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Seediscussions,stats,andauthorprofilesforthispublicationat:https://www.
researchgate.
net/publication/7324329Lipid-basednanoparticlesforcontrast-enhancedMRIandmolecularimaging.
NMRBiomedArticleinNMRinBiomedicine·February2006ImpactFactor:3.
04·DOI:10.
1002/nbm.
1011·Source:PubMedCITATIONS386READS5955authors,including:GustavJStrijkersAcademischMedischCentrumUniversiteitv…253PUBLICATIONS6,338CITATIONSSEEPROFILEGeraldaAFvanTilborgUniversityMedicalCenterUtrecht20PUBLICATIONS1,305CITATIONSSEEPROFILEKlaasNicolayTechnischeUniversiteitEindhoven520PUBLICATIONS16,191CITATIONSSEEPROFILEAvailablefrom:KlaasNicolayRetrievedon:09May2016NMRINBIOMEDICINENMRBiomed.
2006;19:142–164PublishedonlineinWileyInterScience(www.
interscience.
wiley.
com).
DOI:10.
1002/nbm.
1011ReviewArticleLipid-basednanoparticlesforcontrast-enhancedMRIandmolecularimagingWillemJ.
M.
Mulder,1*GustavJ.
Strijkers,1GeraldaA.
F.
vanTilborg,1ArjanW.
Grifoen2andKlaasNicolay11BiomedicalNMR,DepartmentofBiomedicalEngineering,EindhovenUniversityofTechnology,P.
O.
Box513,5600MBEindhoven,TheNetherlands2AngiogenesisLaboratory,ResearchInstituteforGrowthandDevelopment,DepartmentofPathology,MaastrichtUniversityandUniversityHospital,P.
O.
Box5800,6202AZMaastricht,TheNetherlandsReceived18July2005;Revised13October2005;Accepted28October2005ABSTRACT:IntheeldofMRimagingandespeciallyintheemergingeldofcellularandmolecularMRimaging,exiblestrategiestosynthesizecontrastagentsthatcanbemanipulatedintermsofsizeandcompositionandthatcanbeeasilyconjugatedwithtargetingligandsarerequired.
Furthermore,therelaxivityofthecontrastagents,especiallyformolecularimagingapplications,shouldbeveryhightodealwiththelowsensitivityofMRI.
Lipid-basednanoparticles,suchasliposomesormicelles,havebeenusedextensivelyinrecentdecadesasdrugcarriervehicles.
ArelativelynewandpromisingapplicationoflipidicnanoparticlesistheiruseasmultimodalMRcontrastagents.
Lipidsareamphiphilicmoleculeswithbothahydrophobicandahydrophilicpart,whichspontaneouslyassembleintoaggregatesinanaqueousenvironment.
Intheseaggregates,theamphiphilesarearrangedsuchthatthehydrophobicpartsclustertogetherandthehydrophilicpartsfacethewater.
Inthelowconcentrationregime,awidevarietyofstructurescanbeformed,rangingfromsphericalmicellestodisksorliposomes.
Furthermore,amonolayeroflipidscanserveasashelltoencloseahydrophobiccore.
Hydrophobicironoxideparticles,quantumdotsorperuorocarbonemulsionscanbesolubilizedusingthisapproach.
MR-detectableanduorescentamphiphilicmoleculescaneasilybeincorporatedinlipidicnanoparticles.
Furthermore,targetingligandscanbeconjugatedtolipidicparticlesbyincorporatinglipidswithafunctionalmoietytoallowaspecicinteractionwithmolecularmarkersandtoachieveaccumulationoftheparticlesatdiseasesites.
Inthisreview,anoverviewofdifferentlipidicnanoparticlesforuseinMRIisgiven,withthemainemphasisonGd–basedcontrastagents.
Themechanismsofparticleformation,conjugationstrategiesandapplicationsintheeldofcontrast-enhanced,cellularandmolecularMRIarediscussed.
Copyright#2006JohnWiley&Sons,Ltd.
KEYWORDS:Lipid-basednanoparticles;contrast-enhancedmagneticresonanceimaging;molecularimaging;micelles;liposomes;microemulsionsINTRODUCTIONMagneticresonanceimaging(MRI)isthemostversatileimagingmethodavailableinbothclinicalandresearchsettings.
ThesignalofMRIisdependentonthelong-itudinal(T1)andtransverse(T2)protonrelaxationtimesofmainlywaterandthereforedifferencesinprotonrelaxationtimesresultindifferencesincontrastinMRimages(1).
Theintrinsicrelaxationtimesoftissuewateraredependentonthephysiologicalenvironmentandmaybealteredinpathologicaltissue.
Thischangemayshowlittlespecicityandoccuratalatestageofthedisease.
Therefore,amorespecicandearlierdetectionofpathologywithMRIishighlydesirable.
Therelaxationtimesoftissuecanbealteredwithcontrastagentsthatdecreasethelongitudinalandtransverserelaxationtime.
TheabilityofacontrastagenttoshortenT1andT2isdenedastherelaxivity(2),r1orr2,andisexpressedinmM1s1.
Ingeneral,therearetwoclassesofMRcontrastagents.
Ontheonehand,thereareagentsthathavealowr2/r1ratioandthereforegeneratepositivecontrastinT1-weightedimages.
Thesepositivecontrastagents(3)usuallyareparamagneticcomplexesofGd3orMn2ions.
Ontheotherhand,therearesuperpara-magneticcontrastagentswithahighr2/r1ratio,whichcausedarkspotsinT2-andT2*-weightedimagesandarethereforereferredtoasnegativecontrastagents(4).
Thesecontrastagentsareusuallybasedonironoxideparticles.
MRIapplicationsarebecomingmoreandmoredepen-dentoncontrastagents.
ThecombinationofMRIandcontrastagentsgreatlyenhancesthepossibilitiestodepictthevascularsystem(5),inamedtissueasinarthritis(6),tumorangiogenesis(7,8),atheroscleroticplaques(9,10)andthebreakdownoftheblood–brainbarrierrelatedtopathologiessuchasmultiplesclerosis(11).
WithintheemergingeldofcellularandmolecularMRI,contrastagentshavebecomeanessentialelementofthetechnique.
Copyright#2006JohnWiley&Sons,Ltd.
NMRBiomed.
2006;19:142–164*Correspondenceto:W.
J.
M.
Mulder,BiomedicalNMR,DepartmentofBiomedicalEngineering,EindhovenUniversityofTechnology,P.
O.
Box513,5600MBEindhoven,TheNetherlands.
E-mail:w.
j.
m.
mulder@tue.
nlTheaimofmolecularandcellularMRimagingistoimagenon-invasivelycellularandmolecularevents,usuallyre-latedtopathologyortransgeneexpression.
Differentimagingmethodshavealreadyshowntheircapabilitytofunctionasamolecularimagingmodality(12).
NuclearmethodssuchasPETandSPECTareespeciallyinterestingbecauseoftheirhighsensitivity.
Ontheotherhand,thespatialresolutionofthesemodalitiesislowandthedenitionofanatomyispoor.
MRIhasgoodspatialresolution,butinordertobecomesuitableasamolecularimagingmodalitytheinherentlylowsensitivityhastobedealtwith.
Thismayberealizedbyusingcontrastagentswithaveryhighrelaxivity,e.
g.
byusingnanoparticlescontainingahighpayloadofGdcomplexesorusingironoxideparticleswithahighpayloadofiron.
Roughly,MRcontrastagentscanbedividedintofourgroupsorclasses.
Therstgroupconsistsofnon-speciccontrastagentsandincludesboththelowmolecularweightcontrastagents,e.
g.
Gd–DTPA,andthehighmolecularweightbloodpoolagents(13),suchashigh-generationdendrimers(14).
TheseagentscanbeusedforMRangiographyandtomeasuretheperfusionandpermeabilitypropertiesoftissue.
Thesecondclassofmoleculesistargetedcontrastagents,whichareactivelydirectedtoaspecicmoleculartargetwithanappropriateligand.
AnearlyreportofsuchacontrastagentbySipkinsetal.
describedthedetectionoftumorangiogenesiswithanv3-specicantibodythatwasconjugatedtopoly-merizedparamagneticliposomes(15).
Thethirdgroupconsistsoftheso-calledsmartcontrastagents,alsoreferredtoasactivatedorresponsiveagents.
AnexampleofsuchanagentisEgadMe,acomplexwhichcontainsasugarmoietythatpreventswatertocoordinatewithGd3.
Enzymaticcleavageofthissugarby-galactosidaseimprovestheaccessibilityofwatertoGd3,whichresultsinanincreaseintherelaxivityofthecomplex(16).
Thefourthclassisthecelllabelingcontrastagents,suchasTAT–peptideconjugatedironoxideparticles(17)orGd–HPDO3A(18).
Inordertomeetthediverserequire-mentssketchedabove,highlypotent,innovative,specicandpreferablymultimodalcontrastagentsarerequired.
Lipid-basedcolloidalaggregates,suchasliposomes,micellesandmicroemulsions,havebeenusedextensivelyrecentdecadesasdrugcarrierstoimprovepharmacoki-neticpropertiesorthebioavailabilityofthedrug,toincreasethetarget-to-backgroundratioofthedrugortodeliverhydrophobicdrugs(19–23).
Theaboveparticlesarecomposedoflipidsand/orotheramphiphilicmole-cules.
Amphiphilesaremoleculeswithbothhydrophobicandhydrophilicpartsthatspontaneouslyassembleintoaggregatesinanaqueousenvironment.
Targetingligandscanbeconjugatedtothecolloidalparticlestoachievebindingtomolecularmarkersthatarespecicfordiseaseprocesses(24,25).
Fluorescentlabelscaneasilybein-corporatedforuorescencemicroscopy(26).
Arelatively,newapplicationsofnanoparticulatecol-loidslieintheemergingeldofcellularandmolecularMRimaging(27,28).
SmallGd-basedcomplexessuchasGd–DTPAandGd–DOTAarewidelyusedascontrastagentsforclinicalMRI(29).
Inordertoimprovephar-macokineticproperties,tointroducetargetspecicity,tomakethecontrastagentmultimodaland,mostimpor-tantly,toimprovetheT1andT2loweringcapability,lipidicaggregatescontainingMRIcontrastagentsareanattractiveoption.
Theaggregatemorphologycanvaryfrommicelles,throughmicroemulsionstoliposomes(30–35).
Colloidalparticlesincontrast-enhancedMRIhavemanyapplications.
Theycanbeusedasbloodpoolagentswithlongcirculationtimesformagneticresonancean-giography(MRA).
Anotherapplicationisthedetectionofpathologicaltissueswithenhancedvascularpermeability,whichoccursininammation,myocardialinfarction,atherosclerosis,breakdownoftheblood–brainbarrierandtumors.
Likelipidiccolloidsusedfordrugdelivery,theparticulatecontrastagentscanalsobeconjugatedtoligandstotargetthemtoamolecularmarkerofinterest,permittingindirectdetectionofmarkerdistributionbyMRI.
Furthermore,pH-andtemperature-sensitivelipo-somescanbeusedtovisualizeregionaldifferencesintheseparameterswithMRI.
Forcelllabelingpurposes,lipidicnanoparticlesalsoholdgreatpromise.
Inthisreview,wewillrstexplainthepropertiesofamphiphilicmoleculesandtheirassemblyintocolloidalaggregates.
Differentstrategiesforconjugationoftarget-ingligandsandpotentialbiologicaltargetswillbere-viewed.
