theranostics in cancer Ferritin nanocages: A biological platform for drug delivery, imagingand Pharmacological Research

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ContentslistsavailableatScienceDirect

Pharmacological

Research

jo u r n al ho me p a g e :ww w . e l s e v i e r . c o m / l o c a t e / y p h r s

Review

Ferritin

nanocages:

A

biological

platform

for

drug

delivery,

imaging

and

theranostics

in

cancer

Marta

Trufﬁ

a

_,

_Luisa

_Fiandra

a

_,

_Luca

_Sorrentino

a,b

_,

_Matteo

_Monieri

a

_,

_Fabio

_Corsi

a,b

_,

Serena

Mazzucchelli

a,∗

a_Laboratory_of_{Nanomedicine,}_Department_of_Biomedical_and_Clinical_Sciences_University_of_Milan,_“Luigi_Sacco”_Hospital,_Via_G._B._Grassi,_74,₂₀₁₅₇ Milano,Italy

b_Surgery_Division,_Department_of_Biomedical_and_Clinical_Sciences_University_of_Milan,_“Luigi_Sacco”_Hospital,_Via_G._B._Grassi,_74,₂₀₁₅₇_Milano,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received13January2016

Receivedinrevisedform24February2016 Accepted2March2016

Availableonline9March2016 Keywords: Protein-basednanocages Ferritin Drugdelivery Imaging Cancer

a

b

s

t

r

a

c

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Nowadayscancerrepresentsaprominentchallengeinclinics.Mainachievementsincancermanagement wouldbethedevelopmentofhighlyaccurateandspeciﬁcdiagnostictoolsforearlydetectionofcancer onset,andthegenerationofsmartdrugdeliverysystemsfortargetedchemotherapyreleaseincancer cells.Inthiscontext,protein-basednanocagesholdatremendouspotentialasdevicesfortheranostics purposes.Inparticular,ferritinhasemergedasanexcellentandpromisingprotein-basednanocagethanks toitsuniquearchitecture,surfacepropertiesandhighbiocompatibility.Byexploitingnaturalrecognition oftheTransferrinReceptor1,whichisoverexpressedontumorcells,ferritinnanocagesmayensurea properdrugdeliveryandrelease.Moreover,researchershaveappliedsurfacefunctionalitiesonferritin cagesforfurtherprovidingactivetumortargeting.Encapsulationstrategiesofnonmetal-containing drugswithinferritincageshavebeenexploredandsuccessfullyperformedwithencouragingresults. Variouspreclinicalstudieshavedemonstratedthatnanoformulationwithinferritinnanocages signiﬁ-cantlyimprovedtargetedtherapyandaccurateimagingofcancercells.Aimsofthisreviewaretodescribe structureandfunctionsofferritinnanocages,andtoprovideanoverviewaboutthenanotechnological approachesimplementedforapplyingthemtocancerdiagnosisandtreatment.

1. Introduction

Cancerisaleadingcauseofdeathworldwide,accountingfor8.2

milliondeathsin2012[1].Amaingoalofcurrentcancerresearchis

earlyrecognitionofcancercellsandselectivetreatment,beforethe

cancercanprogresstoanunfavourablestage.Basedonthis

ratio-nale,theaimofseveralmoderntherapeuticapproachesforcancer

istoselectivelyremovethetumorbeforeitevolvesintoits

supe-riorstages,andtheeventualonsetofmetastases.Forthisscenario

tobesuccessfulthereisagrowingneedtodevelop:(1)noveland

moresensitivediagnostictoolstoimprovescreeningaccuracyand

thedetectionrateofmalignancies,stagingandfollowupsettings,

andtoachievelessextensivesurgeryormedicaltherapy;(2)new

drugformulationswithenhancedefﬁcacytowardcancercellsand

reducedtoxicitytowardhealthycells[2].

Nanotechnologyholdsgreatclinicalpotentialinreachingthese

majorgoalsandanumberofnanodeviceshavebeendesignedto

∗ Correspondingauthor.

E-mailaddress:[email protected](S.Mazzucchelli).

speciﬁcallytargetcancercells.Thesenanodevicescanbeloaded

withdifferentkindsofchemotherapeuticsand/orcontrastagents,

and provide unmatched promise for development of

theranos-tic devices for both cancer diagnosis and treatment. Indeed,

nanoparticleshavedemonstrated theirpotentialtosolvecrucial

issuesinvolvingbioavailability,tissuepenetrationandcirculation

time [3]. Several nanoparticles constructed frommetals,

semi-conductorsor polymers,have beentested inpreclinical studies

[4],butonlyafewofthemhavebeenclinicallyapproveddueto

theirtoxicity,immunogenicityandsequestrationbythe

reticulo-endothelialsystem[5].Therefore,muchefforthasbeendirected

intothedesignandsynthesisofchemicallyengineered

nanostruc-turesthatarebiologicallycompatible.

