Description
m-Clodrosome®isamultilamellarliposomesUSPensioninwhichClodronateisencapsulatedintheaqueouscompartmentsofthemannosylatedliposomes.m-Encapsome®isformulatedandpreparedidenticallytom-Clodrosome®exceptthatclodronateisnotaddedtotheliposomes.Theliposomesarefilteredthrough2μmpolycarbonatemembranestoensurethatlargerparticles,whichmaybetoxictoanimals,areremovedfromthesuspension.Botharepreparedandpackagedundersterileconditions.Whenanimalsorcellsaretreatedwithm-Clodrosome®,phagocyticcellsrecognizetheliposomesasinvADIngforeignparticlesandproceedtoremovetheliposomesfromthelocaltissueorserumviaphagocytosis.Theliposomesthenreleaseclodronateintothecytosolresultingincelldeath.Non-encapsulatedclodronatecannotcrossthecellmembranetoinitiatecelldeath.m-Encapsome®-controlliposomes-arerecognizedandphagocytosedbythesamemechanismasm-Clodrosome®.Sincethecontrolliposomesdonotcontainclodronate,thephagocyticcellsarenotkilled.However,phagocytesdorespondtotheingestionofcontrolliposomesbycytokinesecretion,temporarysuspensionofphagocyticactivityandotherresponsesdescribedintheliterature.
Mannosereceptortargetingbymannosylatedliposomeshasbeendemonstratedforavarietyofmannosylatedlipidconjugatesinavarietyofliposomemorphologiesandcompositionsinseveraldifferentinvitroandinvivomodels.Thereareseveralpublicationsaboutusingahydrophobicderivativeofmannose(4-aminophenylalpha-D-mannopyranoside)ratherthanusingamannosylatedlipidinclodronateliposomes.Thisismainlyduetothehighcostandcomplexityofsynthesizingandconjugatingmannosetolipid.4-aminophenylalpha-D-mannopyranosideiscommerciallyavailableandfarlessexpensivethansynthesizingmannoseconjugatedlipid.
Whymannose?Mannoseisoneofthecarbohydratecomponentsofmanybacterialandviralcellsurfaces,thereforetheever-efficient,highlyredundantimmunesystemhasevolvedmultiplemechanismsforidentifyingpathogensbasedonmannoserecognition.Theanimalandplantkingdomslikewiseutilizecarbohydraterecognitionsignalingmechanismsincludingmannoseresidues.Manypublicationsevaluateothercarbohydratesastargetingmechanismsforvariouscelltypes,howevermannosetargetingtophagocytesappearstobeoneofthemorespecificmechanismsidentifiedtodate.Mammaliancellsurfaceidentificationmoleculesbasedonmannosebinding,suchastheICAMfamilyofleukocyteadhesionmolecules,targettheSIGNfamilyofmannosereceptorstoaccomplishself-recognitioninvivo.
Awell-knownandcitedstudybyUmezawa&Eto [1]demonstratesthatliposomescontainingaminophenylmannosideweremostefficientlyincorporatedintothemousebrainacrossthebloodbrainbarrier.Theradiolabeledliposomesbearingaminophenyl-alpha-D-mannopyranosideweremaximallyincorporatedintothemousebrainafter48hours,whereasinthespleenandliver,theseradioactivitiesweremaximumafter12hours.Thestudiesalsoshowedthatliposomesweremostincorporatedwasglialcellsratherthanneuronalcell.Thesubcellularfractionationstudyindicatesthatmannoselabeledliposomesareincorporatedintolysosomesrichfractionbothinliverandbrain.
Mannosylatedfluorescentliposomes(m-Fluoroliposome®)suitableformacrophagetargetingandtrackingareavailablecontainingfivedifferentfluorescentdyes(DiI,DiO,DiD,DiAandDiR)thatcoverstheentirespectrum.Fluorescentliposomescomeinstandardandmannosylatedform.Formoreinformationsee here.
Clodrosome®andEncapsome®arestandardandnon-mannosylatedreagentsthatareusedfortargetingmacrophagesinorgansandtissuesotherthancentralnervoussystems.Formoreinformationaboutthesereagentseehere.
