Description

Numeroustechniqueshavebeendevelopedtoprepareimmunoliposomesbasedonthenucleophilicreactivityoffreeaminegroupsofproteinsorpeptides.However,themostcommon,versatileandstraightforwardactivationchemistryforcreatingreactiveacylatingreagentsandlabelingpeptides/proteinsistoformNHSesterwithprimaryamines.AsinglestepnucleophilicsubstitutionreactionbetweenNHSesterderivativeandalphaaminesattheNterminalorthebetaaminesoflysinesidechainsleadstotheformationofastableamidebond.

N-Hydroxysuccinimide(NHS)estersofDSPE-PEG-NHSliposomesreactwiththeprimaryaminegroupsonthepeptides,proteins,antibodiesorotherfunctionalligandsfortargeteddrugdeliveryapplications.ThenucleophilicattackoftheaminegroupsontheNHS-activatedcarbonylgroupoftheDSPE-PEG-NHSresultsintheeliminationoftheNHSgroupandformationofamidelinkage.

Immunodox®-NHSisaPEGylatedproduct.Forotheraminereactive(PEGylatedandnon-PEGyalatedproducts)andalsoImmunodox®productssuitableforothertypesconjugationmethodsseehere.

DownloadProductInsertDownloadSafetyDatasheet(SDS)

FORMULATIONINFORMATION

Immunodox®-NHS(PEGylated)(Post-insertion)

Vial2*	Specification
DSPE-PEG(2000)-NHSLipid	ThisvialcontainsreactiveDSPE-PEG(2000)-NHSlipidinpowderform.Thislipidisconjugatedtoareactiveprotein,peptideorligandcontainingamineandthenmixedwithnon-reactiveDSPE-PEG(2000)lipidinaqueoussolutiontoformmicelles.ThePEGylatedlipidmicellesareincubatedwithpreformedliposomesinvial1andPEGlipidswillpost-insertthemselvesintotheliposomes.
*TheamountofthepowderedPEG(2000)-NHSlipidforthe2-mlkitis1.34mgandforthe5-mlkitis3.34mg.

Vial3*	Specification
DSPE-PEG(2000)Lipid	Thisvialcontainsnon-reactiveDSPE-PEG(2000)lipidinpowderform.ThislipidinmixedwithDSPE-PEG(2000)-NHSlipidwhichisalreadyconjugatedtoaligand(protein,peptide,etc.)inaqueoussolutiontoformmicelles.ThePEGylatedlipidmicellesareincubatedwithpreformedliposomesinvial1andPEGlipidswillpost-insertthemselvesintotheliposomes.
*TheamountofthepowderedPEG(2000)-DSPElipidforthe2-mlkitis5mgandforthe5-mlkitis12.5mg.

ConjugationProtocol

MaterialsandEquipment

The3-vialpost-insertionkitcontainspreformedliposomes(vial1),non-reactivePEGylatedlipidinpowderform(vial2)andDSPE-PEG(2000)-NHSlipidinpowderform(vial3). Inordertousethepost-insertionkit,youwillneed:

1. Laboratoryvortexmixerisrecommendedtohave.
2. Laboratorymagneticstirrerisneededfordialysis.
3. Twosmall10mlroundbottomflasksortwosmallglassvials.
4. Arotaryevaporator. Weunderstandthatmanylabsmightnothavearotovap.Alternatively,youcanuseanitrogentankconnectedtoathinhoseforcreatingastreamofnitrogenflowtodrythelipidandmakeathinfilm.
5. Asmallamountofasolventsuchachloroformormethylenechloride(youwillonlyneedafewmilliliters).
6. Phosphatebufferedsaline(PBS).
7. ASonicator.Itisbettertohaveabathsonicator.Ifyoudonot,thatisfine.Youstillcanfollowtheprotocol.Youmayalsouseavortexinsteadofthesonicatorforagitationofthesolutionaswell.
8. Float-A-Lyzer®withaproperMWCOthateasilyallowsthecleanupofyourliposomeconjugatedligandfromfreeandnon-conjugatedprotein,peptideorantibody.YouneedtomakesurethattheMWCOisbelow1,000,000dalton.At1,000,000daltontheporesizeonthedialysismembranegetscloseto100nmandthereforeyourliposomescanbedialyzedout.Youcannotusedialysiscassettesblindly.Pleaseunderstandthetechniquebeforeusingeitherspincolumnordialysiscassette.IfyoudonotusethecorrectMWCOyoucanloseyourentireprep.ForthisprotocolwerecommendMWCOof300,000dalton.