Naturallyoccurringnanoparticleshaveexistedformillionsof

years,andtheyincludeawidevarietyofnanostructures,whichmay

beformedbyinorganic(i.e.magnetosomes)ororganicmaterials.

Thesenanoparticlescanbeintracellularorextracellularandmay

havedifferentbiologicalroles[6].Despitethesedifferences,

natu-ralnanoparticlesarecharmingfromthebiomedicalpointofview

duetotheiruniformstructure,lowtoxicity,andabilitytoevadethe

immunesystem.Moreover,theyareeasilydegradedafterfulﬁlling

http://dx.doi.org/10.1016/j.phrs.2016.03.002

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58 M.Trufﬁetal./PharmacologicalResearch107(2016)57–65

theirfunction[6,7].Thesefeaturesplacethemaheadofinorganic

orsyntheticmaterialsinclinicaltranslation[7].Biological

nanopar-ticlesare assembled frommolecules oratoms synthesized in a

biologicalsystemwithsizesrangingfrom1to100nm.Theyinclude

magnetosomes,lipoproteins,viruses,exosomesandferritins[6].

Magnetosomes are specialized organelles with narrow size

(50–70nm indiameter), uniformmorphologyand low toxicity;

theyhaveevolvedfrommagnetotacticbacteriaandarecomposed

ofalipidbilayersurroundingmagneticiron-containingminerals

[8–10]. Lipoproteinsare self-assemblingstructuresthat contain

phospholipids,non-esteriﬁedcholesterolandspecializedproteins,

formingasphericalordiscoidalshell(from7to>80nm),which

surroundsacoreofnon-polarlipids,triacylglycerolsand

esteri-ﬁedcholesterols[11,12].Exosomesarenanometer-sizedstructures

secretedbycells andconsist ofanexternal lipidbilayer, which

offersanon-immunogenicandnon-toxicmechanismforthe

deliv-ery oftargeted proteinsor nucleicacids, protectingthem from

degradation and macrophage endocytosis [13–16]. Viruses and

virus-likeparticlesmayalsobeconsideredtobebiological

nanopar-ticles.Theyincludeawidevarietyofsizesandmorphologieswith

deﬁnedgeometries,uniformshapeandarobustproteinshell.They

aresuitableforchemical-andbio-conjugation.Althoughtheir

cap-sidsarestableoverawiderangeofpHandtemperatures,theyare

difﬁculttoproduceinbulk,andthisrepresentsaseverelimitation

fortheiruseinnanotechnology[17].

Amongallclassesofbiologicalnanoparticlesherewefocuson

ferritins,whichareironstorageandtransportproteinsfoundin

mostlivingorganisms[18].Recombinantferritinprovidesa

cen-tralcavity,whichcanbeefﬁcientlyloadedwithtransitionmetals,

drugs,ﬂuorescentmoleculesorcontrastagents[19,20].Sinceithas

auniformcage,ferritinallowstheprecisecontroloftheamount

ofencapsulatedmolecules,whichisacriticalfeatureindeﬁning

drugdosage.Moreover,theproteinshellofferritinscanbe

eas-ilymodiﬁedeitherchemicallyorgeneticallytointroducedifferent

functionalities[21,22].Alloftheseattributeshighlightferritinand

itsderivativesaspowerfulsystemswithpotentialapplicationin

nanomedicine.Indeed,theyhavebeeninvestigatedasanoveltype

ofnano-platformforimaginganddrugdeliveryincancer[19,23].

Theaimofthisreviewistodescriberecentﬁndingsaboutferritin

nanocages’structureandfunction,andtheirbiotechnologicaland

biomedicalapplicationsforMRI,opticalimaginganddrugdelivery

incancer.Anoverviewofstrategiesusedforferritinsurface

mod-iﬁcationtoobtainformulationssuitablefordifferentclinicaluses

willbealsoprovided.

2. Ferritinnanocages:structureandphysiologicalrole

Ferritinisprobablythemoststudiedproteinafterhemoglobin.

Ithasbeeninvestigatedsince1937,whenLaufbergerdescribedits

puriﬁcationwithCadmiumsalts[24],andoverthelastdecadeithas

attractedtheinterestofmanynanotechnologistsduetoits

prop-erties[25].Itisaubiquitousproteinfoundineubacteria,archaea,

plantsandanimals,butnotinyeast[25].Ferritinexistsin

extracel-lularandintracellularcompartments,suchasthecytosol,nucleus

andmitochondriaanditsmainrolesareironstorageand

homeosta-sis[26].Therefore,itisoneofthemosthighlyconservedmolecules.