FormulationInformation
m-Clodrosome®LiposomalClodronateSuspension
| LipidComposition | Concentration(mg/ml) | Concentration(mM) | MolarRatioPercentage |
|---|---|---|---|
| Total | 23mg/ml | 35.1mM | 100 |
| L-alpha-Phosphatidylcholine | 18.8 | 24.3 | 70 |
| Cholesterol | 4.2 | 10.9 | 30 |
| EncapsulatedDrug | Concentration |
|---|---|
| Clodronate((Dichloro-phosphono-methyl)phosphonate),DisodiumSalt | 18.4*mM |
| *Dependingonthetypeoftheclodronatesalt,itsconcentration(mg/ml)varies.Iftetrahydratesaltisused,theconcentrationoftheencapsulateddrugwillbe~7mg/ml,andifanon-hydratedsaltisused,theconcentrationwillbe~5mg/ml. | |
| Mannosylation | Concentration |
|---|---|
| 4-Aminophenyl-alpha-D-mannopyranoside | 9.53mol% |
| BufferandLiposomeSize | Specification |
|---|---|
| Buffer | PhosphateBufferedSaline |
| pH | 7.4 |
| LiposomeSize | 1.5-2µm |
TechnicalNotes
- Toreachbloodstream-accessIBLe,mannose-receptorpositivecellsoutsidetheliver,asignificantnumberofliposomeswillhavetoescapefirst-passuptakebytheliverandspleen,sothatthetargetcellsareexposedtoahigherconcentrationofmannosylatedliposomesfromtheblood.Onestrategythathasbeenusedtoensurethatliposomesescapetheliverandspleenisknownasreticuloendothelialsystem(RES)blockadeinwhichanimalsarepre-dosedwithasufficientquantityofliposomestotemporarilysaturatethephagocyticcellsoftheblood,liverandspleen,alsoknownasthereticuloendothelialsystem(RES)orthemononuclearphagocytesystem(MPS).Thissufficientquantityisdependentupontheliposometypeandcompositionaswellasthespeciesbeingdosed;thepre-dosedliposomesdonotnecessarilyneedtobethesametypeorcompositionasthetherapeuticordiagnosticliposomesavoidingtheRES.Soonafterthispre-doseisclearedfromthebloodstream(usuallywithinacoupleofhours),theliposomesofinterestaredosed.SincetheRESisinvolvedindigestingthepreviousdoseofliposomes,thesubsequentlydosedliposomeswillremaininthecirculationmuchlongerthusbemuchmorelikelytobindtotheirtargetsiteoutsidetheRESincludingthosephagocyticcellswhichareaccessible,butarenotusuallyexposedtoahigherconcentrationofliposomes.
- WhileRESblockadeisusuallythoughtofassaturatingphagocyticcells,ithasbeenshownthatopsonin-bindingbyliposomesisasaturablephenomenon.Therefore,partofRESblockademayinvolveserumdepletionofcomplementandotheropsoninsknowntocoatliposomes.Inthecurrentapplication,removalorreductionintheconcentrationofsolublemannose-receptorsmayfurtherincreasetheprobABIlityofamannosylatedliposomebeingabletointeractwithmannosereceptorsonthetargetcell.Therefore,ifthegoalistodepleteatargetsubsetofmannose-receptor+cellswhichmaynotnormallybeexposedtoasubstantialnumberofmannosylatedliposomes,pre-dosingwithmannosylatedclodronateliposomes,inordertobothsaturatetheblood,liverandspleenphagocytesandreducetheconcentrationofopsoninsincludingsolublemannosereceptors,shouldincreasethenumberofsubsequentlydosedmannosylatedclodronateliposomesavailabletothistargetsubsethypotheticallyresultinginincreaseduptakeanddepletionbythesetargetedcells.
- WhenanimalsorcellsaretreatedwithClodrosome®,phagocyticcellsrecognizetheliposomesasinvadingforeignparticlesandproceedtoremovetheliposomesfromthelocaltissueorserumviaphagocytosis.Theliposomesthenreleaseclodronateintothecytosolresultingincelldeath.Unencapsulatedclodronatecannotcrossthecellmembranetoinitiatecelldeath.
