Description
Biotinylatedliposomescanbeconjugatednoncovalentlywith(strept)avidinthrougheitherdirectinteractionwiththeprotein/antibodyconjugatedto(strept)avidinorbycouplingwithotherbiotinylatedproteinsusing(strept)avidinasabridgingmolecule. Bothavidinand(strept)avidinformstrongnoncovalentbondwithbiotin.Thehighresistancetobreakdownmakesthemveryusefulinbioconjugatechemistry.However,streptavidinhasreplacedavidininmostbioconjugationapplicationsduetoitsenhancedproperties.NeutrAvidin(Thermofisher)isamodifiedavidinwithoutnegativeproperties.Itperformsmuchbetterthanoriginalavidinandsometimesstreptavidin.
Inordertoexploitthehigh-affinityinteractionofbiotinwith(strept)avidin,atwo-step“sandwich”protocol(MethodA)hasbeendevelopedforthepreparationoftargetedimmunoliposomes.Inthismethodology,(strept)avidinisfirstattachedtobiotinylatedliposomes,thenabiotin-modifiedprotein/antibodyisintroducedintothebiotinylated(strept)avidin-labeledliposomes.Thisnoncovalentapproachisrapid,extremelyversatileandapplicabletonumeroustargetingligandsofinterestwithrespecttoinvitroandinvivoapplications.Alternatively,insteadofforminga(strept)avidinbridge,(strept)avidinmoleculecanalsobecovalentlyconjugatedtoantibodyorligand(MethodB)andnon-covalentlyboundtoliposomescontainingbiotinonsurfaceinordertoformimmunoliposomes.
Immunodox®-BiotinisaPEGylatedproduct.Forotheraminereactive(PEGylatedandnon-PEGyalatedproducts)andalsoImmunodox®productssuitableforothertypesconjugationmethodsseehere.
FormulationInformation
Immunodox®-Biotin(PEGylated)
| LipidComposition | Concentration(mg/ml) | Concentration(mM) | MolarRatioPercentage |
|---|---|---|---|
| Total | 15.61mg/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)-Biotin | 0.34 | 0.22 | 1 |
| Buffers,LiposomeSizeandEncapsulatedDrugConcentration | Specification |
|---|---|
| InsideBuffer | AmmoniumSulfate |
| OutsideBuffer | PhosphateBufferedSaline |
| pH | 7.4 |
| LiposomeSize | 100nm |
| EncapsulatedDoxorubicin | 2mg/ml(3.45mM) |
ConjugationProtocol
MaterialsandEquipment
InordertoconjugateyourantibodyorproteintaggedwithbiotintoImmunodox®-Biotinliposomesyouwillneed:
- Laboratorymagneticstirrerisneededfordialysis.
- Vortexlaboratorymixerisrecommendedtohave.
- Float-A-Lyzer®withaproperMWCOthateasilyallowsthecleanupofyourliposomeconjugatedligandfromfreeandnon-conjugatedprotein/peptide/ligand.YouneedtomakesurethattheMWCOisbelow1,000,000dalton.At1,000,000daltontheporesizeonthedialysismembranegetscloseto100nmandthereforeyourliposomescanbedialyzedout.Youcannotusedialysiscassettesblindly.Pleaseunderstandthetechniquebeforeusingeitherspincolumnordialysiscassette.IfyoudonotusethecorrectMWCOyoucanloseyourentireprep.ForthisprotocolwerecommendMWCOof300,000dalton.
PreparationMethods
MethodA.Twostep“Sandwich”protocol;Creating(strept)avidinbridge
- ThetotallipidconcentrationinImmunodox®-Biotinis21.58mM.1%molofthelipidinliposomescontainsPEG-Biotingroupandonlyhalfofthemareexposedtotheoutsideoftheliposomes,whichisequalto0.11mMofreactiveconjugablelipid.For2mlvolumeliposome,thisisequalto2.20×10-7mol,andfor5mlvolumeliposome,thisisequalto5.50×10-7molofPEG-Biotin.PourImmunodox®-Biotininaconicaltubeandvortexitgentlywithonehand.Usetheotherhandandslowlyaddthe(strept)avidinsolutionuntilthetwosolutionaremixed.Youneedtouse10-foldmolarexcessof(strept)avidintoPEG-Biotinlipid.Incubatethesolutionfor1hatroomtemperature.
- Removetheunbound(strept)avidinfromtheprepbydialysis.Wepreferdialysistosizeexclusioncolumns.DialysisisamuchslowerprocessbuttherewillbeminimumlossofImmunodox®-Biotinaftertheprepiscleanedfromunbound(strept)avidin.Spincolumnsaremuchfaster;however,youcaneasilyloseover50%oftheliposomesonthespincolumn.WerecommendusingFloat-A-Lyzer®dialysiscassettewith300KMWCOfromSpectrumLabs.DialyzetheImmunodox®-Biotin/(strept)avidinsolutionin1literofPBSatpH7.4for8hours.Changethedialysisbufferwithafresh1literofPBSandletitdialyzeforanother8hours.Afterthisstep,Immunodox®-Biotin/(strept)avidinisseparatedfromunbound(strept)avidin.
- PourImmunodox®-Biotin/(strept)avidininaconicaltubeandvortexitgentlywithonehand.Usetheotherhandandslowlyaddthebiotinylatedantibodyorbiotinylatedligandsolutionuntilthetwosolutionaremixed.Youneedtouse2-foldmolarexcessofbiotinylatedantibody(ligand)toPEG-Biotinlipid.Incubatethesolutionfor1hatroomtemperature.