PreparationMethod

1. Thepost-insertionkitscomeintwosizes;2mland5ml.Forthe2mlkitsize,dissolvethecontentofvial3in100µlofchloroformormethylenechloride.Forthe5mlkitsize,thecontentofvial3shouldbedissolvedin250µlofchloroformormethylenechloride.Transferthesolutiontoa10mlroundbottomflask.Drythechloroformusingarotaryevaporatororunderastreamofnitrogenandmakeadriedlipidfilm.
2. Forthe2mlkit,add100µlofPBSbuffertothedriedlipidfilm.Forthe5mlkit,theamountoftheaddedbufferis250µl.Itispreferredtosonicatethehydratedlipidfilmusingabathsonicatorandsonicatethemicellesolutionfor5minutes.IfyoudonothaveabathsonicatorthenhydratethedriedlipidfilmwithPBSforatleast1hourandconstantlyrotatethesolutionintheroundbottomflaskusingarotavap(notconnectedtovacuum)orbyhandtomakesurethatallthedriedlipidonthewalloftheroundbottomflaskwillgotothesolutionandformmicelles.Alternatively,youcanuseavortextoagitatethesolution.Thegoalistohaveallthedriedlipidonthewalloftheroundbottomglasstogothemicellesolution.CoverthemouthoftheroundbottomflaskwithParafilm.Refrigeratethemicellesolutionofnon-reactivePEGlipidsuntilitisreadytobemixedwithmicellesformedinstep4.
3. For2mlpost-insertionkit,theamountofDSPE-PEG-NHSis1.34mg(0.22µmol)andfor5mlofpost-insertionkittheamountofDSPE-PEG-NHSis3.34mg(0.55µmol).Forthe2mlkitsize,dissolvethecontentofvial2in100µlofchloroformormethylenechloride.Forthe5mlkitsize,thecontentofvial2shouldbedissolvedin250µlofchloroformormethylenechloride.Transferthesolutiontoa10mlroundbottomflask.Drythechloroformusingarotaryevaporatororunderastreamofnitrogen.
4. Addthewater-solubleprotein,peptideorligandat1:2molarratioofligandtodriedfilmofDSPE-PEG-NHS.For2mlkitadd300µlofwater-solublesolutionofproteinofligandinPBS(pH7.4)tothedriedlipidfilmandfor5mlkitadd750ulofwater-solublesolutionofproteinofligandinPBS(pH7.4)tothedriedlipidfilm.Itispreferredtosonicatethehydratedlipidfilmusingabathsonicatorandsonicatethemicellesolutionfor5minutes.IfyoudonothaveabathsonicatorthenhydratethedriedlipidfilmwithPBSforatleast1hourandconstantlyrotatethesolutionintheroundbottomflaskusingarotavap(notconnectedtovacuum)orbyhandtomakesurethatallthedriedlipidonthewalloftheroundbottomflaskwillgotothesolutionandformmicelles.Alternatively,youcanuseavortextoagitatethesolution.Thegoalistohaveallthedriedlipidonthewalloftheroundbottomglasstogothemicellesolution.Thesolutionisincubatedatroomtemperaturefor6hoursandinrefrigeratorfor24hours.ThereactionispHsensitive.Readthetechnicalnotebelowformoreinformation.
5. Mixthemicellesinstep2tothemicellesinstep4.Thetotalvolumeofmicellesfor2mlkitshouldbe400µlandfor5mlkitshouldbe1000µl.
6. Toconductpost-insertion,themicellardispersionisthenco-incubatedwithpreformedplainliposomesat60℃for30min.
7. Removethenon-conjugatedprotein,peptideorantibodyfromtheimmunoliposomesbydialysis.Wepreferdialysistosizeexclusioncolumns.Dialysisisamuchslowerprocessbuttherewillbeminimumlossofimmunoliposomesaftertheprepiscleanedfromnon-conjugatedprotein/peptide/ligand.Spincolumnsaremuchfaster;however,youcaneasilyloseover50%oftheliposomesonthespincolumn.WerecommendusingFloat-A-Lyzer® dialysiscassettefromSpectrumLabs.YouwillneedtochooseacassettewithproperMWCOdependingontheMWofyourprotein,peptide,antibodyorantibodyfragment.NOTE:Ifyoudecidetouseadialysiscassette,youwillneedtomakesurethattheMWCOisbelow1,000,000dalton.At1,000,000dalton,theporesizeonthedialysismembranegetscloseto100nmandtherefore,yourliposomescanbedialyzedout.Youcannotusedialysiscassettesandspincolumnsblindly.Theycomeinvarioussizesandyouneedtochoosethecorrectsizewisely.Dialyzetheimmunoliposomesolutionin1literofPBSatpH7.4for8hours.Changethedialysisbufferwithafresh1literofPBSandletisdialyzeforanother8hours.Afterthisstep,yourcleanedupimmunoliposomeisreadytobeused.