ThefocusofthisreviewisonGd-containinglipidicnanoparticlesandtheiruseforcontrast-enhancedandmolecularMRI.
AMPHIPHILICAGGREGATESAmphiphilesAmphiphiles,alsoreferredtoassurfactants,aremole-culesthatcontainbothahydrophobic(non-polartail)andahydrophilic(polarhead)part[Fig.
1(A),I].
Becauseofthisdualcharacterandtheenergeticallyunfavorablecontactbetweenthenon-polarpartandwater,amphi-philesself-associateintoaggregatesofdifferentsizesandgeometries.
Thereisawidevariabilityinboththehydrophobicandhydrophilicpartsofamphiphilicmole-cules.
Thehydrophobicpartcanvaryinlengthandcanconsistofmultiplechains,creatingdifferentratiosbe-tweenthesizeofthehydrophobicandhydrophilicpart[Fig.
1(A)].
Forthepolarheadgroup,boththesizeandchargecanvary,dividingthesemoleculesintoionic(anionicorcationic)ornon-ionicamphiphiles.
ThesecharacteristicsandparameterssuchaspH,temperatureandconcentration,eventuallydeterminethegeometryoftheaggregatethatisformedinaqueoussolution.
Phos-pholipidsandcholesterol[Fig.
1(A)]arenaturallyoccur-ringamphiphilicmoleculesthatareimportantstructuralLIPID-BASEDNANOPARTICLESFORCONTRAST-ENHANCEDMRI143Copyright#2006JohnWiley&Sons,Ltd.
NMRBiomed.
2006;19:142–164elementsofbiologicalmembranes.
Inrecentyears,manyphospholipid-likestructureshavebeensynthesizedtobenetfromtheamphiphiliccharacterandusedtoachieveawidevarietyofaggregates.
AmphiphileaggregationThemajorforcesthatdirecttheself-assemblyofamphi-philicmoleculesintowell-denedstructuresinwaterderivefromthehydrophobicassociativeinteractionsofthetailsandtherepulsiveinteractionsbetweenthehydrophilicheadgroups(36,37).
Intheseaggregates,theamphiphilesareorganizedinsuchawaythatthehydro-phobicpartsclustertogetherandthehydrophilichead-groupsfacethewater.
Thelengthofthehydrophobicchain(s)andthesizeoftheheadgroupinrelationtothechaindeterminethecurvatureoftheaggregateandwhetheramicelle-likestructureorabilayer-likestructurewillbeformed.
Awidevarietyofstructuresarepossible.
Inthelowconcentrationrange,sphericalmicelles,cy-lindricalmicellesandbilayeredvesiclesareamongtheaggregatesformed.
Athigherconcentrations,cubic,la-mellarandhexagonalphasesmayoccur(36).
Further-more,microemulsionscanbeformedfromwater,oilandanamphiphile.
Thesearestableisotropicdispersionsofoilcoveredbyalipidmonolayer(38).
Anotherclassoflipidaggregatesisself-assembliesoflipidmono-orbilayersthatcontainasolidcore.
Hydrophobicnanopar-ticlessuchasironoxide(35)andquantumdots(39)canbeentrappedinamicellularshellofPEG–lipids,whileglass,silicaandmica,butalsoironoxidenanoparticles(40),canbecoveredbyalipidbilayer[Fig.
1(B),VI].
Forinvivoapplications,theamphiphilicaggregatesshouldbe(i)stableand(ii)biocompatibleand(iii)haveexcellentpharmacokineticproperties.
PossiblestructuresforinvivousearedepictedinFig.
1(B).
Inthecaseofmicellesthehydrophobicpartoftheamphiphilicmole-culesformsthecoreofthemicelleandthehydrophilicpartformsthemicellecorona.
Micellescanbeformedfromlipidswitharelativelylargeheadgroup,suchaslipidswithasinglefattyacylchain[Fig.
1(B),I].
Furthermore,phospholipidmixtureswithahighpropor-tionofPEG–lipidwillassembleintomicelles,owingtosterichindranceofthePEG–lipidheadgroups(41).
Lipo-somesarecreatedfrombilayer-forminglipids,whichareusuallycomprisedofapolarheadgroupandtwofattyacylchains[Fig.
1(B),II].
Thehydrophobicpartoftheselipidsoccupiesmorespacethanthatofmicelle-forminglipidsandthereforebilayerformationisenergeticallyfavorable.
Forstabilizationofthelipidbilayer,choles-terolisoftenincluded(42).
Inaddition,PEG–lipids(3–7%)maybeincorporatedinthebilayer[Fig.
1(B),III]toincreasecirculationhalf-livesinvivoandtoreduceFigure1.
(A)Schematicrepresentationofamphiphiliclipids.
(I)Amphiphilesconsistofahydrophilicheadandahydrophobictail.
(II)Micelle-forminglipidshavearelativelylargeheadcomparedwiththehydrophobicpart,whereas(III)bilayer-forminglipidsusuallyhavetwohydrophobictails.
(IV)PEG–lipidsareusedtoimprovepharmacokineticpropertiesand(V)cholesterolisusedtostabilizeliposomes.
(B)Possiblelipidaggregatesforinvivouse.
(I)MicellescanbepreparedfrommicellesforminglipidsandfromPEG–lipids.
(II)Aconventionalliposomeconsistsofaphospholipidbilayer.
(III)ImprovedstabilizationofliposomescanbeachievedbyincorporatingasmallamountofPEG–lipidsandcholesterol.
(IV)Microemulsionsconsistofasurfactant(amphiphile)monolayercoveringoil.
(V)Micellescancontainahydrophobicnanoparticle.
(VI)Bilayeronnanoparticlesofsilica,mica,glassorironoxide144W.
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MULDERETAL.
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2006;19:142–164interactionsoftheliposomeswithplasmaproteins(42).
Microemulsionsorhydrophobicallycoatednanoparticles(e.
g.
ironoxideparticlesorquantumdots)inmicellesarealsounderinvestigationforinvivouse.
Drugtargeting/deliveryLiposomeshavebeenstudiedextensivelytoimprovethepharmacokineticpropertiesofmainlywater-solubledrugs,whilemicellesandmicroemulsionsmaybeusedtodeliverdrugswithpoorwatersolubility(43–47).
Thishasresultedintheapprovalofseveralliposomaldrugformulations(48,49),whichhaveprovenespeciallysuc-cessfulintumortargeting(50).
Doxorubicinisthemostcommonlyusedanticanceragentinliposomalformula-tions(51).
Encapsulatingthisdruginliposomeshasledtoimproveddeliverytothetumorandareducedexposureofothertissues.
Cisplatin,anotherdrugwhichisusedinthetreatmentofsolidtumors,hasalsobeenencapsulatedinliposomalformulations.
Liposomalcisplatinhasnotbeenshowntobeveryeffectivethusfar(52),whichispartlyascribedtothelowwatersolubilityofcisplatinthatcausesalowencapsulationefciency.
Recently,anovellipidformulationwithanimprovedcisplatin-to-lipidratioandimprovedcytotoxicityhasbeendescribed(53).
Accumulationofliposomesatthedesiredsitecanbeimprovedbyprolongingthecirculationtime.
Conven-tionalliposomesarerapidlyeliminatedfromthecircula-tionbycellsofthereticulo-endothelialsystemintheliver.
TheclearancerateisenormouslydecreasedwhenliposomalsystemsarecoatedwithahydrophilicpolymersuchasPEG(54,55),whichresultsinimprovedbioavail-ability(56).
Manydrugsarepoorlywatersoluble,whichresultsinalowbioavailability.
Micellesarecurrentlyunderinvesti-gationascarriervehiclesofsuchhydrophobicdrugs(57,58).
Micellessolubilizethesedrugsbyincorporatingthemintotheirhydrophobiccoreandthusincreasethebioavailability.
Microemulsionshavealsobeeninvesti-gatedfortheirpotentialtoserveasadrugcarriervehicle.
Theyareinteresting,sincetheoilphasecancontainahighpayloadofhydrophobicdrugs(38,59).
Specicityforthedesiredtargettissueorcellscanbeobtainedbyconjugatingthesystemsportrayedabovewithligandssuchasantibodies,antibodyfrag-mentsandpeptides(58,60–62).
Conjugationstrategiesand(potential)targetsaredescribedinthefollowingsections.
CONJUGATIONSTRATEGIESInthispart,conjugationstrategieswillbrieybehigh-lighted.
Formorein-depthinformationwerefertoexcellentreviewsdescribingdifferentconjugationmeth-ods(63–65).
Therearetwomainoptionsfortheconjugationofatargetingligandtolipidicparticles:non-covalentlinkage,suchastheavidin–biotininteraction,orcovalentbinding.
Lessfrequentlyusedmethodsaretheincorporationofamphiphilictargetingproteinsinthelipidbilayerofliposomes(66)ortheuseofamhiphileswithafunctionalmoiety,suchasapeptide(67)orapeptidomimetic(68,69).
Lipidicnanoparticlescanbepreparedcontainingawidevarietyoffunctionalizedlipids.
Forthatpurpose,phosphatidylethanolamine(PE)withthefunctionalmoi-etyattachedtothephosphategroupviaaspacerisoftenused.
ThespacercanvaryinlengthandlipidscontainingaPEGspacerwithadistalfunctionalmoietyarealsoavailable.
Amongthefunctionalgroupsarebiotin,mal-eimide,PDP,carboxylicacidandamine.
Viathesefunc-tionalgroups,severaltargetingligands,suchasmAb,Fab,proteinsandpeptides,canbeconjugatedusingdifferentcouplingstrategies.
Twopopularcouplingmeth-odswillbeexplainedinmoredetail.
Avidin-biotinlinkageTheavidin–biotinlinkageistypiedbyitseleganceandsimplicity.
Avidin,atetramericproteinwithamolecularweightof68kDa,iscapableofstronglybindingfourbiotins(KA%1.
71015M1).
Thebiotin–avidininterac-tionhasbeenexploitedforconjugatingliposomeswithbiotinylatedproteins.
ThisstrategyhasalsobeenusedtolinkMRcontrastagentstoantibodies(15,70–72).
ThisconjugationmethodisdepictedschematicallyinFig.
2(B).
Alipidicnanoparticlecarryingalipidwithadistalbiotinisrstincubatedwithavidin.
Inasecondstep,theparticle–avidinconjugateisincubatedwithabiotinylatedligand,e.
g.
apeptideorantibody.
Althoughthismethodissimpleandeffective,theintroductionofavidinintotheconjugatehascertaindrawbacks.
First,thesizeofavidinwillconsiderablyincreasethesizeoftheconjugate.
Moreimportantly,avidinisknown(73)tobeimmunogenicandtoberapidlyclearedbytheliver.
Infact,thispropertyofavidinhasbeenexploitedtochaseandclearantibodies(74)andMRIcontrastagentsfromthecirculation(8).
Covalentlylinkingtheligandtothelipidicparticledirectlywouldleadtoasmallerconjugate,whichhasmorefavorablepharmacokineticproperties.
CovalentbindingCovalentconjugationofligandstoalipidicparticlecanbeachievedwithseveralmethods.
Roughly,thesemeth-odscanbedividedintheformationof(i)anamidebond,betweenactivatedcarboxylgroupsandaminogroups,(ii)adisuldebondand(iii)athioetherbond,betweenmaleimideandthiol.
Thelastapproachwillbediscussedinmoredetail[Fig.
2(C)],sinceitisbroadlyapplicable.