Ferritinisalargeproteinof450kDacomposedof24subunits

thatself-assembleintoasphericalcage-likestructurewithinner

and outerdimensions of 8 and 12nm, respectively.Eukaryotes

havetwoferritingenesencodingtheheavy(H;21kDa)andthe

light(L; 19kDa)chains.The H-chainisresponsible forthe

oxi-dationof Fe(II)to Fe(III)and includes theferroxidase catalytic

site,whiletheL-chainplaysaroleinironnucleation[27].Hand

L chainsco-assemble intoa 24-merheteropolymer, where the

H-chaintoL-chainratiovariesaccordingtoatissuespeciﬁc

dis-tribution[25].Ferritinprotectscellsfromthedamagecausedby

theFentonreactionduringoxidativestress,bysequesteringFe2+_,

asourceoftoxicreactiveoxygenspecies,andconvertingitinto

harmlessFe3+_,_which_is_stored_as_ferrihydrite_crystals._The_ferritin

proteincagecanaccommodateupto4500ironatoms[25].This

protectiveactioniscrucialinthenucleus,wheretheDNAhastobe

particularlypreservedfromiron-inducedoxidativedamage[28].

FerritintranslocatesintothenucleusthroughanATP-dependent

mechanism[28,29],andO-glycosylationoftheH-chainseemsto

beinvolvedin itsnucleartranslocation, buta Nuclear

Localiza-tionSequence(NLS)isnotrequired[29].However,itwasnotclear

whethertheintegrityofferritinwasmaintainedduring

translo-cation.Recently,theKnezgrouphasshedlight onthisissueby

demonstratingthatferritinnanoparticlesareintactduring

translo-cation[30].

Theferritinquaternarystructurehaseighthydrophilicchannels

thatseemtomediateirontransitinandoutoftheproteincage.

Therearealsosixhydrophobicchannels,whichdonotseemtobe

involvedinironexchangealthoughtheycouldmediatethe

tran-sitofprotons[31].Inspiteofthestructuralrigidityofferritinsin

thephysiologicalenvironment,theproteincagescanbereversibly

disassembledwhenthepHbecomesextremelyacidic(pH2–3)or

basic(pH10–12)[27,32].WhenthepHreturnstoneutralityferritin

monomersareabletoself-assembleinashapememoryfashion.

Moreover,theferritin cageisresistanttodenaturants,including

heatingtohightemperatures(>80◦C)[25].

Ferritin features have been extensively studied and

imple-mentedbyresearchers,thusallowingtheuseofferritinnanocages

asbiologicalnanoparticlesfornanomedicalapplications.

3. Surfacemodiﬁcationofferritinnanoparticlesand cellularinteractions

Extracellularferritininteractswithcellsthroughthereceptor

oftransferrin1(TfR1),whichwasidentiﬁedin2010bySeaman’s

group [33]. They demonstrated that TfR1 speciﬁcally binds

H-ferritin,whileL-ferritinshowslow interaction.Afterbindingon

thecellsurface,theH-ferritin-TfR1complexisinternalized,and

canbefoundinearlyandrecyclingendosomes[33,27],

demon-stratingthatTfR1coordinatestheprocessingandtheuseofironby

bindingbothTransferrinandH-ferritin[34].H-ferritinisalsoable

tointeractwiththeT-cellImmunoglobulinandMucindomain-2

(TIM-2)[35,36],whichisoverexpressedinoligodendrocytesand

B-cells [37,38], and is internalized in endosomes upon binding

[39].Otherwise,L-ferritinbindstotheScavengerReceptorClassA,

Member5(SCARA-5)[40],whichisfoundinmacrophagesandthe

retina.BothHandLferritinnanoparticleshavebeenproducedvia

DNA-recombinanttechnology.H-ferritin,whichnaturallyinteracts

withtheTfR1,arethemostextensivelystudied[33].By

exploit-ing theTfR1 overexpression in many typesof tumor cells, Fan

andcoworkershavedevelopedaferritin-basedsystemfor

specif-icallyvisualizingtumorsamongnormalcellsandhealthytissues

[41].Thatstudydemonstratedthattheintrinsictargetingof

fer-ritintowardscancercellscouldbeusedtodesignnaturallytargeted

nanoparticlesfortheranosticpurposes.

Despitethenaturaltargetingofferritin towardscancercells,

several research groups have modiﬁed the surface of ferritin

nanocages by inserting a number of target motifs, such as

antibodies, peptides and antibody fragments, in order to drive

nanoparticlestowardsspeciﬁccellsbyselectiverecognition(Fig.1).

