedthroughamodifiednanoprecipitation/self-assemblymethodwithwell-definedsphericalshape,uniformparticlesizedistribution,gooddispersion,andph-sensitiveproperty.npscoatedwithdifferentcontentsofdopashowedlowtoxicityfordifferentcells.thisresultconfirmedthatdopadidnotincreasethecytotoxicityofnps,offeringnpswithgoodbiocompatibility.3.ac-bcdnpsmodifiedwithdopashowedsignificantlyenhancedendocytosis,andnpswith60%dopaaffordedthebesteffect.4.ptx-loadednpswith0%dopaand60%dopawerepreparedwithwell-definedsphericalshape,uniformparticlesizedistribution,gooddispersion,andrelativelyhighdrugloading.invitroanti-tumorefficacyevaluationsuggestedthattheeffectofptx-loadednpswith60%dopawasbetterthanthatofnpswith0%dopaandfreeptx.5.npswith60%dopadisplayedmoreeffectiveadhesioneffectintumorandthegastrointestinaltract,whencomparedwithnpswith0%dopa. 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