LIPID-BASEDNANOPARTICLESFORCONTRAST-ENHANCEDMRI145Copyright#2006JohnWiley&Sons,Ltd.
NMRBiomed.
2006;19:142–164First,theligandshouldexposeafreethiolgroup,neces-saryforbondformation.
Proteins,antibody(fragments)andpeptidesexposingafreecysteinecandirectlybeusedforcouplingtomaleimide[Fig.
2(A),III].
Peptidessynthesizedwithaprotectiveterminalthioacetategroupcanbeactivatedupondeacetylationwithhydroxylamine[Fig.
2(A),II].
Thisresultsintheconversionofthethioacetateintoathiolgroup.
Thesamestrategyisusedforproteinsthatareactivatedwithsuccinimidyl-S-acetylthioacetate(SATA)[Fig.
2(A),I].
SATAiscoupledtofreeaminogroupspresentintheproteinandwithhydroxyla-minethethioacetatemoietyisconvertedintoafreethiolgroup.
Thethiolligandsreactwithmaleimide-containingparticlesandformacovalentthioetherlinkage,asde-pictedschematicallyinFig.
2(C).
RELAXIVITYOFMACROMOLECULARCONTRASTAGENTSTherelaxivity,i.
e.
thepotencytoshortentheT1andT2relaxationtimesofwater,ofanMRIcontrastagentisdenedbythechangeinlongitudinalortransversalrelaxationratesperunitconcentrationofthecontrastagent(2,3).
Theconstantofproportionalityiscalledtherelaxivity,r1orr2,andisexpressedinmM1s1.
Furthermore,theratiobetweenr2andr1determineswhetheracontrastagentissuitableforcontrast-enhancedT1-weightedimagingorwhetheritcanbetterbeusedforcontrast-enhancedT2-andT2*-weightedimaging.
Theso-calledT1agents,typicallychelatesofGd3ions,havealowratioofr2tor1(usuallybetween1.
1and2)andgeneratepositivecontrast(bright/hotspotsinT1-weightedMRI),whereastheT2agentshavealarger2andgeneratenegativecontrast(dark/coldspotsinT2-andT2*-weightedMRI).
TherelaxivityofparamagneticGd3chelatesisde-terminedbythecomplexinterplayofmanyparametersgoverningthedipolarinteractionsbetweenwaterandtheparamagneticGdentity.
Acompletetreatmentwouldbebeyondthescopeofthisreviewandwethereforerestrictourselvestoaqualitativedescriptionofsomecommonobservations.
Themostimportantparametersforunder-standingtherelaxivityofmacromolecularcontrastagentsaretheexchangeratem,thecoordinationnumberandtherotationalcorrelationtimer.
Thecoordinationnum-berandtheexchangeratemdeterminetheamountofwatermoleculesthatcaneffectivelycoordinatewithFigure2.
(A)Introducingthiolgroupsin(I)proteinsorantibodiesand(II)peptides.
(III)Proteinandpeptidewithfreecystein.
(IV)Targetingligandswithafunctionalgroup,i.
e.
thiolorbiotin.
(B)Schematicrepresentationofavidin–biotinlinkageofaligandtoalipidicnanoparticle.
(C)Schematicrepresentationofmaleimide–thiollinkageofaligandtoalipidicnanoparticle146W.
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MULDERETAL.
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NMRBiomed.
2006;19:142–164Gd3andtherebyincreasetherelaxationrate.
Therota-tionalcorrelationtimerisimportantbecausethelowertumblingratesofmacromoleculesareresponsiblefortheincreaseinr1thatisobservedinatypicalrangeofeldstrengths.
Ausefulwaytogaininsightintotherelaxationbehaviorofmacromolecularcontrastagentsistorecordther1relaxivityasfunctionoffrequency,so-callednuclearmagneticrelaxationdispersion(NMRD).
TheNMRDproleofamacromolecularcontrastagentshowsatypicalpeakathigherfrequencies,inagreementwiththeincreaseintherotationalcorrelationtimesascom-paredwithlow-molecularweightGd3chelates.
Lipid-basedcontrastagentscanbeconsideredmacromolecularcontrastagentsandthetumblingrateoftheGdchelatesinsuchstructuresisstronglydecreased.
Asanexample,Fig.
3showstheNMRDprolesofGd–DTPAandatypicalGd-basedliposomalandmicellularcontrastagent.
TheNMRDprolesoftheliposomesandmicellesdisplaythetypicalpeakathigherfrequencies.
Thismeansthatattheclinicallyrelevanteldstrengthsthesecontrastagentshavethehighestionicrelaxivity.
Furthermore,theamountofGdchelatesperparticleishigh(varyingfrom50forsmallmicellestoseveralhundredthousandforliposomes).
Thisenhancestherelaxivitypercontrastagentparticleenormously.
T2contrastagentsusuallyaresuperparamagneticironoxideparticles.
ThemagneticmomentsofsuchparticlesaremuchlargerthanthatofGd3-containingchelates,typicallyuptomorethanthreeordersofmagnitudedependingontheirsize.
Asaconsequence,superpara-magneticparticleshaveasubstantiallylargerr2relaxivitycomparedwithparamagneticcontrastagents.
Theoriginofthisenhancedrelaxivityliesinthestronglocaleldgradientssurroundingthesuperparamagneticparticles,whichgiverisetoacceleratedlossofphasecoherenceofthesurroundingwaterprotonspins(4).
Increasedrelax-ivitycanbeobservedataconsiderabledistancefromthenanoparticlesince,incontrasttothedipolarrelaxation,thissusceptibility-inducedrelaxationdoesnotdependonadirectphysicalcontactbetweenprotonsandthepara-magneticentity.
Foramorequantitativeinsightintotherelaxationcharacteristicsofdifferentlipidiccontrastagentsdescribedintheliteraturewehavecompiledaselectionofreportedvaluesofr1and/orr2(Table1).
Directcomparisonsbetweendifferentagentsaredifcultbecauseofdifferenteldstrengthsandtemperaturesused.
Therefore,wehavegiventherelaxivityofGd–DTPAmeasuredunderthesameconditionsasareference.
InsomestudiesGd–DTPAwasnotmeasuredasareference.
Asareferencerelaxivityforthesecontrastagents,thevalueofGd–DTPAat298KaspresentedbyAimeetal.
(3)isgiven.
IncaseswhereonlyanNMRDprolewasavailableforagivencontrastagent,wereporttherelaxivityat20and60MHz.
Furthermore,itshouldbetakenintoconsiderationthattherelaxivityisexpressedasfunctionoftheGdconcentration.
Therelaxivityperparticleismuchhigher,sincetheparticlesdepictedinthetablecarryhighpayloadsofGdorFe.
BIOLOGICALTARGETSInthissection,anumberofpathologieswillbebrieydiscussedforwhichMRIandstate-of-the-artcontrastagentscanbeusedtoimageinvivotheinltrationofcellsandtheexpressionofbiologicalmarkerstoimprovethediagnosisofdiseaseandtodeveloptherapeuticstrategies.
InammationInammationisthebody'sresponsetodamage,infection,allergyorchemicalirritation.
Manydisordersareasso-ciatedwithinammation.
Inammationiscausativeandsymptomaticinthedestructionofcartilageinrheumatoidarthritis,typeIdiabetesandlossofintestinalfunctioninCrohn'sdisease.
Inammatoryresponsesarealsoob-servedincancer,whichisaccompaniedbythemassiverecruitmentofleukocytesingrowingtumors(75).
Incardiovasculardiseasessuchasmyocardialinfarctionandatherosclerosis,chronicinammationiscausallyrelatedtothepathology(76,77).
Furthermore,multiplesclerosis,ischemia-reperfusioninjuryaftercerebralstrokeandAlzheimer'sdiseasearecausedbyorasso-ciatedwithinammatoryprocesses(78).
Theinammatoryresponseinvolvesthemigrationofleukocytes,thecellsoftheimmunesystemsuchasneu-trophils,monocytes/macrophagesandlymphocytes,intodamagedorinfectedtissues.
RecruitmentofleukocytesisFigure3.
NMRDprolesofGd–DTPA-andGd–DTPA-BSAcontainingliposomesandmicellesLIPID-BASEDNANOPARTICLESFORCONTRAST-ENHANCEDMRI147Copyright#2006JohnWiley&Sons,Ltd.
NMRBiomed.
2006;19:142–164intricatelyregulatedbyinammatorycytokinessuchasinterferon-,tumornecrosisfactor-andinterleukin-1.
Inresponsetothesecytokines,endothelialcellsoverexpresscellsurfaceadhesionmolecules,includingtheselectins(e.
g.
E-selectin)thatareinvolvedinrollingofleukocytesalongthevascularwall,immunoglobulin-likeadhesionmoleculessuchasintercellularadhesionmolecule-1(ICAM-1)andvascularcelladhesionmolecule(VCAM-1)thatsupportrmadhesionandextravasationofleukocytesintothetissue.
Imagingofinammatorysitescanbeachievedbymakinguseofseveraldifferentcharacteristicsofaffectedtissues.
First,thespecicoverexpressionofendothelialadhesionmoleculescausedbytheexposureTable1.
OverviewoftherelaxivitiesofseveraldifferentlipidicnanoparticulateMRIcontrastagentsaAggregateReferenceDescriptionFieldstrengthTemperature(K)r1Gd–DTPAbr1br2btypeLiposomesTilcocketal.
(112)Gd–DTPAinlumen1.
5T2.
790.
42liposomes400nmGd–DTPAinlumen1.
5T2.
791.
60liposomes70nmKimetal.
(130)MHE–DTTAin0.
47T4.
131.
9bilayerliposomesBME–DTTAin0.
47T4.
127.
1bilayerliposomesTilcocketal.
(132)Stearylester–DTPA–20MHzc30823GdinbilayerliposomesStearylamide–DTPA–20MHzc30813GdinbilayerliposomesStorrsetal.
(30)Polymerizedliposomes2.
0T4.
2412.
2longspacerPolymerized2.
0T4.
245.
7liposomesshortspacerGlogardetal.
(34)AmphiphilicGd20MHzc3004d47chelatesinbilayerAmphiphilicGd60MHzc3004.
5d25chelatesinbilayerBulteandDeMagnetoliposomes1.
5T3103210Cuyper(40)Stealth1.
5T3103240magnetoliposomesBertinietal.
(137)Paramagnetic20MHzc2984d12liposomesParamagnetic60MHzc2984.
5d12.
5liposomesStrijkersetal.
(149)Gd–DTPA–20MHz3103.
88.
2BSA-containingliposomesMicellesNicolleetal.
(193)Gadouorine20MHzc2984d22Gadouorine60MHzc2984.
5d12Hovlandetal.
(135)Amphiphilic20MHzc2984d26GdPCTA-[12]60MHzc2984.
5d28Accardoetal.
(156)Mixedmicellar20MHzc2984d18aggregates60MHzc2984.
5d18MulderandMicellulariron20MHzc2984d15200vanTilborgoxideMCIO(unpublisheddata)MicroemulsionWinteretal.
(194)NanoparticlesGd–1.
5T3134.
5d17.
725.
3DTPA–BOANanoparticles1.
5T3134.
5d33.
750Gd–DTPA–PEHigh-densityFriasetal.
(102)HDL-likenanoparticles65MHz2984.
5d10.
4lipoproteinaNotethattheionicrelaxivityisgiven.