Both lysines and cysteines on the ferritin surface have been

exploitedforchemicalconjugationusingdifferent

heterobifunc-tionalcross-linkerswithN-hydroxysuccinimide(NHS) esterand

maleimidegroups[42–44].LysandCysresidueshavealsobeen

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Fig.1.Schematicrepresentationofstrategiesadoptedtoprovidesurfacefunctionalizationofferritinwithtargetingpeptides,antibodies,siRNAandﬂuorescentdyes.

polyethylene glycol (PEG) molecules [22,45]. Most conjugation

strategies havebeendesigned and realizedbygenetically

engi-neeringferritin monomers.Targetingmoieties,suchastheRGD

peptide[46,47],theanti-melanocytestimulatinghormonepeptide

[48,45]ortheextracellulardomainofmyelinoligodendrocyte

gly-coprotein(MOG) [49]havebeenexpressedasN-terminalfusion

proteins,allowing24targetingdomainstobeexposedonthe

fer-ritincagesurface,withuniformandpreciseorientation.Inthelast

year,thesurfaceconjugationofferritinnanoparticleswitha

com-positeoffourdifferentpeptideswasreported.Thatwasundertaken

byN-terminalcloningfourpeptidesontotheferritinsequence:an

enzymaticallycleavedpeptide(ECP)toreleasesiRNA,acationic

peptide(CAP)tocapturesiRNA,acellpenetratingpeptide(CPP)

anda tumorcelltargeting(CTP)moiety [50].N-terminal fusion

proteinshavealsobeenproducedtodisplayviralhemagglutinin

ontheferritinsurface,withtheresultingneutralizationofH1N1

viruses[51].Alternatively,theproductionofferritinnanoparticles

withpeptidesfusedattheC-terminalhasbeenreportedforthe

developmentofdendriticcell-basedvaccines[52].Beyondthese

strategies,otherapproachesfortheconjugationoftargeting

moi-etiesonferritinsurfacehavealsobeenexplored,includingferritin

nanocagebiotinylationinordertoinserttargetingfunctionalities

[53],orthegeneticengineeringofferritinforgeneratingafusion

protein,whichexploitsN-terminalproteinGforantibody

immo-bilization[54].

4. Nanotechnologicalapplicationsofferritin

Ferritinhasattracted much interestdue to itspotential use

asreaction chamber for theproduction of metal nanoparticles,

or as a template for semi-conductor production [55]. Ferritin

nanocagesallowchemical synthesis tooccurin restricted

cavi-tieswithhomogenousshapeandatomiccomposition[56].Ferritin

nanocageshavebeenextensivelyexploitedforthe

biomineraliza-tionofmetaloxides,suchasiron[57],manganese[58],cobalt[59],

chromiumandnickeloxides[60].Amongthedifferentprotocols,

thesimplestandmostsuitablemethodofmass-productionisthe

one-potsynthesisstrategydevelopedbytheYamashitaetal.[56],

whichdevelopedmetal-loadedferritinnanocageswithpotential

fornanoelectronicalapplication[56].

Nanoparticlesobtainedfrommineralizationofsemiconductors

areveryattractivefromthenanotechnologicalpointofview,since

theirﬂuorescencepropertiesarecloselyrelatedtonanoparticlesize

andshape[56].Inthiscontext,ferritinnanocagecavitieshavebeen

usedforthesynthesisofsemi-conductornanoparticles.However,

themaindrawbackofthisprocessisthationaggregationisinduced

byhighconcentrationsofCd2+_or_Zn2+_and_Se2−_during_the_chemical

reaction[56,61],andbecauseofthisonlyafewresearchgroupsare

engagedinitsdevelopment[56,61].ThesynthesisofCdSeandZnSe

usingferritinsasareactionchamberhasbeensuccessfullyobtained

byYamashitaet al.[62,63]whohave designeda newchemical

approachcalledtheSlowChemicalReactionSystem.Thismethod

avoidsionaggregationsinceCd2+_or_Zn2+_are_attracted_inside_the

apoferritinchamberbyinnernegativeresidues,thuspreventingthe

aggregationprocess;therefore,mineralizationinsideanapoferritin

cavityslowsdownthenucleationofCdSeorZnSe[62–65].

5. Potentialofferritinnanoparticlesincancer

Whileferritinnanoparticleshavebeenwidelyinvestigatedin

thecontext ofnanotechnology,theirmain applicationconcerns

theﬁeldofnanomedicine.Indeed,apartfromtheirapplicationin

thedevelopmentofvaccines[51,52],severalresearchgroupsare

engagedinthestudyofferritinasimaginganddrugdelivery

sys-temsforthediagnosisandtreatmentoftumors,assummarizedin

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60 M.Trufﬁetal./PharmacologicalResearch107(2016)57–65 Table1

Mainapplicationprospectsofferritinnanocagesincancerdiagnosisandtreatment.

