Bovine serum albumin BSA and cleaved BSA

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ContentslistsavailableatScienceDirect

Colloids

and

Surfaces

A:

Physicochemical

and

Engineering

Aspects

j o u r n al ho me p a g e :w w w . e l s e v i e r . c o m / l o c a t e / c o l s u r f a

Bovine

serum

albumin

(BSA)

and

cleaved-BSA

conjugated

ultrasmall

Gd

2

O

3

nanoparticles:

Synthesis,

characterization,

and

application

to

MRI

contrast

agents

Md.

Wasi

Ahmad

a

,

Cho

Rong

Kim

a

,

Jong

Su

Baeck

b

,

Yongmin

Chang

b,d,∗

,

Tae

Jeong

Kim

c,d

,

Ji

Eun

Bae

d

,

Kwon

Seok

Chae

d,e

,

Gang

Ho

Lee

a,d,∗

a_Department_of_Chemistry,_College_of_Natural_Sciences,_Kyungpook_National_University_(KNU),_Taegu_702-701,_South_Korea

b_Department_of_Molecular_Medicine_and_Medical_&_Biological_Engineering,_School_of_Medicine,_KNU,_Taegu_702-701,_South_Korea

c_Department_of_Applied_Chemistry,_College_of_Engineering,_KNU,_Taegu_702-701,_South_Korea

d_Department_of_Nanoscience_and_{Nanotechnology,}_KNU,_Taegu_702-701,_South_Korea

e_Department_of_Biology_Education,_Teacher’s_College,_KNU,_Taegu_702-701,_South_Korea

h

i

g

h

l

i

g

h

t

s

•BSAandC-BSAconjugatedultrasmall

Gd2O3 nanoparticles were

synthe-sized.

•BSAcanbindmanyultrasmallGd2O3

nanoparticleswhereasC-BSAcannot.

•Largewaterprotonrelaxivitieswere

observed.

•HighcontrastMRimagesinamouse

liverafterintravenousinjectionwere

observed.

•These nanoparticles are potential

MRIcontrastagents.

g

r

a

p

h

i

c

a

l

a

b

s

t

r

a

c

t

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received15November2013

Receivedinrevisedform23February2014 Accepted1March2014

Availableonline12March2014

Keywords:

BSA C-BSA

UltrasmallGd2O3nanoparticle

Nanoparticlecarrier MRI

Contrastagent

a

b

s

t

r

a

c

t

Bovineserumalbumin(BSA)(Mn=66.5kD,size=14×4×4nm)isanattractivebiologicalmoleculefor

biomedicalapplicationsbecauseofitswater-solubilityandbio-compatibility.Itcanalsobindmany

ultrasmallnanoparticles(NPs)asconfirmedinthisstudy.Wesynthesizedpolyethyleneglycoldiacid

(PEGD)coatedultrasmallGd2O3nanoparticles(PEGD-GNPs,thecoredavg=2.0nm),whichwerethen

con-jugatedtoBSAandcleaved-BSA(C-BSA)(i.e.BSA-PEGD-GNPsandC-BSA-PEGD-GNPs)throughamide

bonding.Largerelaxivities wereobservedinbothaqueous samplesolutions(r1=6.0s−1mM−1 and

r2=28.0s−1mM−1 forBSA-PEGD-GNPsandr1=7.6s−1mM−1 andr2=22.0s−1mM−1 for

C-BSA-PEGD-GNPs).ThreeteslaT2magneticresonanceimaging(MRI)inamouseaftertheinjectionofanaqueous

samplesolutionofBSA-PEGD-GNPsintoamousetailveinrevealedsignificantnegativecontrast

enhance-ments.LargerelaxivitiesandinvivoMRimagesprovethatBSA-PEGD-GNPsandC-BSA-PEGD-GNPsare

potentialMRIcontrastagents.

Abbreviations:BSA,bovineserumalbumin;C-BSA,cleavedBSA;PEGD,polyethyleneglycoldiacid;NP,nanoparticle;GNP,Gd2O3NP;BRB,Britton–Robinsonbuffer;PBS,

phosphatebuffersolution;MRI,magneticresonanceimaging;NCT,neutroncapturetherapy;CT,X-raycomputedtomography;DTPA,diethylenetriaminepentaaceticacid;PBS, phosphatebuffersolution;NHS,N-hydroxysuccinimide;EDC,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;GPC,gelpermeationchromatograph;HVEM,highvoltage electronmicroscope;XRD,X-raydiffraction;ICPAES,inductivelycoupledplasmaatomicemissionspectrometer;FT-IR,Fouriertransform-infrared;TGA,thermo-gravimetric analyzer;DU145,humanprostatecancercell;NCTC1469,normalmousehepatocytecell.