Therelaxivityperparticleismuchhigher,sincetheparticlesdepictedinthetablecarryhighpayloadsofGdorFe.
br1Gd–DTPA:T1relaxivityofGd–DTPAexpressedinmM1s1.
r1:T1relaxivityofthecontrastagentreferredtoexpressedinmM1s1.
r2:T2relaxivityofthecontrastagentreferredtoexpressedinmM1s1.
cNMRDprolewasgiveninthereference.
dDatafromAimeetal.
(3).
148W.
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NMRBiomed.
2006;19:142–164toinammatorycytokinescanbeusedtotargetcontrastagents.
Second,becauseoftheenhancedpermeabilityofbloodvesselsduetoanongoingangiogenicresponse,injectionofanon-speciccontrastagent,preferablyalong-circulatingcontrastagentofrelativelyhighmole-cularweight,mayresultintheaccumulationofthecontrastagentattheinamedsite.
Furthermore,migra-tionofcellsinvolvedininammationcanbefollowedafterlabelingthecellswithanappropriatecontrastmaterial.
Thiscanbeachievedinvivo,whenacontrastagentistakenupbycells(79)(e.
g.
monocytes)inthecirculationorexvivo,whencellsarelabeledoutsidethebodyandsubsequentlyinjected(80).
Currently,mucheffortisbeingputintoresearchonthetargetedimagingofcelladhesionmoleculesinvolvedininammation.
Tar-getingoftheadhesionmoleculemaybedonewithantibodies,proteins,peptidesorsmallmoleculesconju-gatedtoanMRIcontrastagent.
Lipid-basedcontrastagentshavebeenusedforallthreestrategies(31,81).
AngiogenesisAngiogenesis,theformationofnewbloodvesselsfrompre-existingbloodvessels,isasequenceofeventsthatiskeyinmanypathologicalprocesses(14,82,83).
MRcontrastagentscancontributetothedetectionofangio-genicareasandvesselsviatwostrategies.
Intherststrategy,thepermeabilityoftheangiogenicvasculatureisdeterminedwithdynamiccontrast-enhancedMRI.
Re-cently,ithasbeendemonstratedthattheuseofcontrastagentsofhighmolecularweightgivesmostinsightintovasculaturepermeabilityinangiogenesis(14).
Asanexample,albumintriplylabeledwithauorescentlabel,Gd–DTPAandbiotinhasbeenusedtoinvestigateangio-genesisbymeasuringvascularpermeabilitywithacom-binationofuorescencemicroscopyandcontrast-enhancedMRI(8).
Therateofclearanceofthelatteralbumincontrastmaterialfromthecirculationcanbemanipulatedbyactivelyremovingitwithanavidinchase.
Lipid-basedcontrastagentscanbesynthesizedinawiderangeofsizes,usefulforprobingvascularpermeabilityandwithdifferentfunctionalmoieties,e.
g.
abiotinylatedlipidforanavidinchaseandauorescentlipidandthereforewouldbeverysuitablefortheapplicationsjustdescribed.
Thesecondstrategyistotargetcontrastagentstomarkersthatarespecicallyassociatedwithangiogenicallyactivatedendothelialcells.
Manycellsurfacereceptorsarestronglyexpressedonactivatedendothelialcellsofangiogenicvessels,ascomparedwithrestingendothelialcellsofbloodvesselsinnon-diseasedtissue.
Thesereceptorsincludev3-and1-integrins,vascularendothelialgrowthfactorreceptor(82),CD36andCD44(84).
Especiallythev3-integrinhasbeenshowntobeveryusefulasatargetfortherapiesandmolecularimagingcontrastagents.
Inadditiontov3-specicantibodies,thev3-specicRGDpeptideandpeptidomimeticshavebeenusedinseveralstudies.
Theexpressionofthisintegrinhasbeennon-invasivelyvisualizedintumor-bearingmicewithacombinationofradiolabeledRGDandpositronemissiontomography(85).
Genedeliverywithv3-targetedlipidicnanopar-ticlesintumor-bearingmiceresultedintumorcellapoptosisandsustainedregressionofthetumors(68).
ApoptosisApoptosis,orprogrammedcelldeath,isessentialfortissuedevelopmentandhomeostasis.
Deregulationoftheapoptoticprogramisoftenfoundtoplayacriticalroleintheetiologyofvariouspathologicalconditions,includingneurodegenerativediseases,autoimmunediseases,cardi-ovasculardiseases,tumordevelopmentandorgantrans-plantrejection.
Inaddition,chemotherapeuticdrugsorradiationtherapyoftenrelyontheinductionofapoptoticcelldeath.
Therefore,theinvivodetectionofapoptosiscouldbeofgreatimportancefortheevaluationofdiseaseprogressionordiseasetreatment.
Severalstudieshavebeenperformedinwhichthedetectionofapoptosiswasbasedontheexpressionofthelipidphosphatidylserine(PS)ontheouterlayeroftheapoptoticcellmembrane.
TheexpressionofthisphospholipidcanbedetectedwithannexinVorsynaptotagminIconjugates.
BothproteinsbindwithhighafnitytoPSinaCa2-dependentmanner.
Koopmanetal.
(86)werethersttodescribetheuseofFITC-conjugatedannexinVforthedetectionofapoptoticBcellswithowcytometry.
Therstinvivovisualizationofprogrammedcelldeathwascarriedoutwithradiola-beledannexinV(87)invariousanimalmodelsandthereafterinpatientswithacutemyocardialinfarction(88).
AnnexinV,conjugatedtouorescentdyes,hasbeenusedforinvivoopticalimaginginthebeatingmurineheart(89)andrecentlyseveralCy5.
5-conjugatedannexinVprobeshavebeenintroducedfornear-infrareduor-escentimaging(90).
AnnexinV-functionalizedcross-linkedironoxide(CLIO)wasdesignedasacontrastagentforMRI,whichwasadditionallylabeledwithCy5.
5toallowco-localizationwithopticalimagingtechniques(91).
Alternatively,conjugationofmultipleGd–DTPAmoleculesorsuperparamagneticironoxideparticles(SPIO)totheC2domainofsynaptotagminIwasshowntoallowthedetectionofapoptoticcellsinvitro(92).
Zhaoetal.
(93)werethersttoapplyaC2domain-functionalizedSPIOandshowedverypromisingresultsforfutureinvivoapplicationsofMRcontrastagentsforthedetectionofapoptoticsites.
TumorsAlthoughtheabove-describedprocessesofangiogenesis,apoptosisandinammationaretargetsthemselvesinthedevelopmentofanti-cancertherapies,alotofresearchisLIPID-BASEDNANOPARTICLESFORCONTRAST-ENHANCEDMRI149Copyright#2006JohnWiley&Sons,Ltd.
NMRBiomed.
2006;19:142–164currentlybeingperformedonthedevelopmentoftargetedtherapiesdirectedattumorcells.
Signaltransductionresearchhasshowntheimportanceofthehumanepider-malgrowthfactorreceptor(HER)familyoftransmem-branetyrosinekinasesinanumberofsolidtumortypes.
OnememberofthisfamilyisHER-2(ErbB-2),whichisoverexpressedinseveraltypesofcancers,includingbreast,lung,gastricandbladdercarcinomas.
HER-2expressionissimilarinprimarytumorsandcorrespond-ingmetastases(94).
Theexpressioninnormaltissuesisverylow,makingHER-2acandidatefortargetingstra-tegies.
Also,theepidermalgrowthfactorreceptor(EGFR,alsoknownasErbB-1orHER-1),animportantmoleculeintheproliferationandmetastasisoftumorcells,isfrequentlyoverexpressedincommonsolidtu-morsandhasbecomeafavoredtargetfortherapywithmonoclonalantibodiesdirectedattheextracellulardo-mainofthereceptorandfortherapywithsmallmoleculeinhibitorsofthereceptor'styrosinekinaseactivity(95).
Inadditiontotherapybasedonblockingthereceptorandtherebyachievinginhibitionoftumorcellgrowthandmetastaticpotential,thesereceptorscanalsobeusedfortargeteddeliveryofdrugsordiagnosticimagingagents(96).
TargetsexpressedattumorcellsaremoredifculttouseformolecularMRIsincetheycannotbereacheddirectlyviathecirculation,butrequirethecontrastagenttoleakfromthebloodvesselsintotheextravascularcompartment.
SincemostmolecularMRIcontrastagentsarenanoparticulatematerials,thisposesalimitonimagingsuchextravascularreceptors.
Nevertheless,mo-lecularMRIoftheHER-2/neureceptor,expressedattumorcells,withavidin–Gdcomplexesafterprelabelingthereceptorswithbiotinylatedanti-HER-2/neuantibodyhasbeendemonstrated(97).
Furthermore,HER-2/neu-targetedimmunoliposomeseffectivelyassociatewithtumorcellsandshowantitumorefcacy(98).
AtherosclerosisAtherosclerosisisaprogressivedisease,whichcanbeconsideredachronicinammationofthelargearteries(99,100).
Thediseasestartswithinammation-likeendothelialdysfunctioncausedbylocalinjuryorbytheretentionofatherogeniclipoproteins.
Theprocessisinitiatedbyoxidizedlipoproteinsinthevesselwall,e.
g.
oxLDL,whichtriggerstheendotheliumtoexpressmonocyterecruitingendothelialcellreceptorssuchasVCAM-1,P-selectin,E-selectinandICAM-1.
Mono-cytesaccumulateinthesubendothelialspaceanddiffer-entiateintomacrophagesthattakeupoxLDL.
Eventually,themacrophagesareconvertedintofoamcells.
Atthisstageofthediseasethelesioniscalledanearlylesionorafattystreak.
Withcontinuinglipoproteinaccumulationandfoamcellformation,theearlylesionprogressesintoanatheromatouscore.
Theatheroscleroticplaquethusformedisstabilizedbythemigrationofsmoothmusclecells,resultingintheformationofabrouscap.
Bloodsupplyintotheheavilythickenedvesselwallismain-tainedbyanangiogenicexpansionofthevasavasorum(vesselswithinthewalloflargerbloodvessels)(101).
Theadvancedplaque,orcomplexlesion,maybeatriskofrupture,whichtriggersthrombusformation.
Thisprocessisthemaincauseofacuteclinicalcomplicationssuchasstrokeandmyocardialinfarction.
Inthiscascadeofevents,severalmarkersareofinterestfortarget-specicMRI.
LDLorHDLcanbeparamag-neticallylabeled(102,103)foridenticationofathero-scleroticplaquesafteruptakeofthesecontrastagentsbytheplaque.
Allinammatorymarkers,e.
g.
E-selectin,P-selectinorVCAM-1,expressedontheendothelialcellarepotentialtargetsformolecularMRIofatherosclero-sis.
Fluorescentandsuperparamagneticironoxidenano-particleshavebeensuccessfullytargetedtoplaquesintransgenicmiceusingaVCAM-1-specicpeptide(104).
Furthermore,monocytesandothercellsrelatedtoinammationcanbemagneticallylabeled,invivoandexvivo,tofollowtheirfateinrelationtoatherosclerosis.
Sincestrongangiogenicactivitymayoccurinthevasavasorum,strategiessketchedinthesectionaboutangio-genesisalsohavepotentialinidentifyingandcharacter-izingatheroscleroticplaques.
Inadvancedstagesofplaqueformation,apoptoticactivityiscommonandcontributestoplaqueinstability.
DetectionofapoptoticplaqueswithMRIwouldthereforegivetheopportunitytodeterminetheriskofplaqueruptureinvivo.