Loadedwith... Functionalizedwith... Application Ref.number

Cisplatin – Chemotherapy [68–70]

Cisplatin antibodyagainstthemelanomaantigenCSPG4 Chemotherapy [42]

Doxorubicin RGDpeptide Chemotherapy [47]

Doxorubicin – Chemotherapy [27,72,73,75,30]

Curcumin – Therapy [74]

235_U _– _Radionuclide_Therapy _[75]

– anti-redﬂuorescentprotein(RFP)siRNA RNAinterferenceTherapy [50]

Magnetitecrystal MRImaging [78,79,80]

Gadolinium MRImaging [81,83] Mn(II) MRImaging [82] Gadolinium C3d MRImaging [53] – EGF AlexaFluor750 FluorescenceImaging [85] – RGDpeptide Cy5.5 FluorescenceImaging [22] – Cy5.5 GPLGVRGpeptide

blackholequencher-3(BHQ-3)

FluorescenceImaging

64_Cu _RGD_peptide

Cy5.5

PositronEmissionTomography [22]

NIR – PhotothermalTherapy [90,91]

Cobalt-dopedferritenanoparticles Melanomatargetingpeptide(MSH) MagneticFluidHyperthermia [45]

5.1. Ferritinnanoparticlesasadrugdeliveryplatform

Theirhighstability,biocompatibility,abilitytodisassembleand

reassembleina shapememoryfashionand dispositionfor

sur-facemodiﬁcation make ferritin nanoparticles anideal platform

for drug delivery [55]. Indeed, in physiological conditions

fer-ritinhasastable24-mercagearchitecture,whileinhighlyacidic

andbasic conditionsitsquaternary structure disassembles, and

thenreassembleswhenthepHofthesolutionisbroughtto

neu-trality[66].Thisfeaturehasbeenusedtoencapsulatemolecules

insolutionwithinferritin cavities,simplybydisassembling and

reassemblingthe24-merarchitecture(Fig.2).Drugswitha

natu-raltendencytobindmetals,suchascisplatinanddesferrioxamine

B[67],havebeeneasilyentrappedintheferritinshell.Cisplatin

encapsulation was ﬁrst reported in 2007 by Yang et al. [68],

whoalsostudiedthecellularuptakeofthesenanoparticlesand

severalapplicationsin tumortreatment[69]. TheHuang group

hasusedcisplatin-loadedapoferritintostudy,withaproteomic

approach,theapoptoticprocessinducedbynanoparticlesin

gas-triccancercells[70].Moreover,a drugdeliverydevicetargeted

tomelanomashasbeendevelopedusingcisplatin-loadedferritin,

chemicallyfunctionalizedwithanantibodyagainstthemelanoma

antigenCSPG4[42].Thesestudieshaveprovidedstrongevidenceof

anenhancedefﬁcacyofantiblastictherapywhenitisencapsulated

inferritin-basednanoparticles,particularlyintermsoftargeting

cancercellssuchasmelanoma,atypeofcancertotallyrefractory

tochemotherapyinlaterstages[42].

However,mostofthecurrentlyusedchemotherapiesarenot

basedonmetalssuchascisplatin,andtheincorporationof

non-metal-containing drugs within ferritin is complicated by their

limitedinteractionwiththeproteincage.Toovercomethis

draw-back,differentapproacheshavebeenevaluated,mainlyfocusedon

formingcomplexes ofdrugswithtransitionmetalsorthe

addi-tion of charged accessorymolecules [3,71]. Doxorubicin (DOX)

encapsulationin ferritin nanocagesrepresents themost

exten-sivelyinvestigatedsystemfordeliveryofanticancerdrugs [47].

DOXisawidelyusedcytotoxicdrugforseveraltypesofsolidtumors

andhasanexcellentanticanceractivity;however,itsuseis

dose-limitingsinceitisassociatedwithseveralmajortoxicities,when

administeredathighdoses[47].DOXpre-complexedwithCu(II)

hasbeenloadedinsideferritinnanocagesandevaluatedinvitro

andinvivoinU87MGglioblastomatumormodels.Eightypercent

ofDOXwasgraduallyreleasedfromRGD-functionalized

apofer-ritinwithin10hofincubationat37◦Cinphosphatebufferedsaline

(PBS),whilethecopperremainedboundtotheinternalsurfacesof

thenanocages.Thistargetednanoformulationhasincreaseddrug

tumoruptakeandaccumulation,tumorgrowthinhibition,

circu-lationhalf-life,andhasreducedthecardiotoxicityinducedbyfree

DOX[47].OthergroupshavesetupDOXloadingstrategiesthatdo

notinvolvecomplexeswithtransitionmetals.Inthesecases,good

encapsulation efﬁciencyhasalso beenobtained[72,73,27], and

excellentresultsintermsoftumorgrowthinhibitionandreduced

toxicityhavebeenreported,bothinvitro[27]andinvivo[72].Invivo

resultsclearlydemonstratedthatferritinnanocagesimproveDOX

bioavailability,tumoraccumulationandclearance,andsuggested

thatferritin-mediatedactivetargetingprovidesamajor

contribu-tion(Fig.4)[72].Themechanismsandkineticsofdrugreleasefrom

ferritinshellhasnotbeencompletelyelucidated,althoughreported

datawithDOXsuggestthatencapsulationisstableinserumand

thatapH-triggeredreleasetakesplaceinvitro[75].Moreover,the

Knezgroupdemonstratedthatferritintranslocatesintothenucleus

mediatingthenuclearreleaseofDOX,whileourgrouphas

hypoth-esizedthatDOXloadedferritinactsasaTrojanhorsebecauseithas

beentranslocatedintothenucleusfollowingDNAdamagecaused

bythepartialrelease inthecytoplasmofencapsulatedDOX,by

aself-triggeredtranslocationmechanismwhichreleasesthedrug

directlyintothenuclearcompartment[27,30].