∗Correspondingauthors.Tel.:+82539505340;fax:+82539506330.

E-mailaddresses:[email protected](Y.Chang),[email protected](G.H.Lee).

http://dx.doi.org/10.1016/j.colsurfa.2014.03.011

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68 Md.W.Ahmadetal./ColloidsandSurfacesA:Physicochem.Eng.Aspects450(2014)67–75

1. Introduction

Biologicalmoleculeshave attracted considerableinterest for applicationtonanomedicinebecausetheyarefullybiocompatible

andwater-soluble.Bovineserumalbumin(BSA)isanimportant

carrierproteininbloodplasmaforseveralionsandmolecules.Ithas alargedimension(14×4×4nm)andheavymass(Mn=∼66.5kD).

Thus,itmayalsocarryseveralultrasmallnanoparticles(NPs)with asmallersizeandmassthanBSA.Biologicalmoleculeshave sev-eraladvantagesoversmallmoleculesandpolymersforbiomedical applications.First, the water-solubility of surface-modifiedNPs generallyincreaseswithincreasingmassoftheligands[1,2]and thus,biologicalmoleculeswillprovideenhancedwater-solubility forNPs.Second,theNPsconjugatedtobiologicalmoleculescan

remain in the blood for a longer duration than free NPs and

Gd-chelates,allowinglongerimagingtimes(socalledblood-pool imagingagents)andahigherlikelihoodofdeliveringNPstothe targetedareasinabody[3–6].

ThisstudymakesuseofBSAandultrasmallGd2O3NPs(GNPs)

for magnetic resonance imaging (MRI). Ultrasmall GNPs have

shownlongitudinal(r1)andtransverse(r2)waterproton

relax-ivities larger than those of Gd-chelates because of the dense

populationofGd(III)inNPs[7,8].Therefore,BSAwhichcouldbind severalultrasmallGNPs maybeusefulfor MRI.Here, ultrasmall

GNPs can bealso appliedto X-raycomputed tomography (CT)

asCTcontrastagentsandneutroncapturetherapy(NCT)asNCT agentsbecauseGdhasalargeX-rayattenuationpower(∼2.5times

strongerthancommercialiodineCTcontrastagents)[9–12]anda verylargeneutroncapturecrosssection(∼254,000barns)[13–16].

Thisimplies thatBSAconjugatedultrasmallGNPs couldbealso usefulforCTandNCT.

PreviousstudiesonMRIusingBSAincludeBSA[Gd-chelates]n

andBSA-GNPs(d=20–40nm)[3–6,10,17].Enhancedwaterproton relaxivities have beenobserved in both systems after conjuga-tiontoBSA.Inaddition,theinvivoapplicationofBSA[Gd-DTPA]n

(DTPA=diethylenetriaminepentaaceticacid)showedalonger

cir-culationtime intheblood than Gd-DTPA, providing longerMR

imagingtimesinbraintumorsandbloodvesselsinrats[3,4]. ThisstudyexaminedBSAandcleavedBSA(C-BSA)(<7.0kD) con-jugatedultrasmallGNPsforMRIcontrastagents.Here,ultrasmall nanoparticles(d<3nm)areclinicallyimportantbecausetheycan beexcretedthroughrenalsystem[18]andtherefore,haveahigh potentialforbiomedical applications.ToconjugateGNPstoBSA andC-BSA,theGNPsweresurfacemodifiedwith polyethylenegly-coldiacid(PEGD)(Mn=600)andthenconjugatedtoBSAandC-BSA viaamidebonding.Theparticlesizes,surfacemodifications, num-berofultrasmallGNPsconjugatedtoBSAandC-BSA,waterproton relaxivities,cellulartoxicities,andinvivoMRimagesusingamouse werecharacterized.TheBSAconjugatedGNPswerefurtherapplied toMRIinamouseandnegativecontrastenhancementsin3TT2MR imageswereclearlyobserved.

