Description

Numeroustechniqueshavebeendevelopedtoprepareimmunoliposomesbasedonthenucleophilicreactivityoffreeaminegroupsofproteinsorpeptides.OneofthemostpopularandcommonlyusedmethodsistocovalentlycouplefreecarboxylicgroupstoprimaryaminesthroughactivationofthecarboxylgroupswithEDC(1-ethyl-3-[3-dimethylaminopropyl]carbodiimide).EDC,whichisaso-calledzero-lengthcrosslinkingagent,reactswiththecarboxyltoformanaminereactiveintermediate(O-acylisourea).TheproducedO-acylisoureacanbeeasilydisplacedbynucleophilicattackfromprimaryaminogroupsinthereactionmixture.However,thisintermediateisunstableandhydrolyzedinaqueoussolutions.Inordertopreventtheintermediatehydrolysis,sulfo-NHS(N-hydroxysulfosuccinimide)isaddedtoEDCtoproduceasignificantlymorestableandmoresolubleactiveintermediate(NHSester).

Consequently,theimmunoliposomesarepreparedbyatwo-stepcouplingprocedure:first,activatingthefreecarboxylgroupofthelinkerlipidincorporatedintheliposomeswithEDCandsulfo-NHS,andthencovalentlyconjugatingantibodiestothelipidsthroughdisplacementofsulfo-NHSgroupsbyantibodyamines,asdepictedbelow.EDC/sulfo-NHScouplingreactionsarehighlyselectiveandhighlyefficient,andtheBIOLOGicalactivityoftheproteinorpeptideispreserved.

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

DownloadProductInsertDownloadSafetyDatasheet(SDS)

FormulationInformation

Immunodox®-Succinyl(PEGylated)

LipidComposition	Concentration(mg/ml)	Concentration(mM)	MolarRatioPercentage
Total	15.91mg/ml	21.58mM	100
HydrogenatedSoyPC	9.58	12.22	57
Cholesterol	3.19	8.25	38
DSPE-PEG(2000)	2.5	0.89	4
DSPE-PEG(2000)-Succinyl	0.64	0.22	1

ConjugationProtocol

MaterialsandEquipment

Inordertoconjugatetheamineonyourantibody, proteinorpeptidetoImmunodox®-Succinyl(PEGylated)liposomesyouwillneed:

1. EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride).Thesolutionshouldbemadefreshmomentsbeforeuse.
2. Sulfo-NHS(N-hydroxysulfosuccinimide).Thesolutionshouldbemadefreshmomentsbeforeuse.
3. Sephadex®spincolumn.SephadexsizeexclusionspincolumncanbeusedforseparationofliposomesformfreeEDC(MW:191.70).SinceEDCisbeingseparatedfromlargeliposomeparticlesthenanysizesofSephadex®spincolumnsuchasG-10,G-15,G-25,G50canbeused.However,keepinmindthatyouwilllosealargepercentageofyourliposomesonthespincolumn.Alternatively,insteadofremovingtheEDCbyspincolumnyoucanquenchitbyusing2-mercaptoethanol.
4. 2-Mercaptoethanol.ToquenchtheunreactedEDC,2-mercaptoethanolisaddedtoformastablecomplexwiththeremainingcarbodiimide.The2-mercaptoethanolmightnotbenecessaryifyouprefertocleanupyourliposomefromfreeEDCusingaspincolumn.
5. Float-A-Lyzer®withaproperMWCOthateasilyallowsthecleanupofyourliposomeconjugatedligandfromfreeandnon-conjugatedprotein,peptideorantibody.YouneedtomakesurethattheMWCOisbelow1,000,000dalton.At1,000,000dalton,theporesizeonthedialysismembranegetscloseto100nmandthereforeyourliposomescanbedialyzedout.Youcannotusedialysiscassettesblindly.Pleaseunderstandthetechniquebeforeusingeitherspincolumnordialysiscassette.IfyoudonotusethecorrectMWCO,youcanloseyourentireprep.Forthisprotocol,werecommendMWCOof300,000dalton.

PreparationMethod

Thetwo-stepprotocolincludestheactivationofcarboxylgroup-containingliposomeswithEDC/sulfo-NHS,andsubsequentconjugationwiththeaminegroupontheproteins,peptidesorantibodies:

1. Inordertoactivatethecarboxylgroupsontheliposomes,EDCandsulfo-NHSshouldbeaddedtotheliposomes.ThetotallipidconcentrationinImmunodox®-Succinyl(PEGylated)is21.58mM.1%molofthelipidinliposomescontainsPEG-COOHgroupandonlyhalfofthemareexposedtotheoutsideoftheliposomes,whichisequalto0.11mMofreactiveconjugablelipid.Fora2mlvolumeliposomethisisequalto2.20×10^-7molandfor5mlvolumeliposomesthisisequalof5.50×10^-7molofPEG-COOH.Add10-foldmolarexcessofEDCand25-foldmolarexcessofsulfo-NHStoImmunodox®-Succinyl(PEGylated).Toaidinaliquotingthecorrectamountofthesereagents,theymaybequicklydissolvedinthePBSbufferatahigherconcentration,andthenapropervolumeimmediatelyPipettedintotheproteinsolutiontoobtainthepropermolarquantities. Mixwellandallowthereactiontoproceedfor15minatroomtemperature.
2. Beforeaddingtheprotein,peptideorantibody,removetheexcessEDCeitherusingasizeexclusionspincolumn,suchasSephadex®spincolumnorthroughquenchingby2-mercaptoethanolata20mMfinalconcentration.Additionof2-mercaptoethanolwillnotimpacttheliposomes.
3. Dissolvetheprotein,peptideorantibodyat1-10mg/ml,dependingontheantibody,proteinorpeptide,inPBSorotheramine-free,carboxylatefreebuffer,pH7-8.
4. Addtheprotein,peptideorantibodytotheEDC/Sulfo-NHSactivatedImmundox®-Succinyl(PEGylated)liposomes.Themolarratioofthereactivecarboxyllipidtoprotein,peptideorantibodyispreferredtobearound10:1.Thetotallipidconcentrationinourliposomesis21.58mM.1%molofthelipidinliposomescontainsPEG-COOHgroupandonlyhalfofthemareexposedtotheoutsideoftheliposomes,whichisequalto0.11mMofreactiveconjugablelipid.Foramlvolumeliposome,thisisequalto2.20×10^-7mol,andfor5mlvolumeliposome,thisisequalto5.50×10^-7molofPEG-COOH.Youwillneedtocalculatethetotalmolofyourpeptide,proteinorligandinyoursolutionandadd1:10molarratioofligandtolipid. Mixwellandallowtoreactfor2hatroomtemperature.
5. 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.Themolarconcentrationofliposomeis21.58mM.Byhaving liposomediameter(nm)andlipidconcentration(µM),youcancalculatethetotalnumberofthelipidsinoneliposomeandthenumberoftheliposomesinonemilliliteroftheliposomesolution.Tousethecalculatorclick here.

TechnicalNotes

• Doxorubicinisafluorescentmoleculewithλ_ex470nmandλ_em585nm.Ifyouareusingafluorescenttagonyourantibodyorligand,thenyouneedtomakesurethattheywillnotinterferewitheachother.
• EDCandsulfo-NHSshouldbepreparedimmediatelybeforeuseandkeptatroomtemperature.
• TheactivationreactionwithEDCandSulfo-NHSismostefficientatpH4.5-7.2,andEDCreactionsareoftenperformedinatpH4.7-6.0.Forthisreason,wehaveformulatedtheliposomesinPBSbufferandadjustedthepHto6.
• ReactionofSulfo-NHS-activatedmoleculeswithprimaryaminesismostefficientatpH7-8,andSulfo-NHS-esterreactionsareusuallyperformedinphosphate-bufferedsaline(PBS)atpH7.2-7.5.
• Trisbuffershouldneverbeusedinanystepoftheprocesssinceitcontainsamine.
• IfyouareusingaligandorpeptidethatishydrophobicthenitisrecommendedtosolubilizeitinDMSOorDMFandthenaddthebuffertoit.Itisrecommendednottousemorethan5%volumeofDMSOorDMFinthesolution.DMFandDMSOarebothcompatIBLewithliposomesandtheyarealsomiscibleinwater.Otherorganicsolventsuchasethanolandchloroformarenotcompatiblewithliposomesandwillcausetheliposomestolyse.IfyouendupusingDMSOorDMFthenaftertheconjugationreactionisdone,youneedtoremoveDMSOandDMFfromtheliposomes.InordertodothatyouneedtouseadialysiscassettethatismadefromREGENERATEDCELLULOSEMEMBRANE.NOTE:NotallmembranesarecompatiblewithDMFandDMSO.WerecommendusingaSlide-A-Lyzer™MINIDialysisDevicewithMWCOof2KmadefromregeneratedcellulosemembranemanufacturedbyThermofisher.AfterDMSOorDMFisremoved,youcanuseFloat-A-Lyzer®dialysisdeviceforthefinalstepofcleaninguptheprep.
• Liposomesshouldbekeptat4°CandNEVERbefrozen.

Database

Directlinktothedatabasepageforeasynavigation:ImmunoliposomesConjugationDatabase

Appearance

Immunodox®-Succinylisaredtranslucentliquidmadeofnanosizeunilamellarliposomes.Usuallyduetothesmallsizeofliposomesnosettlingwilloccurinthebottomofthevial.Theliposomesarepackagedinanambervial.

EducationalVideo

Ordering/ShippingInformation

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

StorageandShelfLife

Storage

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

ShelfLife

Immunodox®-Succinylismadeondailybasis.Thebatchthatisshippedismanufacturedonthesameday.Itisadvisedtousetheproductswithin2monthsofthemanufacturingdate.

ReferencesandbackgroundreADIng

1.HermansonGT.Bioconjugatetechniques.Academicpress;2013Jul25.

2.TorchilinV,WeissigV,editors.Liposomes:apracticalapproach.OxfordUniversityPress;2003Jun5.

3.GrabarekZ,GergelyJ.Zero-lengthcrosslinkingprocedurewiththeuseofactiveesters.Analyticalbiochemistry.1990Feb15;185(1):131-5.

4.YanL,CraytonSH,ThawaniJP,AmirshaghaghiA,TsourkasA,ChengZ.ApH‐ResponsiveDrug‐DeliveryPlatformBasedonGlycolChitosan–CoatedLiposomes.Small.2015Oct1;11(37):4870-4.

5. Silva-LópezEI,EdensLE,BardenAO,KellerDJ,BrozikJA.ConditionsforliposomeadsorptionandbilayerformationonBSApassivatedsolidsupports.Chemistryandphysicsoflipids.2014Oct31;183:91-9.

6.HazraM,SinghSK,andRayS.SurfaceModificationofLiposomalVaccinesbyPeptideConjugation.JournalofPharmaSciTech,2011;1(1):41-47.