LiposomeParticleCalculator

Immunodox®liposomesareunilamellarandsizedto100nm.Themolarconcentrationofliposomeis24.56mM.Byhaving liposomediameter(nm)andlipidconcentration(µM),youcancalculatethetotalnumberofthelipidsinoneliposomeandthenumberoftheliposomesinonemilliliteroftheliposomesolution.Tousethecalculatorclick here.

TechnicalNotes

• Doxorubicinisafluorescentmoleculewithλ_ex470nmandλ_em585nm.Ifyouareusingafluorescenttagonyourantibodyorligand,thenyouneedtomakesurethattheywillnotinterferewitheachother.
• HydrolysisofDSPE-PEG-NHSinaqueoussolutionscompeteswiththeprimaryaminereaction,resultingintheeliminationoftheNHSgroup,whichcanconsequentlydecreasethecouplingyieldpriortothereactionwiththeprotein/antibody.InordertominimizetheimpactofthehydrolysisofNHSester,useahighconcentrationofprotein/antibodytoincreasetheefficiencyofthecross-linking.
• ThereactionofNHSesterswithaminesisstronglypH-dependent:atlowpH,theaminogroupisprotonated,andnomodificationtakesplace.Athigher-than-optimalpH,hydrolysisofNHSesterisquick,andmodificationyielddiminishes.Thehalf-lifeofNHSestersatpH7and8is4-5hoursand1hour,respectively.WhilstNHSestershaveahalf-lifeofonly10minutesatpH8.6.Therefore,toavoidthehydrolysisofNHSester,DSPE-PEG-NHSlipidshouldbeusedimmediatelyforconjugationtoantibodies,proteinsorpeptidescontainingfreeamines.NHSesterreactionsareconductedincommonbuffersatpH7-8.
• PrimaryaminebufferssuchasTrisshouldNOTbeusedbecausetheycompeteforreaction;however,insomeprocedures,itisusefultoaddTrisorglycinebufferattheendofaconjugationproceduretoquench(stop)thereaction.
• IfyouareusingaligandorpeptidethatishydrophobicthenitisrecommendedtosolubilizeitinDMSOorDMFandthenaddthebuffertoit.Itisrecommendednottousemorethan5%volumeofDMSOorDMFinthesolution.DMFandDMSOarebothcompatIBLewithliposomesandtheyarealsomiscibleinwater.Otherorganicsolventsuchasethanolandchloroformarenotcompatiblewithliposomesandwillcausetheliposomestolyse.IfyouendupusingDMSOorDMFthenaftertheconjugationreactionisdone,youneedtoremoveDMSOandDMFfromtheliposomes.InordertodothatyouneedtouseadialysiscassettethatismadefromREGENERATEDCELLULOSEMEMBRANE.NOTE:NotallmembranesarecompatiblewithDMFandDMSO.WerecommendusingaSlide-A-Lyzer™MINIDialysisDevicewithMWCOof2KmadefromregeneratedcellulosemembranemanufacturedbyThermofisher.AfterDMSOorDMFisremovedyoucanuseFloat-A-Lyzer®dialysisdeviceforthefinalstepofcleaninguptheprep.
• Liposomesshouldbekeptat4°CandNEVERbefrozen.

Database

Directlinktothedatabasepageforeasynavigation:ImmunoliposomesConjugationDatabase

Appearance

Immunodox®-NHS(PEGylated)post-insertionkitcomesinthreevials:vial1isaredtranslucentliquidmadeofnanosizeunilamellarliposomeswhichdoesnotcontainanyreactiveofnon-reactivePEGylatedlipid. Usuallyduetothesmallsizeofliposomesnosettlingwilloccurinthebottomofthevial.Vial2containsreactiveDSPE-PEG(2000)-NHSlipidinwhitepowderform.Vial3containsnon-reactiveDSPE-PEG(2000)lipidinwhitepowderform.