2. Experimentals

2.1. Chemicals

AllchemicalssuchasGdCl3·xH2O(99.9%),NaOH(>99.9%), tri-ethyleneglycol(99%),polyethyleneglycoldiacid(PEGD)(Mn=600), BSA (Mn=∼66.5kD), phosphate buffersolution (PBS) (pH=7.2),

HCl (>99%), N-hydroxysuccinimide (NHS) (98%),

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (>97%), boric acid

(>98%),phosphoricacid(>97%),andacetic acid(98%)were pur-chasedfromSigma-Aldrichandusedasreceived.Triplydistilled waterwasusedforbothwashingtheproductsandpreparingthe MRIsamplesolutions.

2.2. SynthesisofPEGDcoatedultrasmallGNPs(PEGD-GNPs)

PEGD-coatedultrasmallGNPsweresynthesizedusingone-pot

process(Scheme1a).Twoseparatesolutionswereprepared.One isaprecursorsolutionmadefrom5mmolofGdCl3·xH2Oin25mL oftriethyleneglycol,andtheotherisaNaOHsolutionmadefrom 15mmolofNaOHin10mLoftriethyleneglycol.Theprecursor solu-tionwasheatedto60◦_C_with_magnetic_stirring_under_atmospheric conditionsuntiltheprecursorwasdissolvedcompletelyin triethy-leneglycol.ANaOHsolutionwasaddedtotheprecursorsolution. Themixedsolutionwasstirredmagneticallyat180◦_C_for₄_h._The solutiontemperaturewasreducedto80◦_C_and₈_mmol_of_PEGD_was addedtothesolutionforsurfacecoating.AnexcessofPEGDwas

usedtoensurethatonlyone–COOHgroupamongthetwo–COOH

groupsinPEGDcouldbeusedforconjugationtoultrasmallGNPs becausetheother–COOHgroupshouldbefreeforconjugationto BSA(orC-BSA).Thesolutiontemperaturewasagainincreasedto 180◦_C_and_stirred_for_an_additional₄_h._The_solution_was_cooled toroomtemperatureandtransferredtoa1Lbeakercontaining 500mLoftriplydistilledwater.Thiswasstirredmagneticallyfor 10minandstoredforaweekorsountilthePEGD-coatedultrasmall

GNPssettledinabeakerbottom.Thesupernatantwasdecanted

andtheremainingsamplesolutionwaswashedagainwithtriply distilledwater.Thisprocedurewasrepeatedthreetimes.

2.3. SynthesisofC-BSA

To prepare C-BSA, 10mmol of l_-serine _and ₁₀_mmol _of l

-histidineweredissolvedin 300mLofaBritton–Robinsonbuffer

(BRB) (40mM phosphate, 40mM acetate, and 40mM borate)

(pH=5–6)at60◦_C_and_under_atmospheric_conditions₍_Scheme₁_b)

[19].Here,apHof5–6ofthebuffersolutionwasachievedbyadding NaOHslowlytotheoriginalbuffersolution.Aftermagneticstirring for30min,156mgofBSAwasaddedtothesolution,andthe solu-tionwasstirredmagneticallyfor36hat60◦_C._After_the_reaction wascomplete,thewaterwasevaporated.Thiscleavagereaction

wasrepeated10timestoobtainenoughC-BSA.Themassesof

C-BSAswerecharacterizedbygelpermeationchromatography(GPC) andtheresultissummarizedinSupportingInformation.Twomajor

C-BSAswithmassesof6.67and2.01kDwereobservedfromGPC

analysis.TheC-BSAwasusedwithoutfurtherpurificationbecause thewater-solubleC-BSAcouldnotbeseparatedfromother water-solublereagentsusedinthereaction.Ontheotherhand,thewater solublereagentswerelaterremovedafterconjugationofC-BSAsto

PEGD-GNPsthroughamidebonding.

2.4. SynthesisofBSA-PEGD-GNPsandC-BSA-PEGD-GNPs

BSA-PEGD-GNPs and C-BSA-PEGD-GNPs were synthesized

usinganEDC/NHScouplingmethod(Scheme1c)[20,21].Inthis

reactionamidebondswereformedbetween–COOHofPEGD-GNPs

and–NH2ofBSA(orC-BSA).Thereactionwascarriedoutatroom

temperatureandunderatmosphericconditions.SolutionpHwas

fixedat6.0byadding1mMHCltotheoriginalPBSwithpHof7.2.