- Encapsome®controlliposomesarerecognizedandphagocytosedbythesamemechanismasClodrosome®.Sincethecontrolliposomesdonotcontainclodronate,thephagocyticcellsarenotkilled.However,phagocytesdorespondtotheingestionofthecontrolliposomesbycytokinesecretion,temporarysuspensionofphagocyticactivityandotherresponsesdescribedintheliterature.
- Theproductmustberemovedfromthevialusingsteriletechnique.Donotuseifsterilityiscompromised.Thisisparticularlyimportantifasinglevialisaccessedmultipletimesoverseveralweeks.Theproductshouldnotbeusedmorethan60daysafterreceipt,evenifunopened.
- Liposomesmaysettlewhenleftundisturbedformorethanafewhours.Immediatelypriortouse,inordertoensureahomogeneousliposomesuspension,slowlyinvertthevialseveraltimesuntilthesuspensionappearshomogeneousbyvisualinspection.Vigorousorerraticshakingwillnotdamagetheliposomesbutmayinducefoamingandbubbleformationmakingitmoredifficulttoaccuratelymeasurethedesireddosage.
- Ifthepersonnelperformingintravenousinjectionsarenotexperiencedinorfamiliarwith,precautionsforinjectinglargervolumes(~10%animalweightinml),viscousliquidsorparticulatesuspensions,considerhavingextraanimalsavailableincaseseriousinjection-relatedadverseeventsoccur.Dosecontrolanimalsfirsttobecomefamiliarwithlargevolumeinjections.
- WithinhoursaftersystemicadmiNISTrationofClodrosome®,animalsbegintoloseimportantcomponentsoftheirimmunesystem.Standardanimalhandlingandhousingprotocolsarenotsuitableforimmunocompromisedanimals.Evenwhensuchprecautionsaretaken,monitorthegeneralhealthofeachanimalforopportunisticinfectionsunrelatedtotheexperimentalprotocol.Thereisnoinherenttoxicitytotheproductattherecommendeddoselevels.
- Whendosingintravenously,usestandardprecautionsfordosinglargervolumestoanimalsincludingthefollowing:a)warmproducttoroomtemperaturepriortodosing;b)ensurethatallairbubblesareremovedfromthesyringepriortodosing.Intravenousinjectionofairbubblesmayresultinairemboliwhichcankillorseriouslyinjureanimals;c)injectproductataslow,steadyrateofnomorethan1ml/min;d)decreaseinfusionrateifanimalsdisplayanyatypicalreactionssuchasunusualagitation.
- Infusion-relatedadversereactionsusuallyinvolvetheanimalgaspingforairorotherseizure-likemovements.Animalsoftenrecoverwithnoapparentpermanentinjury,butanypotentialeffectsonexperimentalresultsmustbeassessedbytheresearcher.
- Liposomesshouldbekeptat4°CandNEVERbefrozen.
Dosage
Appearance
m-Clodrosome®andm-Encapsome®arebothwhitemilkysuspensionsmadeoflargemicrosizemultilamellarliposomes.Duetotheirlargesizesomeliposomesmightsettletothebottomofthevial.Ifleftsittingidleintherefrigerator,m-Encapsome®willphaseseparateandformpelletsinthebottomofthevialleavingaclearsolutionontop.m-Clodrosome®mightdothesameonlynotasseverely.Therefore,bothshouldbegentlyshakennottoformbubblesbuttoformahomogeneoussolutionpriortouse.
EducationalVideos
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
m-Clodrosome®andm-Encapsome®shouldalwaysbestoredatinthedarkat4°C,exceptwhenbroughttoroomtemperatureforbriefperiodspriortoanimaldosing.DONOTFREEZE.Ifthesuspensionisfrozen,clodronatecanbereleasedfromtheliposomesthuslimitingitseffectivenessindepletingmacrophages.ENSisnotresponsibleforresultsgeneratedbyfrozenproduct.