Inthis context,theuseofunfunctionalizedferritin seemsto

beadvantageousfor manyreasons:(1)higherpuriﬁcationyield

[72,27];(2)hightumorrecognition[72]speciﬁctumorrecognition

mediatedbyTfR1;(3)ferritinspeciﬁcphysiologicalbehaviorisnot

prevented,allowingthenucleartranslocationforthereleaseofDOX

tobeexploited[27].Furthermore,ferritinnanoparticleshavebeen

usedtostabilizelipophilicdrugs,suchascurcumin[74].However,

todatefewstudieshavebeenpublishedonthis,probablybecause

lowdrughydrosolubilitystronglyaffectstheencapsulation

reac-tion.

Ferritin-basednanocagescouldbeimplementedforthedelivery

ofradioisotopesincreasingloadingefﬁciencyandimproving

phar-macokinetics.Hainfeldhasdevelopedferritincageswithapayload

of about 800235_U _{atoms/nanoparticle,}_able _to _kill _surrounding

tumorcells[75].Thesamestrategyhasbeensuggestedasbeing

applicablewithotherisotopescurrentlyusedin clinics,suchas

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Fig.2.Schematicrepresentationofferritindevelopedfordrugdeliverytocancer.

Finally,ferritin nanocageshave beenemployed forsiRNA or

miRNAdelivery.Theseshortnon-codingRNAmolecules,havebeen

stronglyrelated toeithercancerprogressionor resistance,

sug-gestingthatmiRNA/siRNA-basedtherapycouldbeassociatedwith

standardtreatment.Leeandcoworkershaverecentlyproposeda

geneticvariantofH-ferritindesignedtomediatetargeted

deliv-eryandinternalizationofsiRNAimmobilizedonthenanoparticle

surfacesthrough charge-chargeinteraction[50]. Invitro results

demonstratedtheefﬁcacyoftheas-designednanoparticlein

tar-geting tumor cells and the nanoparticle-mediated intracellular

delivery of an anti-red ﬂuorescent protein (RFP) siRNA, which

inducedRFPsuppression[50].Thisresultpavesthewayforfuture

applicationofferritinnanoparticlesingenesilencing.

5.2. Ferritinnanoparticlesforinvivoimaging

Theplasticityofferritinasamineralizationchamberforheavy

atoms or complexes, and the possibility of modifying ferritin

nanocagesbyproteinengineeringorbychemicalreactiontoinsert

probemolecules,arefeaturesthat makeferritin anidealdevice

forimaging[53].Inparticular,ferritinshavefoundgreat

applica-tioninmagneticresonanceimaging(MRI)andoptical[44]imaging

(Fig. 3). Indeed, MRI is a powerful diagnostic technique with

wideinvolvementintumorimaging,thankstoitshighsensitivity

andaccuracy[55].However,thecurrentlyusedgadolinium-based

contrastagents indistinctlyenhance allhighly vascularized

tis-sues,andthuslackspeciﬁcityforcancercellsresultinginahigh

rateof false positives.On theotherhand,occult cancer

micro-depositsmaybenotdetectedonMRIduetoaninsufﬁcientspatial

resolution, and oligometastatic disease could be misdiagnosed

[76,77].Bothendogenousand exogenousferritins have

demon-stratedgreatutilityinthisareabyovercomingtheselimitations.

Ferritin nanoparticles loadedwith magnetic species have been

developed as MRI contrast agents for tumor imaging. Cao and

coworkershaveobtainedananoparticlewithtransverserelaxivity

r2of224mM−1s−1,bysynthesizingasinglecrystalofmagnetite

inside theferritin cage [78]. Injection of suchnanoparticles in

MDA-231 tumor-bearing mice caused a drop of the T2 signal

duetoTfR1-dependenttumoraccumulation[78].Moreover,iron

nanoparticleshavebeenproducedintoferritincages,and

optimiz-ingstrategieshavebeendevelopedbyUchidaetal.[79]bysimply

adjustingtheextentofFeloading.Theincreasedamountofiron

inthereactionmixturedetermines anincreasein thecoresize

ofironnanoparticlesencapsulatedintoferritin,whichisdirectly

relatedtoT2contrastpower[79,80].OthertypesofMRI-detectable

nanoparticleshavebeenproducedusingtheferritincavityasa

reac-tionchamber.FivenmGdnanoparticleshavebeenproducedinside

ferritins,obtainingnanoparticleswithlongitudinalandtransverse

relaxivityfrom10to70timeshigherthancommerciallyavailable

Gd-chelates[81].Mn-coupledferritin-basedMRIcontrastagent,

obtainedbyreductionof␤-MnOOH,hasbeenreportedbyKalman

andcoworkers.Thisnanoparticlecontainsupto300–400Mn(II)

ionsandanr1relaxivityrangingbetween4000and7000s−1[82].