5mmolofEDCand5mmolofNHSwereaddedto30mLofPBS

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Scheme1.Synthesesof(a)theultrasmallGNPsandPEGD-GNPs,(b)theC-BSA,and(c)theBSA-PEGD-GNPsandC-BSA-PEGD-GNPs.

otherreagentsusedinsynthesisofC-BSA,wereremovedfromthe

products.Portionsofthesampleswereevaporatedtoapowder

formin airandtheremainingportionsweredilutedwithtriply distilledwatertoprepareaqueoussamplesolutionsfortheMRI experiments.

2.5. Generalcharacterization

TheparticlediametersoftheultrasmallGNPsweremeasured

with a high voltage electron microscope (HVEM) (JEOL

JEM-ARM1300S,1.2MeVaccelerationvoltage).Acoppergrid(PELCO

No. 160, TED PELLA,INC.) covered with an amorphous carbon

membrane was placed onto a filter paper and a sample

solu-tiondilutedintriplydistilledwaterwasdroppedontothecopper gridusingamicropipette (Eppendorf,2–20␮L).Thecoppergrid

wasdried inair for onehour toremove thesolvent.The

crys-tal structure of ultrasmall GNPs was examinedusing an X-ray

diffraction(XRD)spectrometer(Philips,X-PERTPROMRD)with

unfilteredCu-K␣radiationof1.54184 ˚A,scanningstepof0.033◦,

andscanrangeof2=15–100◦_._The_{concentration}_of_Gd_in_the sam-plesolutionwasdeterminedusinganinductivelycoupledplasma atomicemission spectrometer(ICPAES) (ThermoJarrell AshCo.,

IRIS/AP).The surfacecoatingof theultrasmallGNPs withPEGD

andconjugationofPEGD-GNPstoBSA(orC-BSA)wereinvestigated usingaFouriertransform-infrared(FT-IR)absorption spectrome-ter(MattsonInstruments,Inc.,Galaxy7020A).Thepowdersamples

were dried ona hot plate at 50◦_C _in _a _hood _for _one _week _to

minimizethewatercontent.TorecordtheFT-IRabsorption spec-tra(400–4000cm−1_),_pellets_of_the_powder_samples_in_KBr_were

prepared.The amountof surface coatingwasestimated witha

thermo-gravimetricanalyzer(TGA)(TAInstruments,SDT-Q600).

TheTGAcurvesofthepowdersampleswererecordedbetween

roomtemperatureand700◦_C_while _air_flowed._The_amounts_of

PEGD,BSA,andC-BSAperGNPwereestimatedbyrecordingtheTGA curves.Waterdesorptionbetweenroomtemperatureand∼110◦C

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2.6. Relaxivityandmapimagemeasurement

Boththelongitudinal(T1)andtransverse(T2)relaxationtimes and longitudinal (R1) and transverse (R2) map images of the

aqueoussamplesolutions of BSA-PEGD-GNPsand

C-BSA-PEGD-GNPsweremeasuredusing a1.5Tmagneticresonance imaging

(MRI)instrument(GE1.5TSignaAdvantage,GEmedicalsystem) equippedwithaKneecoil(EXTREM).Aqueoussamplesolutionsat differentGdconcentrationswerepreparedbydilutingthe origi-nalsamplesolutionswithtriplydistilledwater.Bothmapimages andrelaxationtimesweremeasuredusingthesesolutions.The lon-gitudinal(r1)and transverse(r2)waterprotonrelaxivitieswere estimatedfrom theslopesin the plots of1/T1 and 1/T2 versus theGdconcentration,respectively.Thetypicalparametersusedto

measuretherelaxation timesandmapimageswereasfollows:

theexternal MRfield (H)=1.5T,thetemperature(T)=22◦_C,_the numberofacquisition(NEX)=1,thefield ofview(FOV)=16cm, the phase FOV=1, the matrix size=512×512, the slice

thick-ness=5mm,thespacinggap=0,thepixelbandwidth=61.0547,the repetitiontime(TR)=2009ms,andtheechotime(TE)=9ms.