EducationalVideo

Ordering/ShippingInformation

• Allliposomebasedformulationsareshippedonblueiceat4°Cininsulatedpackagesusingovernightshippingorinternationalexpressshipping.
• LiposomesshouldNEVERbefrozen.Icecrystalsthatforminthelipidmembranecanrupturethemembrane,changethesizeoftheliposomesandcausetheencapsulateddrugtoleakout.Liposomesinliquidformshouldalwaysbekeptintherefrigerator.
• ClientswhoorderfromoutsideoftheUnitedStatesofAmericaareresponsiblefortheirgovernmentimporttaxesandcustomspaperwork.EncapsulaNanoSciencesisNOTresponsibleforimportationfeestocountriesoutsideoftheUnitedStatesofAmerica.
• WestronglyencouragetheclientsinJapan,Korea,TaiwanandChinatoorderviaadistributor.Toughcustomsclearanceregulationsinthesecountrieswillcausedelayincustomclearanceoftheseperishableformulationsifordereddirectlythroughus.Distributorscaneasilyclearthepackagesfromcustoms.Toseethelistofthedistributorsclickhere.
• ClientsorderingfromuniversitiesandresearchinstitutesinAustraliashouldkeepinmindthattheliposomeformulationsaremadefromsyntheticmaterialandtheformulationsdonotrequirea“permittoimportquarantinematerial”.LiposomesareNOTBIOLOGicalproducts.
• Ifyouwouldlikeyourinstitute’sFedExorDHLaccounttobechargedforshipping,thenpleaseprovidetheaccountnumberatthetimeofordering.
• EncapsulaNanoScienceshasnocontroloverdelaysduetoinclementweatherorcustomsclearancedelays.YouwillreceiveaFedExorDHLtrackingnumberonceyourorderisconfirmed.ContactFedExorDHLinadvanceandmakesurethatthepaperworkforcustomsisdoneontime. AllsubsequentshippinginquiriesshouldbedirectedtoFederalExpressorDHL.

StorageandShelfLife

Storage

Immunodox®productsshouldalwaysbestoredatinthedarkat4°C,exceptwhenbroughttoroomtemperatureforbriefperiodspriortoanimaldosing.DONOTFREEZE.IfthesUSPensionisfrozen,theencapsulateddrugcanbereleasedfromtheliposomesthuslimitingitseffectiveness.Inaddition,thesizeoftheliposomeswillalsochangeuponfreezingandthawing.

ShelfLife

Immunodox®-NHSismadeondailybasis.Thebatchthatisshippedismanufacturedonthesameday.Itisadvisedtousetheproductswithin4monthsofthemanufacturingdate.

ReferencesandbackgroundreADIng

1.NaK,LeeSA,JungSH,HyunJ,ShinBC.Elastin-likepolypeptidemodifiedliposomesforenhancingcellularuptakeintotumorcells.ColloidsandSurfacesB:Biointerfaces.2012Mar1;91:130-6.

2.DaiW,FanY,ZhangH,WangX,ZhangQ,WangX.AcomprehensivestudyofiRGD-modifiedliposomeswithimprovedchemotherapeuticefficacyonB16melanoma.Drugdelivery.2015Jan2;22(1):10-20.

3.FerreiraDdosS,FariaSD,deAraújoLopesSC,TeixeiraCS,MalachiasA,Magalhães-PaniagoR,deSouzaFilhoJD,OliveiraBL,GuimarãesAR,CaravanP,FerreiraLA.Developmentofabone-targetedpH-sensitiveLiposomalformulationcontainingdoxorubicin:physicochemicalcharacterization,cytotoxicity,andbiodistributionevaluationinamousemodelofbonemetastasis.Internationaljournalofnanomedicine.2016;11:3737-51.

4.WangD,FuJ,ShiY,PengD,YuanL,HeB,DaiW,ZhangH,WangX,TianJ,ZhangQ.ThemodulationoftumorvesselpermeABIlitybythalidomideanditsimpactsondifferenttypesoftargeteddrugdeliverysystemsinasarcomamousemodel.JournalofControlledRelease.2016Sep28;238:186-96.

5.FeiW,ZhangY,HanS,TaoJ,ZhengH,WeiY,ZhuJ,LiF,WangX.RGDconjugatedliposome-hollowsilicahybridnanovehiclesfortargetedandcontrolleddeliveryofarsenictrioxideagainsthepaticcarcinoma.Internationaljournalofpharmaceutics.2017Mar15;519(1):250-62.

6.AbuchowskiA,KazoGM,VerhoestJrCR,VanEsT,KafkewitzD,NucciML,ViauAT,DavisFF.Cancertherapywithchemicallymodifiedenzymes.I.Antitumorpropertiesofpolyethyleneglycol-asparaginaseconjugates.Cancerbiochemistrybiophysics.1984Jun;7(2):175-86.

7. SartoreL,CalicetiP,SchiavonO,VeroneseFM.EnzymemodificationbyMPEGwithanaminoacidorpeptideasspacerarms.Appliedbiochemistryandbiotechnology.1991Jan1;27(1):45-54.

8. ShahinM,SoudyR,El-SikhryH,SeubertJM,KaurK,LavasanifarA.Engineeredpeptidesforthedevelopmentofactivelytumortargetedliposomalcarriersofdoxorubicin.Cancerletters.2013Jul1;334(2):284-92.