Aimeetal.[83]exploitedapH-mediatedferritindisassemblyto

loadabout8–10Gd-HPDO3A/nanoparticle,obtainingar1relaxivity

of80mM−1s−1.UponmodiﬁcationwithC3d,apeptidethat

specif-icallyrecognizesvesselendothelium,Gd-nanoparticlesshoweda

30%signalincreaseintumorinamousemodel[53].

Ferritin nanoparticles can also be conjugated with dye

molecules and ﬁnd application as optical imaging probes in

imaging-guidedsurgery for cancer[84].Li etal. [85]employed

a ferritin H-chaingeneticallyfused withepidermal growth

fac-tor(EGF),andsubsequentlylabeledCysresidueswithAlexaFluor

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Fig.3.Schematicrepresentationofferritindevelopedforcancerimaging.

Fig.4.Schematicrepresentationofinvivodrugdeliveryofferritinnanoparticles.

Besides,Linetal.[22]developedaCy5.5-labelledferritinby

react-ingthe dye withtheprotein’s primary amine.Then, using the

pH-disassemblyprocedure,theCy5.5-labelledferritinmonomers

weremixed withRGD-bearingferritinmonomers.Theresulting

hybridferritin,whichdisplaysonthesurfaceboththeCy5.5dye

andtheRGDpeptide,hasbeeninjectedintoU87MGtumor-bearing

micedemonstratinggoodtumoraccumulation.Inanotherstudy,

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Fig.5. Invivoimagingwithferritinnanoparticles.Invivoimagesof(a)positronemissiontomography(PET)and(b)near-infraredﬂuorescenceimagesafteradministration offerritinnanocages.30minbeforeferritinprobeadministration,micewereinjectedwithablockingdoseofc(RGDyK)todemonstratetargetspeciﬁcity.(Reprintedwith permissionfromRef.[22].Copyright2011AmericanChemicalSociety).

Cy5.5-GPLGVRGpeptideorwiththeblackholequencher-3

(BHQ-3),obtainingahybridnanoparticlethatemitsﬂuorescenceonlyina

metalloproteinaserichenvironment,suchasthatfoundintumors

[43]. This nanoparticle was further implemented through the

encapsulationof64_Cu,_a_radioisotope_commonly_used_in_positron

emissiontomography(PET),thusobtainingaferritinplatformfor

multimodalimaging(Fig.5)[22].Combiningopticaland

nanotar-getedstrategiescouldimproveandreﬁneintraoperativeimaging,

toproperlyexciseatumorlesionwithadequatemargins,and

pro-videtargeteddiagnostictoolsforcancerfollowup.

5.3. Ferritinnanoparticlesinphotothermaltherapy

Thankstoitshighbiocompatibility, lowimmunogenicityand

good pharmacokinetics proﬁle ferritin is useful in

photother-maltherapy(PTT)[86].PTTemploysphotothermalagents,which

absorbtheirradiation energyof anoptical laser and convertit

intoheatforkillingcancercells.Indeed,mostofthe

photother-malagentsshowhighimmunogenicity,non-biodegradability,long

termtoxicity and poorpharmacokinetics, which stronglyaffect

their clinical application [87,88]. Haung et al. have reported

thedevelopmentofanearinfra-red(NIR)-loadedferritin,which

displayed strongabsorbance in theNIRregion for

photoacous-tic/ﬂuorescence multimodal imaging-guided PTT [89,90]. The

as-designedferritin nanovector exhibited invivo highPTT

efﬁ-cacyandlowsystemictoxicityincomparisontothefreedye[90].

Anotherkindofferritinnanoparticlehasbeendevelopedbythe

Cecigroupas aplatform for hyperthermia.Theyfunctionalized

theferritincagewiththemelanomatargetingpeptideandused

itasamineralizationchamberforthesynthesisofcobalt-doped

ferritenanoparticles.Theas-synthesizednanoparticleswere

uni-forminsize(6–7nm)andmorphology(spherical).Moreover,they

displayedaninvitrohyperthermicefﬁcacyinverselyrelatedtothe

amountofCousedforthedoping[45].Altogether,thereporteddata

suggestthatferritinnanocagescouldbeapromisingvectorfor

pho-tothermaltherapy,althoughfurtherstudieshavetobeperformed

tobetterinvestigateferritin’spotentialinthisﬁeld.

6. Conclusionsandfutureperspectives

Sofar,nanotechnologyhasemerged aspromisingfrontierin

cancertreatment. Super-paramagnetic iron oxide nanoparticles

(SPIONs)are beingtested in clinicaltrialsas magneticcontrast

agents for MRI and for intraoperative sentinel node

identiﬁca-tionduringbreast cancersurgery [91,92].Liposomeshavebeen

alreadyestablishedinclinicalpracticeforimproving

bioavailabil-ityandreducingtoxicityproﬁleofsomeexcellentcytotoxicdrugs

associatedwithseverecardiotoxicity,suchasanthracyclines[93].