2.7. Invitrocytotoxicitymeasurement

Thein vitro cytotoxicityof theaqueoussample solutions of

BSA-PEGD-GNPsandC-BSA-PEGD-GNPswasmeasuredusingboth

humanprostate cancer(DU145)and normal mousehepatocyte

(NCTC1469)cells.ACellTiter-GloLuminescentCellViabilityAssay (Promega,WI,USA)wasusedtomeasurethecytotoxicity.Inthis

assay,theintracellularATPwasquantifiedusingaluminometer (Victor3,PerkinElmer).Thecellswereseededontoa24-wellcell cultureplateandincubatedfor24h(5×104celldensity,500␮L

cellsperwell,5%CO2,37◦C).Aseriesoftestsamplesolutions(0, 10,100,and200␮M)werepreparedbydilutingtheoriginalsample

solutionswithasterilephosphatebuffersalinesolution.∼2mLof

eachtestsolutionwasaddedtothecellculturemedia.Thetreated cellculturemediawerethenincubatedfor48h.Thecellviabilityof eachcellwasdeterminedandnormalizedwithrespecttothatofthe controlcellwith0.0MGdconcentration.Themeasurementsforall testcellswererepeatedtwicetoobtaintheaveragecellviabilities.

2.8. Invivo3TT2MRimagemeasurement

A3TMRIinstrument(SIEMENS3.0TMAGNETOMTrioaTim)

wasusedtomeasuretheT2 spinecho(SE)imagesof amouse.

Theanimalexperiment inthis studywascarriedoutunderthe

permissionandguidanceoftheKNUanimalcommittee.AnICR

female mouse (ICR—Institute of Cancer Research, USA) with a

weight of ∼100g was used for the MR image measurements.

Themouse wasanesthetized by1.5% isofluranein oxygen. The

measurementsweremadebeforeandafterinjectingthesample

solutionintoamousetailvein.Theinjectiondosewas∼250␮L

(∼0.1mmol Gdkg−1). After the measurement, the mouse was

revivedfromanesthesia,placedintoacage,andgivenafreeaccess

toboth food and water. During themeasurement, each mouse

wasmaintainedat∼37◦Cusingawarmwaterblanket.The

typ-ical measurement parameters were as follows: the H=3T, the

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T=37◦_C,_the_NEX₌_3–4,_the_FOV₌₆₀_mm,_the_phase_FOV₌₃₀_mm, thematrixsize=128×256,theslicethickness=1mm,thespacing

gap=0.1mm,theTR=2690ms,andtheTE=37ms.

3. Resultsanddiscussion

3.1. Particlediameter(d)andcrystalstructureofultrasmallGNPs

Fig. 1a–d shows HVEM images of the as-prepared

ultra-smallGNPs,BSA-PEGD-GNPs,andC-BSA-PEGD-GNPs.Theparticle

diametersoftheultrasmallGNPsrangedfrom1to3nmwiththe

davgof2.0nm(Fig.1aandb).TheHVEMimageofBSA-PEGD-GNPs

indicatedthatmanyultrasmallPEGD-GNPswereconjugatedtoa

BSA(Fig.1c).Ontheotherhand,theHVEMimageof C-BSA-PEGD-GNPsshowedthatmanyC-BSAswereconjugatedtoeachultrasmall PEGD-GNP(Fig.1d).Aswillbediscussedlater,thisisconsistent

withthenumbersof PEGD-GNPs conjugatedtoBSA andC-BSA

estimatedfromTGAanalyses.

TheXRDpatternsofpowdersamplesofbothBSA-PEGD-GNPs

andC-BSA-PEGD-GNPsweremeasuredbeforeandafterTGAand

areprovidedinFig.2.TheXRDpatternsoftheas-prepared pow-dersampleswerebroad,duelikelytoultrasmallparticlediameters

[22].Ontheotherhand,theXRDpatternsoftheTGA-treated

pow-dersamplesrevealedsharp peaks,correspondingtomonoclinic

GdPO4.AllpeaksafterTGAanalysiscouldbeassignedtomonoclinic GdPO4andthepeakpositionswithsufficientintensitiesaremarked with‘*’inFig.2andtheMillerindex(hkl)assignmentsofthese peaksareprovidedinSupportinginformation.Theformationof GdPO4afterTGAanalysisisbecausetheEDC/NHScouplingreaction wascarriedoutinPBS.Thatis,thePO43−ionswerelikelyattached

to amine groups of BSA-PEGD-GNPs and C-BSA-PEGD-GNPs

20 40 60

* : GdPO₄

* * * * * * * * * * * * * * * * * * *

2

In te nsit y (Ar b . U ni ts )

as prepared after TGA

*

(a)