Strategiestoimageapoptosisareoutlinedinthesectiononapoptosis.
Inthenalstageoftheatheroscleroticprogression,thrombi,e.
g.
formedasaresultofplaquerupture,canbetargetedwithbrinorplatelet-specicpeptides(105)orantibodies(72).
LIPID-BASEDMRICONTRASTAGENTS:APPLICATIONSLiposomalcontrastagentsInthissection,anoverviewofliposomalMRIcontrastagentsisgiven.
Abriefoutlineofhistoricaldevelopmentsisfollowedbyasummaryofwhathasbeendonethusfar.
Inthesecond,partanumberofinterestingandinnovativedevelopmentsarereviewedanddiscussedinmoredepth.
Liposomeswerediscoveredintheearly1960sbyBanghametal.
,whofoundthategglecithinphospho-lipidscombinedwithwaterself-organizedintospheresbecauseoftheamphiphiliccharacterofthelipids(106).
Liposomescanbedenedasspherical,self-closedstructuresformedbyoneorseveralconcentriclipidbilayerswithanaqueousphaseinsideandbetweenthelipidbilayers(107).
Liposomeshavebeenusedexten-sivelyasamodeltostudythepropertiesofbiologicalmembranes.
Theycanvaryinsizeandlamellarityandarethereforesubdividedintomultilamellarvesicles(MLV),consistingofseveralconcentricbilayers,large150W.
J.
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2006;19:142–164unilamellarvesicles(LUV),inthesizerange200–800nm),andsmallunilamellarvesicles(SUV),inthesizerange50–150nm.
Soonafterthediscoveryofliposomes,theyweresuggestedforuseasdrugcarriers,becauseoftheirstrikingbiologicalproperties.
First,liposomesarecomposedofnaturallyoccurringlipidsorcloselyrelatedsyntheticlipidsandthereforearebio-compatible.
Liposomescancarrywater-solublephar-maceuticalagentsintheaqueousinteriorandamphiphilicorhydrophobicagentsinthelipidbilayer.
Furthermore,liposomalpharmaceuticalagentsarepro-tectedfrominteractionswithplasmaproteinsanddeactivationandexhibitprolongedcirculationtimesandfavorablebiodistributionproperties.
Alteringtheirsurfacepropertiesmakespossibleimproveddeliveryofliposomestodiseasedtissueandintocells.
ThesepropertiesalsomakeliposomesexcellentcandidatestocarryordelivercontrastagentsforMRIandinthe1980stherststudiesabouttheuseofliposomesascarriersofMRcontrastagentsappearedintheliterature.
ThersttypeofliposomalMRIcontrastagentsde-scribedwereliposomesentrappingparamagneticagentsintheaqueouslumen.
ParamagneticagentssuchasMnCl2,(108,109)Gd–DTPA(110–112),Mn–DTPA(113),Gd–DTPA–BMA(114)andGd–HP–DO3A(115)andmacromolecularcontrastagentssuchasMn2boundtoserumproteins(116)canbepreparedinaliposomalformulation.
Contrastagentsofthistypehavebeenusedsuccessfullytoimprovethedetectionoftumorsintheliverofratswithhepaticmetastases(117–119).
Recently,gadodiamide-anddoxorubicin-containingliposomeswereusedtostudytheliposomaldistributionafterconvection-enhanceddeliverytobraintumorsinrats(120)andmice(121).
Furthermore,gadobutrol-containingliposomalcarriersequippedwithRGDligandsweretargetedtov3-integrin-expressingcellsinvitro(122).
Althoughtheliposomalformulationscontainingaparamagneticpayloadintheaqueouslumenhavebeenusedsuccess-fully,theutilityofthesecontrastagentsislimited.
First,therelaxivityoftheentrappedparamagneticspeciesislowered,becauseofthelimitedexchangeofbulkwaterwiththecontrastagents(112,123).
Thisexchangeisdependentonthepermeabilityoftheliposomalmem-branetowater(124).
Thepermeabilityoftheliposomalmembranedependsonthelipidcompositionandcanbealteredbyvaryingthesaturationlevelofthelipidchainsandthelengthofthelipidchainsandbyincorporationofcholesterol.
Themorepermeablethemembrane,thebetteristhewateruxacrossthebilayerandthebettertherelaxivity(112).
Furthermore,thecontrastefciencycanbeimprovedbyusingsmallerliposomes.
Thevo-lume-to-surfaceratioofsmallliposomesislowerandthereforetheexchangewithexternalbulkwaterisbetter(112,123).
Thisimpliesthatintermsofrelaxationproper-ties,anoptimalformationwouldbeliposomesofsmallsizewithapermeablebilayer.
Unfortunately,permeableliposomesusuallyarelessstableinserumthanliposomeswithamorerigidbilayer.
Aseconddrawbackisthatupondegradationtheliposomesmaylosetheirparamagneticcontent,whichmakestheinterpretationofobservedcontrastenhancementproblematic.
Nowadays,suchsys-temsarestillindevelopmentandthedrawbacksjustdescribedhavealsobeenusedbenecially,e.
g.
formonitoringdrugreleasefromliposomes(125)andtousemembranetransitionpropertiestomonitorlocaltissuetemperature(126)orpH(127).
Moreaboutthissubjectwillbediscussedinalatersectionaboutsmartcontrastagents.
Asecondclassofliposomalcontrastagentscarriestheparamagneticmoleculeinthelipidbilayer,whichmakestheamphiphilicparamagneticcomplexesanintegralpartoftheliposomalsurface(128,129).
Thisapproachresultsinanimprovedionicrelaxivityofthemetalcomparedwiththeapproachofencapsulatingtheparamagneticmoleculesintheaqueousinteriorandcomparedwithlowmolecularweightcomplexes(130).
TheamphiphilesusedcanconsistofGd–DTPAasthehydrophilicpartattachedtoahydrophobicpart.
DTPA–stearate(131,132)orDTPAattachedtoalkylchainsviaamidelinkersareexamplesofsuchmolecules(133,134).
Phosphatidy-lethanolamine(PE)hasbeenlinkedtoDTPAtoobtainPE–DTPA,whichcanformvesicleswhenmixedwithnaturalphospholipids(135).
TheNMRDproles,eld-dependentrelaxationmeasurements,closelyresemblethoseofmacromolecularcontrastagents,withatypicalpeakathighereldstrengths(34,132),becauseofthelongerrotationalcorrelationtime.
Theapplicabilityofsuchliposomalcontrastagentsisbroad.
Pegylatedlipo-someshavebeenusedasabloodpoolagent(136),forthedetectionoflymphnodes(136)andtoachievesustainedcontrastenhancementoftumors(137).
Regionsofen-hancedpermeabilitycanbedetecteduponinjectionofparamagneticliposomes,whichisusefule.
g.
aftermyo-cardialinfarction(138)andfortumordetection.
Re-cently,thebiodistributionoflipoplexes,cationicliposomesboundtoDNA,wasevaluatedovertimebyincorporatingaGd–lipidamphiphile(139).
Uponintra-tumorinjectionofthelipoplexes,astrongandpersistentT1-weightedMRIsignalincreasewasobserved.
Polymerizedliposomes.
In1995,Storrsetal.
intro-ducedanovelliposomalMRIcontrastagent(30).
ADTPA-basedlipid,amphiphiliccarrierlipidsandabio-tinylatedamphiphile,allcontainingadiacetylenetriplebondinthefattyacylchains,weresynthesized.
Fromtheselipids,liposomeswerepreparedandforfurtherstabilizationthetriplebond-containinglipidsinthelipo-someswereilluminatedwithUVradiationtoinducepolymerizationviathetriplebonds.
Thebiotingroupinthesepolymerizedvesicleswasusedforconjugationofbiotinylatedantibodiesviaanavidin–biotinlinkingpro-cedure.
Inrats,thehalf-lifeofthiscontrastagentisalmost20h(140).
Sipkinsetal.
usedthevesiclescoupledLIPID-BASEDNANOPARTICLESFORCONTRAST-ENHANCEDMRI151Copyright#2006JohnWiley&Sons,Ltd.
NMRBiomed.
2006;19:142–164tov3-specicLM609antibodiestodetectangiogen-esisintumor-bearingrabbits(15).
SinceMRIhasaninherentlylowsensitivitycomparedwithnuclearmeth-ods,thedetectionoftheexpressionofsparseepitopesshouldbedonewithapowerfultargetedcontrastagent.
Thepolymerizedliposomesusedinthisstudywere300–350nminsizeandcontained30%oftheGd–lipid.
Thismeansthattheseliposomeshaveanextremelyhighpayloadofgadoliniumperparticle,whichmakesthemverypotent.
Thecontrastagentwasappliedintravenouslyintumor-bearingrabbits,whichresultedinastatisticallysignicantsignalintensityenhancement24hafterinjec-tion.
Importantly,thesignalenhancementcorrelatedwiththeimmunohistochemicaldeterminationofv3-integrindistribution.
Inlaterstudies,thispolymerizedliposomalsystem,targetedtothev3-integrin,wasusedtodeliveramutantRafgene(ATPmu-Raf)toangiogenicbloodvesselsintumor-bearingmice(68).
ATPmu-Rafblocksendothelialsignalingandangiogen-esisinresponsetomultiplegrowthfactors.
Apoptosisofthetumor-associatedendotheliumwasinducedbysys-temicinjectionoftheliposomesintomice,whichre-sultedinsustainedregressionofthetumors.
TheaboveliposomalsystemwasalsousedtodetecttheexpressionofleukocyteadhesionmoleculeswithMRIinthebrainofmicewithexperimentalautoimmuneence-phalitis(EAE)(71),amousemodelofmultiplesclerosis.
Tothatend,thepolymerizedliposomeswereconjugatedtobiotinylatedantibodiesspecicforICAM-1.
MicewereinjectedwithICAM-1-specicliposomesandnon-specicliposomes.
Thebrainsoftheanimalswereremovedandscannedexvivowithhigh-resolutionMRI.
MarkeddifferenceswereobservedbetweenEAEmicethatwereinjectedwiththespeciccontrastagentandthecontrolcontrastagentandhealthymicethatwereinjectedwiththespeciccontrastagent(Fig.
4).
T1-weightedimagesoftheEAEmicethathadreceivedICAM-1specicliposomesdemonstratedwidespreadMRsignalintensityincreasesthroughoutthecentralnervoussys-tem,whichcorrelatedwiththepatternofICAM-1ex-pressionasdeterminedimmunohistochemically.
Magnetoliposomes.
Magnetoliposomesareliposomescontainingsolidironoxideparticlesintheliposomallumen(40).
Magnetoliposomeshavebeenusedoriginallytostudybiologicalmembranes(141)andforcellsorting(142).
Furthermore,theycanbeusedfortargeteddrugdelivery(usingaconstantmagneticeld)andcontrolledrelease(usinghigh-frequencymagneticeldoscillations)ofanentrappeddrug(143,144).
Twotypesofmagneto-liposomeshavebeenusedmostoften.
Thersttypecontainswater-solubleironoxideparticlesintheaqueouslumen(145,146).
Thesecondtype,developedbyDeCuyperandJoniau,(147),isanironoxideparticleof$15nmcoveredwithalipidbilayer.
Thelatterwillbediscussedinmoredetail,sinceithasbeenappliedasanMRIcontrastagentinvivo.
Theformationofthistypeofmagnetoliposomestartswiththesynthesisofamagneticuidofsuperparamagneticironoxideparticles.