- Removethenon-conjugatedantibodyorligandfromtheprepbydialysisbyusingFloat-A-Lyzer®dialysiscassettewith300KMWCOfromSpectrumLabs.Dialyzetheimmunoliposomesolutionin1literofPBSatpH7.4for8hours.Changethedialysisbufferwithafresh1literofPBSandletitdialyzeforanother8hours.Afterthisstep,yourcleanedupimmunoliposomesolutionisreadytouse.
MethodB.Usingaantibody/protein/ligandwhichisalreadycovalentlyattachedto(strept)avidin(lesscommonmethod)
- ThetotallipidconcentrationinImmunodox®-Biotinis21.58mM.1%molofthelipidinliposomescontainsPEG-Biotingroupandonlyhalfofthemareexposedtotheoutsideoftheliposomes,whichisequalto0.11mMofreactiveconjugablelipid.Fora2mlvolumeliposomethisisequalto2.20×10-7molandfora5mlvolumeliposomesthisisequalof5.50×10-7molofPEG-Biotin.PourImmunodox®-Biotininaconicaltubeandvortexitgentlywithonehand.Usetheotherhandandslowlyaddtheantibodyconjugated(strept)avidinuntilthetwosolutionaremixed.Youneedtouse2-foldmolarexcessofantibodyconjugated(strept)avidin.Incubatethesolutionfor1hatroomtemperature.
- Removethenon-conjugatedantibodyorligandfromtheprepbydialysisbyusingFloat-A-Lyzer®dialysiscassettewith300KMWCOfromSpectrumLabs.Dialyzetheimmunoliposomesolutionin1literofPBSatpH7.4for8hours.Changethedialysisbufferwithafresh1literofPBSandletitdialyzeforanother8hours.Afterthisstep,yourcleanedupimmunoliposomesolutionisreadytouse.
LiposomeParticleCalculator
Immunodox®liposomesareunilamellarandsizedto100nm.Themolarconcentrationofliposomeis21.58mM.Byhaving liposomediameter(nm)andlipidconcentration(µM),youcancalculatethetotalnumberofthelipidsinoneliposomeandthenumberoftheliposomesinonemilliliteroftheliposomesolution.Tousethecalculatorclick here.
TechnicalNotes
- Doxorubicinisafluorescentmoleculewithλex470nmandλem585nm.Ifyouareusingafluorescenttagonyourantibodyorligand,thenyouneedtomakesurethattheywillnotinterferewitheachother.
- Toavoidprecipitationoflipidinthenoncovalentapproach,careneedstobeemployedinmaintainingahighratioof(strept)avidintobiotin-liposomes.Otherwise,thecouplingefficiencieswouldberelativelylow.
- Alternatively, Sepharose®CL-4BsizeexclusionspincolumncanbeusedinsteadofFloat-A-Lyzer®.However,keepinmindthatalargeamountofliposomeswillbelossonthecolumnduringtheprocess.Dialysisisamuchslowerprocessthatsizeexclusionhowevertherewillbeminimallossofliposomes.
- Ifyoudecidetouseadialysiscassette,youwillneedtomakesurethattheMWCOisbelow1,000,000dalton.At1,000,000dalton,theporesizeonthedialysismembranegetscloseto100nmandtherefore,yourliposomescanbedialyzedout.Youcannotusedialysiscassettesandspincolumnsblindly.Theycomeinvarioussizesandyouneedtochoosethecorrectsizewisely.
- IfyouareusingaligandorpeptidethatishydrophobicthenitisrecommendedtosolubilizeitinDMSOorDMFandthenaddthebuffertoit.Itisrecommendednottousemorethan5%volumeofDMSOorDMFinthesolution.DMFandDMSOarebothcompatIBLewithliposomesandtheyarealsomiscibleinwater.Otherorganicsolventsuchasethanolandchloroformarenotcompatiblewithliposomesandwillcausetheliposomestolyse.IfyouendupusingDMSOorDMFthenaftertheconjugationreactionisdone,youneedtoremoveDMSOandDMFfromtheliposomes.InordertodothatyouneedtouseadialysiscassettethatismadefromREGENERATEDCELLULOSEMEMBRANE. NOTE:NotallmembranesarecompatiblewithDMFandDMSO.Werecommendusinga Slide-A-Lyzer™MINIDialysisDevice withMWCOof2KmadefromregeneratedcellulosemembranemanufacturedbyThermoFisher.AfterDMSOorDMFisremoved,youcanuse Float-A-Lyzer® dialysisdeviceforthefinalstepofcleaninguptheprep
- Liposomesshouldbekeptat4°CandNEVERbefrozen.
Database
Directlinktothedatabasepageforeasynavigation:ImmunoliposomesConjugationDatabase
Appearance
Immunodox®-Biotinisaredtranslucentliquidmadeofnanosizeunilamellarliposomes.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®-Biotin(PEGylated)ismadeondailybasis.Thebatchthatisshippedismanufacturedonthesameday.Itisadvisedtousetheproductswithin4monthsofthemanufacturingdate.
ReferencesandbackgroundreADIng
1.HermansonGT.Bioconjugatetechniques.Academicpress;2013Jul25.
2.LoughreyHC,ChoiLS,WongKF,CullisPR,BallyMB.Preparationofstreptavidin-liposomesforuseinligand-specifictargetingapplications.Liposometechnology.1993;3:163-78.
3.HauglandRP,BhalgatMK.Preparationofavidinconjugates.ImmunochemicalProtocols.1998:185-96.