20 40 60

* * *

*

* : GdPO₄

* * * * * * * * * * * * * *

* _after_TGA

as prepared

2

Inte n sity (Arb. Uni ts ) (b) *

Fig.2. XRDpatternsofpowdersamplesof(a)BSA-PEGD-GNPsand(b) C-BSA-PEGD-GNPsbefore(i.e.as-prepared)andafterTGAanalysis.AllpeaksafterTGAanalysis couldbeassignedtomonoclinicGdPO4andonlythepeakpositionswith

suffi-cientintensitiesaremarkedwith‘*’(Millerindex(hkl)assignmentsareprovided inSupportinginformation).

throughhydrogenbonding[23–25].Theestimatedcellconstants area=6.644 ˚A,b=6.841 ˚A,c=6.328 ˚A,andˇ=103.976◦ _for TGA-treatedBSA-PEGD-GNPsanda=6.643 ˚A,b=6.839 ˚A,c=6.326 ˚A,and ˇ=104.001◦_for_TGA-treated_{C-BSA-PEGD-GNPs}_which_are consis-tentwiththevaluesreportedinPCPDFWIN[26].

3.2. SurfacemodificationofultrasmallGNPs

ThesurfacecoatingofultrasmallGNPswithPEGDfollowedby conjugationtoBSAorC-BSAwasinvestigatedbyFT-IRabsorption spectroscopy.Asmentionedpreviously,onegroupamongthetwo

–COOHgroupsineachPEGDwasconjugatedtoanultrasmallGNP

andtheotherwasleftfree foramidebondingtoBSA orC-BSA.

These were confirmed from the two different C=O stretching

vibrations in theFT-IR absorption spectrum of thePEGD-GNPs

(Fig. 3a). The free –COOH was observed at 1730cm−1 _but _the –COOHbondedtoGNPs,at1620cm−1_._The_peaks_at₂₉₁₀_cm−1 (C–Hstretch)and1110cm−1_(C–O_stretch)_also_confirmed_that_the

PEGDswerebondedtoultrasmallGNPs.Thepeakat3420cm−1

in thePEGD-GNPs wasassigned tothewater –OH stretch.The

∼110cm−1 red shiftof the C=O stretch after bonding to GNPs

from that of thefree –COOH had been observed in a range of

themetaloxideNPscoatedwiththeligandswith–COOHgroups

[22,27–30], supporting this result. The successful amide bond

formationbetweenPEGD-GNPsandBSA(orC-BSA)wasconfirmed

fromthedisappearanceofafreeC=Ostretchat1730cm−1_in_both BSA-PEGD-GNPsandC-BSA-PEGD-GNPs(Fig.3b).Instead,theN–H stretchat3440cm−1_(overlapped_with_water_–OH_stretch)_and_the N–Hbendat1530cm−1_were_observed_[31–33]_._The_reduced

inten-sityinN–HbendinbothBSA-PEGD-GNPsandC-BSA-PEGD-GNPs

was,however, observed owingtothe amidebonding of amine

groups of BSA and C-BSA with PEGD-GNPs and the hydrogen

bondingofaminegroupsofBSAandC-BSAwithPO43−ions.

4000

300

0

200

0

100

0

PEGD-G

NP

Waven

umber

(c

m

-1

)

Transm

ittance

(%

)

3420 2910

1730

1620

1110

(a)

PEGD

4000

300

0

200

0

100

0

C-BSA-PEGD

-GNP

BSA-PEGD-

GNP

BSA

Wavenu

mber

(

cm

-1)

Tr

ansmittance

(%

)

3440

1620 1530

(b)

2910

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0 100 200 300 400 500 600 700

0 20 40 60 80

100 Water desorption = 9.8%

Temperature (oC)

We

ight (%)

28.0% (a)

0 100 200 300 400 500 600 700

0 20 40 60 80 100

47.4%

Water desorption = 4.9%

Weight (%

)

(b)

0 100 200 300 400 500 600 700

0 20 40 60 80 100

(c)

61.7%

Water desorption = 7.3%

W

eig

h

t (%

)

Fig.4. TGAcurvesofpowdersamplesof(a)PEGD-GNPs,(b)BSA-PEGD-GNPs,and (c)C-BSA-PEGD-GNPs.