Theironoxideparticlesarestabilizedandsolubilizedwithlaurate.
Whentheparticlesareincubatedwithanexcessofphospholipidvesiclesanddialyzedforanum-berofdays,thephospholipidsfromthevesiclestransferandabsorbonthesolidsurface,ultimatelyformingabilayerofphospholipidsaroundtheironoxideparticles.
Forimprovedpharmacokineticproperties,PEG–lipidscanbeintroducedbysimplymixingthemagnetolipo-someswithdonorvesiclescontainingtheselipids.
ThePEG–lipidstransferspontaneouslyfromthedonormem-branestothebilayerofthemagnetoliposomes.
Bulteetal.
demonstratedtheapplicabilityofthisnanoparticleasabonemarrowMRcontrastagent(148).
Furthermore,thepegylatedmagnetoliposomescanbefunctionalizedthroughincorporationofaPEG–lipidwithadistalfunc-tionalmoiety,whichcanbeusedforconjugationtoachievespecicityforthebiologicalmarkerofinterest.
Figure4.
High-resolutionMRimagesoftheEAEmousebrainwithanti-ICAM-1PVcontrastenhancementvscontrols.
(A)T2-weightedMRimageofexvivoEAEmousebrain.
T1-weightedMRimageofexvivomousebrain(B)withand(D)withoutEAEafterinjectionofanti-ICAM-1antibody-conjugatedparamagneticliposomes.
(C)EAEmousebrainafterinjectionofcontrolisotypeantibody-conjugatedparamagneticliposomes.
WidespreadMRsignalintensityenhance-mentthroughoutthebrainwithEAEcanbeobservedfor(B)only.
AdaptedfromFig.
4ofSipkinsetal.
,ICAM-1expressioninautoimmuneencephalitisvisualizedusingmagneticresonanceimaging.
J.
Neuroimmunol.
2000;104:1–9,withpermissionfromElsevierScience152W.
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MULDERETAL.
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NMRBiomed.
2006;19:142–164Bimodalliposomes.
Mulderetal.
introducedabimo-daltargetedliposomalcontrastagentforthedetectionofmolecularmarkerswithbothMRIanduorescencemicro-scopy(31,149).
TheliposomesconsistofGd–DTPAat-tachedtotwostearylchains,auorescentlipid,DSPC,cholesterolandPEG–DSPE.
Thelastcomponentprovidestheliposomeswithahydrophiliccoatingforimprovedstabilityinvivo.
Invitro,thiscontrastagentconjugatedwithE-selectin-specicantibodieswastestedonhumanen-dothelialcells(HUVEC)stimulatedwithtumornecrosisfactor(TNF).
Apronouncedcontrastagentassociationwasobservedwithuorescencemicroscopyatthesubcel-lularlevelandwithMRIoncellpellets(31).
Furthermore,apoptoticjurkatcellsweresuccessfullytargetedandimagedinvitrowiththebimodalliposomalcontrastagentconjugatedwithAnnexin-V(150).
Follow-ingincubationwiththecontrastagent,pelletsofapopto-ticcellsshowedincreasedsignalsinT1-weightedimages,whereasitwasrevealedwithconfocallaserscanninguorescencemicroscopythatthecontrastagentwasboundtothecellsurface.
TheliposomalcontrastagentconjugatedwithcyclicRGD–peptideswasusedtoidentifytheangiogenicendotheliumintumorbearingmicewithinvivoMRIandexvivouorescencemicroscopy(151,152).
ThecyclicRGD–peptidehashighafnityforthev3-integrin,whichisupregulatedatendothelialcellsofangiogenicbloodvessels.
MRIrevealedthatuponintravenousinjectionofthecontrastagent,theRGD–liposomeslocalizedtoalargeextentinthetumorrim,whichisknowntohavethehighestangiogenicactivity[Fig.
5(A)].
Non-specicRAD-liposomesalsotargetedthetumor,butshowedadiffusedistributionpattern[Fig.
5(B)].
Thedifferentmechanismsofaccumulationwereestablishedwithuorescencemicroscopy,whichrevealedthatRGD–LNPwereexclusivelyassociatedwithtumorbloodves-sels[Fig.
5(C)]whereasRAD–LNPwere,toalargeextent,localizedintheextravascularcompartment[Fig.
5(D)].
ThisstudydemonstratedthecriticalimportanceofvalidatingtheMRIndingswithacomplementarytech-niquesuchasuorescencemicroscopy.
MicellularcontrastagentsMicellesareinparticularinterestingforcarryingpoorlysolublepharmaceuticalagents(153).
Furthermore,theyFigure5.
MRimagesoftumorsofmiceaftertheywereinjectedwith(A)paramagneticv3-specicRGD–liposomesand(B)non-specicparamagneticRAD–liposomes.
Fluor-escencemicroscopyof10msectionsfromdissectedtumorsrevealedadistinctdifferencebetweentumorsofmicethatwereinjectedwithRGD–liposomes(C)orRAD–liposomes(D).
Vesselstainingwasdonewithanendothelialcell-specicFITC–CD31antibody.
Thereduorescencerepresentstheliposomes(C)andthegreenuorescencerepresentsbloodvessels.
RGD–liposomeswereexclusivelyfoundwithinthevessellumenorassociatedwithvesselendothelialcells(C),whereasRAD–liposomes(D)werealsofoundoutsidebloodvesselswithinthetumorLIPID-BASEDNANOPARTICLESFORCONTRAST-ENHANCEDMRI153Copyright#2006JohnWiley&Sons,Ltd.
NMRBiomed.
2006;19:142–164canbepreparedfromlipidsthatcontainaPEGmoietytomakethesurfaceofthemicelleinerttobloodcompo-nents.
Thisletsmicellescirculateinthebloodforafairlylongtimewithoutbeingrecognizedbycertainplasmaproteinsand/orphagocyticcells.
Inthisway,theycanbeusedaslong-circulatingbloodpoolagents,whichnon-specicallytargettoareaswithaleakyvasculature.
Ligandscanalsobecoupledtothesurfaceofthemicellesfortargetingtospecicsites(154).
ThesepropertiesmakemicellesexcellentcandidatestofunctionasMRIcontrastagents.
Themostcommonstrategytopreparemicelleswithparamagneticpropertiesistouseamphi-philicmoleculeswithaGd3chelatingandhydrophilicheadgroupandonehydrophobicchain.
AmixtureofparamagneticamphiphilicmoleculesandphospholipidsorPEG–lipidsmayalsobeemployed.
TheNMRDproleofamicellularcontrastagentshowsthetypicalpeakintheT1relaxivityr1athighereldstrengths(155),sincemicelleshavearelativelylongrotationalcorrelationtime,typicalformacromolecularcontrastagents.
TheionicrelaxivityofGd-basedmicellularcontrastagentsatclinicallyrelevanteldstrengthsisthereforesuperiortothatoflowmolecularweightcomplexes.
Recently,Accardoetal.
describedthesynthesisandphysiochem-icalcharacterizationofatarget-specicmicellularcon-trastagentwhichwascomposedofamixtureoftwoamphiphilicmolecules(156).
TherstmoleculeisaC18hydrophobicmoietyboundtoanoctapeptide.
ThesecondmoleculecontainsthesameC18moiety,butiscoupledtoDTPA.
Thepeptidesequenceuseddisplayshighafnityforcholecystokininreceptors,whicharelocalizedinthecellmembraneandoverexpressedinmanytumors.
Theauthorsinferredthatthistypeofmixedmicellularcon-trastagentisapromisingtoolfortarget-specicMRimaging.
Micellescomposedofphospholipids,asurfactantandanamphiphilicGdcontrastagentwererecentlyusedfordetectingmacrophages(157).
Macrophagesareknowntoplayacentralroleinthepathogenesisandevolutionofatheroscleroticplaques.
Thecontrastagentwasemployedtomacrophagesinvitroandinvivoinamousemodelofatherosclerosisandshowedmacrophage-specicuptake.
Immunomicelleswerepreparedbycouplingbiotinylatedantibodies,specicforthemacrophagescavengerrecep-tor,tothemicellesviaanavidin–biotinlinkage.
Animproveduptakeoftheantibodyconjugatedmicellescomparedwithbaremicelleswasobserved,whichwasestablishedwithMRIanduorescencemicroscopy.
Gadouorine.
GadouorineisamicellularcontrastagentcomposedofasurfactantwithahighlyhydrophobicperuoroalkyltailandahydrophilicGd3chelatingheadgroupandhasbeenshowntobeespeciallysuccessfulforMRlymphography(158–160)andimproveddetectionofatheroscleroticplaques(81,161).
Barkenhausenetal.
studiedtheaorticarchofhyperlipidemicrabbitswithMRIbeforeandaftertheadministrationofgadouorine(81).
Enhancementoccurredintheaorticwallofallhyperlipidemicrabbits.
InFig.
6,imagesbefore(AandB)and48hafter(CandD)applicationofthecontrastagentaredepicted.
Aring-shapedcontrastenhancementoftheaorticwallwasobserved.
TheMRIndingswerevalidatedwithSudanRedstainingandmatchedwithexvivoMRscansofthesamespecimen.
Siroletal.
demonstratedthatgadouorineisespeciallyusefulfordetectinglipid-richplaques(162).
Furthermore,usinganimprovedscanningprotocoltheauthorswereabletodetectatheroscleroticplaquewithin1haftergadouorineinjection.
AnotherstudybySiroletal.
demonstratedthatearlyandadvancedlesionscanbediscriminatedbygadouorine-enhancedimaging(163).
Uponinjectionofgadouorine,earlylesionsshowedlowerenhancementthanmoreadvancedlesions.
Thiscorrelatedwiththedensityofthevasavasorum,suggestingthatplaqueenhancementwithgadouorineisdependentonneoves-seldensity.
Gadouorinehasalsobeenshowntobeusefulfortheassessmentofnervedegeneration,sinceitselec-tivelyaccumulatedandretainedinnervebersunder-goingWalleriandegenerationinthelegofratscausingbrightcontrastonT1-weightedMRimages(164).
Nanoparticle-containingmicelles.
Nanoparticles,suchasironoxideparticlesandquantumdots,aremostlysynthesizedinnon-polarorganicsolventsandcappedFigure6.
HASTE(A,C)andIRturboFLASH(B,D)imagesbefore(A,B)and48hafter(C,D)applicationofgado-uorinetoan18-month-oldWHHLrabbitatidenticalslicepositions.
Wallofdescendingaorta(arrows)showsmarkedenhancementaftercontrastinjection(D).
AdaptedfromFig.
2ofBarkhausenetal.
,Detectionofatheroscleroticplaquewithgadouorine-enhancedmagneticresonanceimaging.
Circulation2003;108:605–609,withpermissionfromLippincottWilliams&Wilkins154W.
J.
M.
MULDERETAL.
Copyright#2006JohnWiley&Sons,Ltd.
NMRBiomed.
2006;19:142–164withasurfactant.
Iftheyaretobesolubilizedinaqueousbuffers,theirhydrophobicsurfacecomponentsmustbereplacedbyamphiphilicones.
AnalternativestrategywasdevelopedbyDubertretetal.
forTOPO-coatedquantumdots(39).
Thehydrophobicparticlesweredissolvedinchloroformtogetherwithpegylatedphospholipids.
Afterevaporatingthesolventandhydratingthemixedlmofquantumdotsandlipids,quantumdot-containingmicelleswereformed.
Thesamemethodcanbeusedforencapsulatinghydrophobicironoxideparticlesinmicelles.