ShelfLife
m-Clodrosome®andm-Encapsome®aremadeondailybasis.Thebatchthatisshippedismanufacturedonthesameday.Itisadvisedtousetheproductswithin60daysofthemanufacturingdate.
Referencesandbackgroundreading
1.UmezawaFA,EtoY.Liposometargetingtomousebrain:mannoseasarecognitionMarker.Biochemicalandbiophysicalresearchcommunications.1988Jun30;153(3):1038-44.
2.ItoT,IshigamiM,MatsushitaY,HirataM,MatsubaraK,IshikawaT,HibiH,UedaM,HirookaY,GotoH,YamamotoA.SecretedEctodomainofSIGLEC-9andMCP-1SynergisticallyImproveAcuteLiverFailureinRatsbyAlteringMacrophagePolarity.Scientificreports.2017Mar8;7:44043.
3.MironVE,BoydA,ZhaoJW,YuenTJ,RuckhJM,ShadrachJL,vanWijngaardenP,WagersAJ,WilliamsA,FranklinRJ.M2microgliaandmacrophagesdriveoligodendrocytedifferentiationduringCNSremyelination.Natureneuroscience.2013Sep;16(9):1211.
4.AndreouK,SarmientoSotoM,AllenD,EconomopoulosV,deBernardiA,LarkinJ,SibsonNR.Anti-InflammatoryMicroglia/MacrophagesasaPotentialTherapeuticTargetinBrainMetastasis.Frontiersinoncology.2017;7:251.
5.AlishekevitzD,Gingis-VelitskiS,Kaidar-PersonO,Gutter-KaponL,SchererSD,RavivZ,MerquiolE,Ben-NunY,MillerV,Rachman-TzemahC,TimanerM.Macrophage-inducedlymphangiogenesisandmetastasisfollowingpaclitaxelchemotherapyisregulatedbyVEGFR3.Cellreports.2016Oct25;17(5):1344-56.
6.OhSH,KimHN,ParkHJ,ShinJY,BaeEJ,SunwooMK,LeeSJ,LeePH.Mesenchymalstemcellsinhibittransmissionofalpha-synucleinbymodulatingclathrin-mediatedendocytosisinaParkinsonianmodel.Cellreports.2016Feb2;14(4):835-49.
7.KanoF,MatsubaraK,UedaM,HibiH,YamamotoA.SecretedEctodomainofSialicAcid‐BindingIg‐LikeLectin‐9andMonocyteChemoattractantProtein‐1SynergisticallyRegenerateTransectedRatPeripheralNervesbyAlteringMacrophagePolarity.STEMCELLS.2017Mar1;35(3):641-53.
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逆转录（reverse transcription）是以RNA为模板合成DNA的过程，即RNA指导下的DNA合成。此过程中，核酸合成与转录（DNA到RNA）过程与遗传信息的流动方向（RNA到DNA）相反，故称为逆转录。逆转录过程是RNA病毒的复制形式之一，需逆转录酶的催化。 逆转录过程的揭示是分子生物学研究中的重大发现，是对中心法则的重要修正和补充。人们通过体外模拟该过程，以样本中提取的mRNA为模板，在逆转录酶的作用下，合成出互补的cDNA，构建cDNA文库，并从中筛选特异的目的基因。该方法已成为基因工程技术中最常用的获得目的基因的策略之一。
1.模板提取（一般为RNA）：Trizol、氯仿、异丙醇、无水乙醇、DEPC处理水
2.模板浓度测定：分光光度计或NanoDrop
3.逆转录：逆转录试剂盒（或者一步法试剂盒），这一步可以用普通PCR做，也可以用水域做。
4.荧光定量PCR试剂：通常有用SYBR Green Mix做的，但是这里建议你用EvaGreen做，灵敏度和平行性都要好于SYBR Green，并且如果你那是ABI或者Stratagene的PCR如果用SYBR Green还需要加一步Rox很麻烦。
5.其他：除了以上的那些还需要离心管、PCR管或板（Axygen反应比较好）、移液枪等，暂时就想到这么多。
向左转|向右转
大家miRNA逆转录和qPCR的试剂盒用的是那个公司的啊，求推荐！