Nanoformulatedalbumin-bound paclitaxelhasbeennotoriously

introducedinclinicalmanagementofvarioustypesofcancerwith

encouraging results in terms of anticancer efﬁcacy [94].

How-ever,thesebasicnanoparticleslacka speciﬁcmethodofcancer

targeting, and despite theirbeneﬁt in reducing side effects of

cytotoxicdrugsin clinicaltrials,nosubstantialimprovementin

long-termoutcomeshasbeendocumented[95].Inthiscontext,

ferritinnanocagesappeartobepowerfulandfascinatingtoolsfor

bothimagingandtherapeuticpurposes,inparticularconsidering

thatferritinisaproteinnormallypresentinphysiologicalsystems.

Different strategies ofloading ferritin withdrugs,molecules

andmetalshavebeensuccessfullyexplored;nevertheless,some

aspects particularly regarding their kinetic release from the

nanovector still need to be investigated in detail. Much work

hasalreadybeendonetoincreasethetypesofmolecules

encap-sulated,particularly inthe case of non-metal-containingdrugs,

whichincludethemajorityofchemotherapeutics.However,afew

questionsremain:(1)thefateofferritinanditsderivativesafter

sys-temicinjectionand(2)thepossibilitytosuppressferritinuptakein

off-targetorgansand/orsequestrationbythereticulo-endothelial

system,and thereby(3)thehopetoextenda drug’shalf-lifeby

modifyingtheferritinsurfacewithstealthmoieties.Someresearch

groupshavetakenadvantageoftheabilityofH-ferritinto

specif-icallyrecognizeTfR1[33],areceptorexpressedonthesurfaceof

manytypesofcancercells[41].Othergroupshavechemicallyor

geneticallyinsertedtargetingmotifsontotheferritinsurface,to

improvetumorhoming[42–54].Thisstrategyhasbeen

demon-stratedtobeverypromising,sincetargetedtherapiesareprobably

thekeyforcancertreatment[96].Speciﬁctargetingofcancercell

receptorsenhancesanticanceractivitybyselectiveinhibitionofthe

underlyingcellularpathways,andmediatesaselectivereleaseof

cytotoxicdrugs.However,despitethefactthatsurface

modiﬁca-tionofferritin impartstargetspeciﬁcity,it isnotclearwhether

ornotthesemodiﬁcationsalterthenanoparticle’s

immunogenic-ity. Anotheraspectwhich shouldnot beunderestimated is the

possibilityofcombinationtherapy.Encapsulationofvariousdrugs

into thesame nanocage would provide contemporary multiple

anticancer activitiestowards various cancer survival pathways.

Finally, theintrinsic capabilityof ferritinnanocages toundergo

nucleartranslocationcanbeexploitedtodesignanovelclassof

smartnanodrugs,whichcouldactas“Trojanhorses”,by

releas-ingchemotherapyintothenucleusofcancercells,thusstrongly

improving theircytotoxic activity.At present, unfunctionalized

DOX-loadedferritinrepresentsthemostextensivelyinvestigated

oftheferritin-basednanoparticles,andseveralinvitroandinvivo

(8)

nanoparticlesarecurrentlynotusedinclinicalpractice,and

fur-therresearchisrequiredbeforeclinicaltranslation.Ferritinhasalso

foundanintriguingapplicationintheﬁeldofinvivoimaging.

Partic-ularly,ferritin-basedcontrastagentsforMRIhavebeendeveloped,

providinganimagingplatformwithincreasedtumor

accumula-tionincomparisontothecontrastagentscurrentlyusedinclinical

practice.However,toxicity,biodistributionandclearanceofthese

nanocompoundsneedstobemoreclearlyunderstoodbeforetheir

clinicaltranslation.Overall,ferritinnanocagesarepromising

nano-platformsforimagingandtherapyofcancer.Someissuesstillneed

tobethoroughlyinvestigated,butmanysigniﬁcantresultshave

alreadybeenobtained.Physiologicalfeaturesandthe

bioengineer-ingversatilityofferritin-basednanocagesmaketheirtranslation

from bench to bedside a reasonable possibility. Moreover, the

combinationofimagingandtherapeuticfunctionalityintoferritin

nanocagesseemstobethemostpromisingandfascinatingfrontier

incancertheranostics.

Conﬂictofinterest

Theauthorsdeclarethatthereisnoconﬂictofinterest.

Acknowledgements

Theresearchwassupportedby FondazioneRegionaleper la

RicercaBiomedica(NANODRUGplatformproject).FRRBsupported

S.M,L.F.andM.T.WethankChiaraSRLandE.Fezzuoglioforﬁgures

andgraphicalabstract.

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