TodeterminetheamountsofBSAintheBSA-PEGD-GNPsand

C-BSAintheC-BSA-PEGD-GNPs,theTGAcurvesof PEGD-GNPs,

BSA-PEGD-GNPs,andC-BSA-PEGD-GNPswererecorded(Fig.4a–c).

Waterdesorptionbetweenroomtemperature and∼110◦C was

consideredintheseestimations.TheamountofPEGDwas

esti-matedtobe28.0% fromtheTGAcurveof PEGD-GNPs(Fig.4a).

TheamountsofBSA-PEGDandC-BSA-PEGDwereestimatedtobe

47.4and 61.7%fromtheTGAcurves ofBSA-PEGD-GNPsand

C-BSA-PEGD-GNPs,respectively(Fig.4bandc).TheamountsofBSA

andC-BSAwereestimated tobe19.4and33.7% bysubtracting

theamountofPEGDfromthose ofPEGD-BSAandC-BSA-PEGD,

respectively.Usingthedavgof2.0nmfortheultrasmallGNPs

esti-matedfromtheHVEMimageandassumingthattheirdensityis

thesameasthat (=7.407gmL−1₎_[34]_of _bulk _Gd

2O3,the

num-berofGNPsconjugatedtoeachBSA andC-BSAwereestimated

tobe8.8and0.2,respectively,whichwereconsistentwithHVEM observations(Fig.1bandc).Therefore,BSAisagood nanoparti-clecarrier,butC-BSAisnot.Thiscanbeexplainedusingthesizes andmassesofBSAandC-BSA.Thatis,themassofultrasmallGNPs withthedavg=2.0nmwasestimatedtobe10–20kDbycalculating thevolumeoftheultrasmallGNPsandusingthebulkdensityof Gd2O3[34],whicharesmallerthanthoseofBSA(mass=66.5kD andsize=14×4×4nm).Ontheotherhand,C-BSAswithmasses

of6.67and2.01kDestimatedfromGPChavesizesof6.5×1.9×1.9

0.00 0.25 0.50 0.75 1.00

0 10 20 30

r₁ = 6.0 s-1mM-1

r₂ = 28.0 s-1mM-1

Concentration Gd (mM)

1/T

(s

-1 )

(a)

0.00 0.04 0.08 0.12 0.16 0.20

0 1 2 3 4 5 6

r₁ = 7.6 s-1mM-1

r₂ = 22.0 s-1mM-1

(b)

1/

T

(

s

-1 )

Concentration Gd (mM)

Fig.5.Plotsof1/T1and1/T2oftheaqueoussamplesolutionsof(a)

BSA-PEGD-GNPsand(b)C-BSA-PEGD-GNPsasafunctionoftheGdconcentration.Theslopes correspondtother1andr2values,respectively.

and4.4×1.2×1.2,respectively,assumingacuberootdependence

ofthesizeonthemass.Therefore,C-BSAisnotlargeenoughin massandsizetobindmanyultrasmallGNPs,whichissimilarto thepolymers.

3.3. SuggestedstructuresofBSA-PEGD-GNPand C-BSA-PEGD-GNP

Asmentionedbefore,theconjugationbetweenPEGD-GNPand

BSA(orC-BSA)isanamidebondbetween–COOHofPEGD-GNP

and –NH2 of BSA (or C-BSA). The BSA consists of 607 amino

acids and has amino acids with a free –NH2 [35,36] that can

beusedfortheamidebondingtoPEGD-GNP.In fact,60lysines witha free–NH2 areinBSA.Therefore,thereareplentyoffree

–NH2inBSAwhichcanbeconjugatedtoPEGD-GNPsthroughthe

amidebonding.Asdescribedpreviously,∼9PEGD-GNPswere

esti-matedtobeconjugatedtoeachBSA whereas∼0.2PEGD-GNPs,

to each C-BSA. Based on these results, structures of the

BSA-PEGD-GNP and C-BSA-PEGD-GNP were schematically drawn in

Scheme1c.

3.4. Relaxivitiesandmapimages

Magnetic propertiesof gadolinium oxide nanoparticleshave

been well characterized [37]. They are paramagneticbut have

anappreciable magneticmoment at roomtemperature. Thisis

becauseGd(III)hassevenunpaired4f-electrons(8_S

7/2).Therefore, appreciabler1 andr2valuesareexpectedfromsamplesolutions, which were in fact observed in this study. The r1 and r2 val-uesofBSA-PEGD-GNPswere estimatedtobe6.0s−1_mM−1 _and 28.0s−1_mM−1_,_{respectively,}_from_the_slopes_in_the_plot_of_1/_T

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Table1

Waterprotonrelaxivity(r1andr2)aofvariouschemicals.