Withthiselegantlysimpleprocedure,oneobtainsawater-solubleparticleofsmallsize,whichcaneasilybefunctionalizedbyjustmixingtheappropriatelipids.
Furthermore,uorescentlipidscanalsobeincor-poratedforopticalimaging.
InFig.
7,theprocedureisdescribedschematically.
VanTilborgetal.
functionalizedthemicellularironoxideparticleswithAnnexinVforthedetectionofapoptoticcells.
Invitro,theAnnexinV-conjugatedparticlesshowedahighafnityforapopto-ticcells,whichresultedinalargedecreaseinT2ofapelletofthesecells,whereasthecontrolcellsshowedalmostnoT2decrease(165).
Invivo,thiscontrastagentmightbeveryusefulfordetectingapoptoticcellsinpathologicalprocessessuchasischemicreperfusionin-jury,atherosclerosisandtumors.
Nitinetal.
developedasimilarapproachtosolubilizeironoxide(35).
TheyconjugatedTAT–peptidesandauorescentlabeltothedistalendofthePEGchainsofthephospholipidstocoattheironoxideparticles.
TheTAT–peptidehasbeenshowntodelivernanoparticlesintocells,makingitattractiveforintracellularlabeling.
TheuptakeofthisconjugatewasdemonstratedinvitrowithbothMRIanduorescencemicroscopy.
MicroemulsionsMicroemulsionsaremixturesofwater,oilandanamphi-phile,whichresultinanopticallyisotropicandthermo-dynamicallystablesolution(38).
TheusefulnessofmicroemulsionsasMRIcontrastagentwasrecognizedbyLanzaandWickline.
Theirnanoparticlehasaper-uorocarboncore,whichiscoveredwithamonolayeroflipids.
Initiallytheyusedtheirtechnologyincombinationwithultrasound,becauseoftheacousticpropertiesofthecontrastagent.
Intheirrststudy,athree-stepapproachtotargetthrombuswasutilized(166).
Firstabiotinylatedantibrinmonoclonalantibodywasusedtotargetbrin,amaincomponentofthrombus.
Next,avidinwasadminis-teredtobindtotheantibrinantibodiesviaabiotin–avidinlinkage.
Sinceavidinhasfourbindingsitesforbiotinthenon-boundsitesofavidincouldnextbeusedfortargetingthebiotinylatedperuorocarbonnanoparti-cles.
Inthismanner,thrombiweredetectedwithultra-sound.
AsimilarapproachwasusedfordetectingbrinindogswithMRI(72).
Withthataim,Lanzaetal.
slightlymodiedtheirproceduresbyalsoincorporatingparamag-neticlipids(Gd–DTPA–BOA)inthelipidmonolayerofthenanoparticles.
Becauseofthesizeofthenanoparticle,thepayloadofGd–DTPA–BOAwasashighas50000perparticle.
Thisdramaticallyincreasestherelaxivityperparticle,whichisnecessaryfordetectingsparseepitopeswithMRI.
ScanningelectronmicrographsofcontrolbrinclotsandbrinclotstargetedwiththeparamagneticnanoparticlesaredepictedinFig.
8(A)and(B).
Anextensiveassociationofthecontrastagentwiththeclotscanbeobserved.
AninvivoMRimageofadogwiththrombus(intheexternaljugularvein)afterincubationwiththecontrastagentshowsapronouncedcontrastenhancementofthethrombusonT1-weightedimages[Fig.
8(C)and(D)].
Inapaperpublishedin2002,theseauthorsextendedtheirtechnologyforsimultaneousima-ginganddeliveryofanantiproliferativedrugtosmoothmusclecells(167).
Furthermore,theperuorocarboncoreofthenanoparticlesallowsparticledetectionwith19FNMRspectroscopy,whichmakesquanticationofdrugdeliverypossible.
Morawskietal.
usedculturesofFigure7.
Schematicrepresentationoftheencapsulatingprocedureofhydrophobicnanoparticlesinmicelles.
Lipidsaremixedwiththenanoparticlesinanapolarsolvent.
Themixedlmobtainedishydrated.
Thereafter,thenanoparticle-containingmicellesandemptymicellesareseparatedbycentrifugationLIPID-BASEDNANOPARTICLESFORCONTRAST-ENHANCEDMRI155Copyright#2006JohnWiley&Sons,Ltd.
NMRBiomed.
2006;19:142–164smoothmusclecellmonolayerstomodelandvalidatetheMRIdetectionlimitofsparsemolecularepitopeswhentargetedwiththiscontrastagent(168).
Theyshowedthatimagingofcellmonolayerswaspossibleat1.
5Tusingperuorocarbonnanoparticlesof250nm,containing90000Gd–DTPA–BOA,targetedtotissuefactor-expres-singcells.
Furthermore,quanticationwasachievedandpicomolarconcentrationsofthenanoparticlesweresuf-cienttogenerateasignicantchangeincontrasttonoiseratio.
Theperuoronanoparticlestargetedtothev3-integrinhavebeenusedfordetectingangiogenesisinarabbitmodelofatherosclerosis(169)andintumorsimplantedinrabbits(69).
Angiogenesisplaysanimpor-tantroleinprovidingthetumorwithnutrients.
Anti-angiogenictherapiesarethereforebelievedtoprovideapowerfultreatmentoptionforcancer.
Inatherosclerosistheplaquescontainangiogenicmicrovessels,whicharebelievedtoplayanimportantroleinplaquedevelopment.
Imagingplaquesandtumorswithanv3-speciccon-trastagentisthereforeofimportanceforearlydetection,deningtheseverityofthediseaseandfollowingtheeffectoftherapy.
Recently,Winteretal.
presentedresultsofacombinatoryapproachofMRmolecularimaginganddrugtargetingofatherosclerosiswiththiscontrastagent(170).
Tothatendtheyusedthev3-specicnanopar-ticlestotargettheaorticvesselwallafterballooninjury.
Fortherapeuticpurposestheyincludedfumagillininthelipidmonolayerofthenanoparticlesandobservedananti-angiogeniceffectwithMRIthatwasconrmedhistologically.
LipoproteinsLow-densitylipoprotein(LDL)andhigh-densitylipopro-tein(HDL)playanimportantroleinthetransportofcholesterol.
LDLconsistsofalipidcoreofcholesterolestersandtriglyceridescoveredbyaphospholipidmono-layerwhichcontainsalargeapolipoprotein.
LDLbindstotheLDLreceptorexclusivelyviathisapolipoproteinandissubsequentlyinternalized.
TheoverexpressionoftheLDLreceptorisassociatedwithseveralpathologies,includingatherosclerosisandcancer.
Huietal.
labeledLDLbyincubatingitwithPTIR267molecules(171),acontrastagentwithaGd–DTPAmoietyandauorescentlabel.
Invitrouptakeofthiscontrastagentbymelanomacellswasdeterminedwithuorescencemicroscopy.
InvivotheyobservedincreasedcontrastinthemouseB16melanomatumorsandinthemouseliver,whereastheuptakeintheliverofLDLreceptorknockoutmicewasverylow.
AnHDL-likeparticlewasdevelopedbyFriasetal.
forbimodalimaging(102).
Theyconstructedtheparticlefromapo-HDLproteinsandphospholipids,withorwith-outunesteriedcholesterol.
Aparamagneticphospholi-pid,Gd–DTPA–DMPEandauorescentphospholipidwereincorporated(Fig.
9)forimagingpurposes.
Thiscontrastagentwastestedonamousemodelofathero-sclerosisthatwasimagedinvivowithMRI.
Thevesselwalloftheabdominalaortashowedstrongsignalen-hancementwithamaximum24hpost-injection.
Follow-ingaortaexcision,itwasestablishedwithpostmortemconfocaluorescencemicroscopythatthecontrastagentwasmainlylocalizedintheintimallayer.
Thelipoproteincontrastagentsmaybeofgreatuseforthenon-invasivecharacterizationofcancerandatherosclerosis.
SmartcontrastagentsSmartcontrastagents,alsoreferredtoasresponsiveoractivatedcontrastagents,areagentsthatundergoalargechangeinrelaxivityuponactivation.
AkeystudyinthiseldwaspublishedbyLouieetal(16).
TheydevelopedaGd3chelatingcomplex,which,inthepresenceoftheenzyme-galactosidase,undergoesasizableincreaseinFigure8.
Scanningelectronmicrographsofcontrolbrinclot(A)andbrin-targetedparamagneticnanoparticlesboundtoclotsurface(B).
Arrowsindicate(A)brinbriland(B)brin-specicnanoparticle-boundbrinepitopes.
Thrombusinexternaljugularveintargetedwithbrin-specicparamagneticnanoparticlesdemonstratingdramaticT1-weightedcontrastenhancementingradient-echoimage(C)withowdecit(arrow)ofthrombusincorrespond-ingphase-contrastimage(D).
AdaptedfromFigs1and5ofFlackeetal.
,NovelMRIcontrastagentformolecularimagingofbrin:implicationsfordetectingvulnerableplaques.
Circulation2001;104:1280–1285,withpermissionfromLippincottWilliams&Wilkins156W.
J.
M.
MULDERETAL.
Copyright#2006JohnWiley&Sons,Ltd.
NMRBiomed.
2006;19:142–164relaxivity.
InitsinactiveformwaterisnotaccessibletoGd3becauseofblockagewithasugarmoiety.
Whentheenzymecleavesoffthesugarwatercandirectlycoordi-natewithGd3explainingther1increase.
PeroxidaseactivityhasbeendetectedwithMRIbyusingironoxidenanoparticlesconjugatedwithphenolicmoleculesthatcrosslinkinthepresenceofperoxidases.
Thisleadstotheself-assemblyofthenanoparticles(172),whichresultsinaconcentration-dependentdecreaseofT2.
Liposomeshavealsobeenusedassmartcontrastmaterial.
Liposomescan,forexample,bepreparedtoundergoaphasetransitionofthebilayerfollowingaphysiologicaltrigger.
Whenthebilayerisinarigid,gel-likestate,itispoorlypermeabletowater.
Afterthephasetransition,themembranebecomesmoreuidandhencemorepermeabletowater.
Thephasetransitioncanbecausedby,e.
g.
,pHchangesortemperaturechanges.
Gd–DTPA–BMAencapsulatedbypH-sensitiveliposomescomposedofphosphatidylethanolamineandpalmiticacidwerestudiedbyLoklingetal.
(127).
Theuseoftheseliposomeswasinitiallylimitedbecauseoftheirlowstabilityinblood(173).
Exchangingpalmiticacidforthedouble-chainedamphiphiledipalmitoylglycerosuccinateresultedinaformulationwithincreasedstabilityandgoodpHsensitivity(174).
ThiscontrastagentmightbeusefulforpHquantication,whichisclinicallyrelevantforcharacterizingtumors.
Similarliposomalsystemshavebeenusedinseveralstudiesforthenon-invasivedeterminationoflocaltemperature(114).
Therelaxivityoftheseliposomesincreaseswhenthetemperatureisabovethegel-to-liquidcrystallinephasetransition.
Frichetal.
usedthermosensitiveliposomesforimage-guidedthermalablationofrabbitliver(126).
Uponheating,anincreaseintissuesignalwasobservedinT1-weightedimages(Fig.
10),thusallowingon-linemonitoringofthermalablation.
AnotherexampleofasmartimagingstrategyistodeterminedrugreleasefromliposomesusingMRI(125).
