Description

InordertodeveloparapidandstraightforwardcouplingprocedureatthePEGterminus,amethodofdirectcouplingantibodiestothePEGterminusofliposomeswasintroducedbyBendasetal.Inthismethodology,antibodiesaresimplyattachedtothePEGterminusofliposomes,whichhadbeenendgroup-functionalizedwithcyanuricchloride,inmildbasicconditions(pH8.8)withoutpriorantibodyderivatizations.Ithasbeenshownthatinordertoobtainastableattachmentofproteinsonliposome,theDSPE-PEG-cyanurwasaddedintotheliposomestochemicallyconjugatewithproteinstoformastablecomplexandminimizethedenaturationofproteins.

Proteinscanbecovalentlycoupledtotheliposomesviaamine-reactivecyanur-groups,eitherdirectlytothevesiclesurfaceusingcyanuricchloride-activatedDSPE(cyanur-DSPE)ortothedistalendsofPEG-spacersusingactivatedcyanur-PEG-PE(ammoniumsalt).CyanuricchlorideatthePEGterminusfunctionstolinkpeptides,antibodiesandotheramine-containingbiomoleculesornanoparticlesviaanucleophilicsubstitutionreactionunderbasicconditions.Antibodiesorotherproteinscanbeconjugatedwithoutanypreviousderivatization.

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

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FormulationInformation

Immunodox®-Cyanur(PEGylated)

LipidComposition	Concentration(mg/ml)	Concentration(mM)	MolarRatioPercentage
Total	15.92mg/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)-Cyanur	0.65	0.22	1

ConjugationProtocol

MaterialsandEquipment

Inordertoconjugateyourantibody,protein,peptideorligandtoImmunodox®-Cyanur(PEGylated)youwillneed:

1. Float-A-Lyzer®withaproperMWCOthateasilyallowsthecleanupofyourliposomeconjugatedligandfromfreeandnon-conjugatedprotein/peptide/ligand.YouneedtomakesurethattheMWCOisbelow1,000,000dalton.At1,000,000dalton,theporesizeonthedialysismembranegetscloseto100nmandthereforeyourliposomescanbedialyzedout.Youcannotusedialysiscassettesblindly.Pleaseunderstandthetechniquebeforeusingeitherspincolumnsordialysiscassettes.IfyoudonotusethecorrectMWCO,youcanloseyourentireprep.InthiscasewerecommendusingadialysiscassettewithMWCOof300,000dalton.
2. Boratebufferyoucaneithermaketheboratebufferorpurchaseitfromachemicalvendor.Inanycase,youneedtomakesurethatthepHisadjustedto8.8.

PreparationMethod

1. ThetotallipidconcentrationinImmunodox®-Cyanuris21.58mM.1%molofthelipidinliposomescontainsPEG-Cyanurgroupandonlyhalfofthemareexposedtotheoutsideoftheliposomes,whichisequalto0.11mMofreactiveconjugablelipid.Fora2mlvolumeliposomethisisequalto2.2×10^-7molandfor5mlvolumeliposomesthisisequalof5.50×10^-7molofPEG-Cyanur.
2. Add1:1000molarratioofantibody,protein,peptideorligandtototallipid.Thiswillbeequalto1:5molarratioofantibody,protein,peptideorligandtoPEG-Cyanurlipid.Forexampleina2mlkit,for2.2×10^-7molofPEG-Cyanurlipid,4.4×10^-8molofantibody,protein,peptideorligandisneeded.
3. TheconjugationhastobedoneundermildbasicconditionsuchaboratebufferpH8.8.Dissolveyourantibody,protein,peptideorligandinboratebufferpH8.8.
4. IncubateImmunodox®-Cynaurwithantibody,protein,peptideorligandfor16hoursatroomtemperature.
5. Removenon-conjugatedantibody,protein,peptideorligandbydialysis.Wepreferdialysistosizeexclusioncolumns.Dialysisisamuchslowerprocessbuttherewillbeminimumlossofimmunoliposomesaftertheprepiscleanedfromnon-conjugatedprotein/peptide/ligand.Spincolumnsaremuchfaster,butyoucaneasilyloseover50%oftheliposomesonthespincolumn.WerecommendusingFloat-A-Lyzer®dialysiscassettefromSpectrumLabs.YouneedtochooseacassettewithproperMWCOdependingontheMWofyourprotein,ligand,antibodyorantibodyfragment.InthiscasewerecommendusingadialysiscassettewithMWCOof300,000dalton.NOTE:IfyoudecidetouseadialysiscassetteyouneedtomakesurethattheMWCOisbelow1,000,000dalton.At1,000,000dalton,theporesizeonthedialysismembranegetscloseto100nmandthereforeyourliposomescanbedialyzedout.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.
• Trisbuffershouldneverbeusedinanystepoftheprocesssinceitcontainsamine.
• Cyanuricchlorideisconsideredasasensoryrespiratoryirritant.However,despitethenameofthecyanur-modifiedliposomes,theyhavenotshownanysignofacute,chronicorgenotoxicity.
• Cyanurgroupsareamine-reactive,however,somerandomattachmentsoftheantibodiescanbeexpectedsincecyanuricchloridecanreactwithawiderangeofnucleophilicfunctionalities,suchasalcoholsandthiols.Thismayinterferewiththebindingoftheantibodytotheliposome,andtherefore,thebindingaffinitywouldchange.
• SizeexclusionspincolumnssuchasSepharose®CL-4BcanbeusedinsteadofFloat-A-Lyzer®dialysiscassette.However,averylargeamountofliposomeswillsticktothecolumnduringthecleanupprocessandthereforewestronglysuggestusingdialysisthansizeexclusivebeads.
• 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®-Cyanurisaredtranslucentliquidmadeofnanosizeunilamellarliposomes.Usuallyduetothesmallsizeofliposomesnosettlingwilloccurinthebottomofthevial.Theliposomesarepackagedinanambervial.

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®-Cyanurismadeondailybasis.Thebatchthatisshippedismanufacturedonthesameday.Itisadvisedtousetheproductswithin4monthsofthemanufacturingdate.

ReferencesandbackgroundreADIng

1.BendasG,KrauseA,BakowskyU,VogelJ,RotheU.TargetABIlityofnovelimmunoliposomespreparedbyanewantibodyconjugationtechnique.Internationaljournalofpharmaceutics.1999Apr20;181(1):79-93.

2.LeeHY,MohammedKA,KayeF,SharmaP,MoudgilBM,ClappWL,NasreenN.Targeteddeliveryoflet-7amicroRNAencapsulatedephrin-A1conjugatedLiposomalnanoparticlesinhibittumorgrowthinlungcancer.Internationaljournalofnanomedicine.2013;8:4481.

3. NyanhongoGS,SteinerW,GübitzGM,editors.BiofunctionalizationofPolymersandtheirApplications.SpringerScience&BusinessMedia;2011Aug6.