MnSO4-anddoxorubicin-loadedliposomeswereusedformonitoringthereleaseofthedrugfromtheliposomeswithMRI.
Mn2isaparamagneticionwhichshortenstheT1ofwater.
However,whenMn2isencapsulatedinliposomestherelaxivitydecreasesdramatically.
Thereleaseofcontentfromtemperature-sensitiveliposomescouldbefollowedbytheincreaseintheMRIsignalinT1-weightedimages.
Aliposomalcontrastagentresponsivetoradicalshasalsobeendescribed(175).
Inthisagent,Gd3chelatesareconjugatedtotheliposomeviaadisuldelinker.
Therotationalcorrelationtimeofthisconjugateislong,resultinginahighrelaxivity.
Whenthecontrastagentisexposedtoradicals,theparamagneticchelatesarecleavedoff,leadingtofastertumblingandacorrespond-ingdecreaseintherelaxivity.
CelllabelingMRIasacellularimagingmodalitydependsonmeth-odstolabelmagneticallycellsofinterestinordertomonitortheirtrafckingaspartofcell-basedrepair,replacementandtreatmentstrategies.
Efcientmag-neticlabelingofthecellstobetrackedwithMRIisofgreatimportanceforkeepingthedetectionlimitofthecellsaslowaspossible.
Themigrationofmagne-tically-labeledimplantedstemcellswassuccessfullymonitoredinthebrainofexperimentalstrokeratsbyHoehnetal.
(176)andinamyocardialinfarctionpigmodelbyKraitchmanetal.
(177)Differentstrategieshavebeendescribedthatmainlyfocusonloadingcellswithironoxideparticles.
InanearlystudyperformedbyBulteetal.
,liposomescontain-ingdextran-coatedironoxideparticleswereusedforlabelingofhumanperipheralbloodmononuclearcellswithironoxideparticles(145).
Forefcientincorpora-tion,transfectionagents,originallydevelopedtotransfectnon-viralDNA,maybeused.
Amongthedifferenttransfectionagents,asubgrouparethelipid-basedtrans-fectionagents,usuallycationicliposomes.
Labelingcellswithmagneticironoxideparticlescanbedonewithhighefciencyusingtheselipid-basedtransfectionagents.
VandenBosetal.
,forexample,reportedthattheuseofcationicliposomesforironoxidestemcelllabelingincreasedthelabelingefciencyapproximately100-fold(178).
Theabovemethodsintroducesuperparamagneticpropertiestothecells.
Efcientparamagneticlabelingofcellscanbedonewithcationicliposomes(179)ormicroemulsionscontainingahighpayloadofanamphi-philicparamagneticmoiety.
Thesesystemscanalsocarryauorescentlipidforsimultaneousdetectionwithuorescencemicroscopy.
Recently,peruoropolyether-containingmicroemulsionshavebeenusedtolabeldendriticcells(180).
ThecellsweretrackedinmiceFigure9.
DifferentcomponentsoftherecombinantHDL-likeMRIcontrastagent.
AdaptedfromScheme1ofFriasetal.
,RecombinantHDL-likenanoparticles:aspeciccontrastagentforMRIofatheroscleroticplaques.
J.
Am.
Chem.
Soc.
2004;126:16316–16327,withpermissionfromtheAmericanChemicalSocietyLIPID-BASEDNANOPARTICLESFORCONTRAST-ENHANCEDMRI157Copyright#2006JohnWiley&Sons,Ltd.
NMRBiomed.
2006;19:142–164invivousing19FMRI.
Themainadvantageofthistechniqueisthatitallowsimagingofthecellswithoutanybackground,so-calledhot-spotimaging,sincetherearenoendogenousuorineatomspresentinthebody.
Theinformationobtainedwith19FMRIcanbesuper-imposedonhigh-resolutionanatomical1HMRimages,allowingaccuratelocalizationofthecontrastmaterial.
RECENTDEVELOPMENTSInadditiontoMRI,lipidicnanoparticleshavealsobeenemployedascontrastagentsforotherimagingmodal-ities.
Liposomeswithappropriateradiolabelshavebeenusedforpositronemissiontomography(PET)(181),singlephotonemissioncomputedtomography(SPECT)(182)andscintigraphyimaging(183).
Forcomputedtomography(CT),liposomescancarryheavilyiodinatedorganiccompounds,whereasforultrasoundthelipo-somesshouldbeechogenic(184).
Micelleswithanamphiphilicindium-111-labeledmoietyhavebeenap-pliedforlymphography(33).
Inadditiontotheestab-lishedutilityofperuorocarbonnanoparticlesasultrasonicandMRIcontrastagents,thistechnologyhasbeenfurthermodiedforotherimagingmethodssuchasCT(185)andnucleartechniques.
Suchmultimodalnanoparticlesmaybeofuseinchoosingtheoptimaldetectionorimagingmethodwiththesamenanoparticu-lateprobe.
Furthermore,acombinatoryapproachmaybebenecialforimproveddetection.
Asensitiveandlow-resolutiontechnique,suchasthenucleartechniques,canprovideinformationonthebiodistributionofthenano-particles.
Awholebodycanbescannedandareasofinterestcanthusbeidentiedrelativelyrapidly.
There-after,theseareasmaybeinvestigatedinmoredetailwithhigh-resolutionandlow-sensitivitytechniquessuchasCTandMRI.
ThecombinationmayalsobeofusetomergeanatomicalinformationfromMRIorCTwiththesensitivedetectionofthecontrastagentwithnuclearmethods.
Quantumdots,semiconductornanocrystalsafewnan-ometersinsize,haverecentlyattractedmuchattentionforbiologicalimagingpurposes(186)becauseoftheirex-cellentuorescentproperties,buttheirusehasbeenlimitedbydifcultiesinobtainingquantumdotsthatarebiocompatible.
Quantumdotscappedinphospholipidmicelles(39)wereamongthersttobeappliedinvivo.
Mulderetal.
recentlyextendedthisapproachbyapplyingaparamagneticlipidiccoatingtoquantumdotsforsimultaneousdetectionwithMRI(187).
Anotherpromisingapplicationofnanoparticlesistheiruseforcombineddiagnosticsandtreatment.
Asdiscussedpreviously,extensiveresearchhasbeendonewithlipidicaggregatesaspharmaceuticalcarriers,whichmakestheimplementationofcombiningaMRIcontrastagentwithatherapeuticagentrelativelyeasy.
CEST(chemicalexchangesaturationtransfer)agentsareagentsthatcontainatleastonepoolofexchangeableprotons,whichuponirradiationattheirresonancefre-quencytransfermagnetizationtothestrongsignalfrombulkwater.
CESTagentsareinterestingbecausetheycanbeswitchedonandoff,butarelimitedbecauseoftheirlowsensitivity.
Creatingparticleswithalargenumberofexchangeableprotonsmaybedonewithliposomes.
Thisso-calledLIPOCESTapproach,whichwasrecentlypro-posedbyAimeetal.
(188),reliesontheincorporationofanMRshiftreagentintheliposomallumen.
Thiscausestheresonancefrequencyofwaterprotonsinsidetheliposomestobeshiftedfromthatoftheexternalwater.
Inaphantomstudy,theauthorswereabletodetectthecontrastagentataconcentrationaslowas90pM.
Terrenoetal.
presentedinitialresultsofinvitrotargetingofthisLIPOCESTagent(189).
Inthatstudy,theliposomeswerecoatedwithPEGandcoupledtopeptidesviaavidin–biotinbonding.
Inaddition,MRIvisualizationofthepassiveaccumulationofpegylatedLIPOCESTintumor-bearingmicewasdemonstrated.
Figure10.
T1-weightedimagesofradiofrequencyablationinrabbitliverafterinjectionofliposomalcontrast.
Priortoheating(t0),duringheating(t1),afternormalizationoftissuetemperature(t2)and15–20minafternormalizationoftissuetemperature(t3).
Notetheincreasingsignalintensityintheperipheryofthethermallesionintherightliverlobe(whitearrows).
Theradiofrequencyelectrodehasbeenrepositionedintheleftliverlobeatt3(whitearrowhead).
AdaptedfromFig.
4ofFrichetal.
,Experimentalapplicationofthermosensitiveparamagneticliposomesformonitoringmagneticresonanceimagingguidedthermalablation.
Magn.
Reson.
Med.
2004;52:1302–1309,withpermissionfromJohnWiley&Sons,Inc158W.
J.
M.
MULDERETAL.
Copyright#2006JohnWiley&Sons,Ltd.
NMRBiomed.
2006;19:142–164CONCLUSIONSInthisreview,wehaveshownthevarietyofpossibilitiesofusinglipidsandotheramphiphilesasbuildingblocksofnanoparticulateMRIcontrastagents.
Phospholipidsarethemostcommonlyusedamphiphilesforbiomedicalapplications,whichhasresultedinthecommercialavailabilityofmanydifferentlipidsandphospholipid-likestructures.
Magneticresonanceisaleadingdiagnosticimagingmodality,sinceitexcelsindepictingsofttissuewithhighspatialresolution.
Nevertheless,toimprovetheapplicabilityofcontrast-enhancedMRI,thelimitationsofthetechniquehavetobetakenintoconsideration.
First,therelativelylowsensitivityofMRcanbecompensatedbycreatinghighlypotentcontrastagents,whichispossiblebyusinglipidicparticleswithahighpayloadofcontrastmaterial.
Second,althoughthespatialresolutionofMRIissuperiortothatofothernon-invasiveinvivoimagingmodalities,thelevelofdetailthatisobtainedwithimmunohistochemicaltechniqueswillnotbeachieved.
Therefore,intheinitialphaseMRIshouldbecombinedwithacomplementaryimagingtechniquetogainmoreinsightintothepathologystudiedandintothemechanismsofuptakeandfateofthecontrastagent.
Fluorescencemicroscopyissuchatechnique,whichiscapableofstudyingprocesseswithsubcellularresolution.
Fluorescencemicroscopyismainlyappliedpostmortemonsmalltissuesections,becauseofthelimitedpenetrationdepthoflight.
TheavailabilityofuorescentlipidsprovidesopportunitiestocreateeasilybimodalcontrastagentsthatcanbedetectedwithbothMRIanduorescencemicroscopy.
Muchisexpectedfromthisbimodalimagingapproachandinitialstudieshavealreadyrevealedpromisingpossibilities.
Gd3asanMRIcontrastagenthastobechelatedinacomplexwithahighbindingconstant,becauseofitstoxicity.
ThetoxicityofGd–DTPAislowandwelldocumented.
Gd–DTPAthereforeisFDAapprovedandthemostcommonlyusedagent.
Ithasalowmolecularweightandisrapidlysecretedbythekidneys.
Gd–DTPA-basedcontrastagentsofhighmolecularweighthaveprolongedcirculationtimesandaccumulateatspecicsites.
Futurestudieshavetorevealwhetherthiscausesanyadversehealtheffectsandwhetherlipidicnanoparti-culatecontrastagentscanbeusedsafelyandcost-effectivelyinhumans.
Theeaseofpreparation,theexibilityand,mostimportantly,thebiocompatibilityoflipidicnanoparticlesmakethemusefultoolsforbiomedicalimagingpurposes.
Lipidicnanoparticlesalsoprovideanexcellenttemplateforanynew(molecular)MRInanoparticle.
Ongoingdevelopmentsinnanotechnology(190–192)mayleadtothedevelopmentofnanoparticlesforMRIwithbetterrelaxationproperties,improvedstabilityandamoredenedsizeandstructure.
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