Chemical dordavgb Nc r1 r2 Hd Te Ref.

Gd-DTPA – – 4.1 – 0.47 38 [5]

BSA-GNP 20–40 – 6.7 38.5 4.7 37 [10]

BSA-PEGD-GNP 2.0 8.8 6.0 28.0 1.5 22 Thiswork C-BSA-PEGD-GNP 2.0 0.21 7.6 22.0 1.5 22 Thiswork

a_Unit:_s−1_mM−1_.

b_Particle_diameter_or_average_particle_diameter_(nm). c _Number_of_GNPs_conjugated_to_a_BSA_or_C-BSA. d _Applied_MR_field_(T).

e_Sample_solution_temperature₍◦_C).

forcomparison.Ther1andr2valuesofBSA-PEGD-GNPsand C-BSA-PEGD-GNPsarelargerthanthose[5,6]ofmolecularGd-DTPA.These increasedrelaxivitieswereattributedtothehighdensityofGd(III) intheNPs.Theselargervaluesgenerallyleadtoahighersensitivity fordetectingdiseasesinthebodythroughcontrastenhancements andcanalsoprovidethesamequalityMRimagesasthoseofthe Gd-chelatesatreduceddoses.Ther2valuesaresignificantlylargerthan thatofmolecularGd-DTPA,whichiswhyonlyNPsareeligibleas

T2MRIcontrastagents,whereasmolecularagentsareonlysuitable as T1 MRI contrastagents. The r1 and r2 values of BSA-PEGD-GNPsandC-BSA-PEGD-GNPsweresimilartothose[10]ofBSA-GNP (d=20–40nm)measuredatahigherappliedMRfield.Ontheother hand,consideringthatthewaterprotonrelaxivitiesincreasewith

increasingappliedMRfield,thoseoftheBSA-PEGD-GNPsand

C-BSA-PEGD-GNPswillbelargerthanthoseofBSA-GNPatthesame appliedMRfield. Thisisdue likelytotheparticlesizeeffectof

theGNP.BothaqueoussolutionsofBSA-PEGD-GNPsand

C-BSA-PEGD-GNPsshowedcleardose-dependentcontrastenhancements

intheirR1andR2mapimages(Fig.6aandb),suggestingthatthese NPsarepotentialcandidatesforMRIcontrastagents,whichwere confirmedinamouseexperiment.

Fig.6.R1andR2mapimagesofaqueoussamplesolutionsof(a)BSA-PEGD-GNPs

and(b)C-BSA-PEGD-GNPsasafunctionoftheGdconcentration.

3.5. Invitrocytotoxicity

Theinvitrocytotoxicityoftheaqueoussamplesolutionsof

BSA-PEGD-GNPsandC-BSA-PEGD-GNPsweremeasuredusingDU145

andNCTC1469cellswithGdconcentrationsupto200␮M(Fig.7a

andb).TheresultsshowedthatC-BSA-PEGD-GNPswereslightly

lesstoxicthanBSA-PEGD-GNPs.Thisisprobablybecausemany

C-BSAlikepolymersencapsulatedthePEG-GNPs,asshownin the

HVEM image (Fig.1c), whereas many PEGD-GNPs were

conju-gatedtoeachBSAonthesurfaceofBSA,asshownintheHVEM

image(Fig.1b).Therefore,PEGD-GNPswerebetterprotectedin C-BSA-PEGD-GNPsthaninBSA-PEGD-GNPs.Thecellviabilityofboth samplesdecreasedgraduallywithincreasingGdconcentration.The cellviabilityofC-BSA-PEGD-GNPsat100␮MGdreachedmorethan

70%forbothcells,whereasthatofBSA-PEGD-GNPsreached∼60%

forbothcells.Theselevelsofcellulartoxicityaresufficientlylowto carryoutinvivoMRIexperiments.

3.6. Invivo3TT2MRimagesofamouse

BecauseBSAcouldbindmanyultrasmallGd2O3NPs,whereas

C-BSAdidnot,asmeasuredbyTGA,aninvivoMRIexperiment

0 20 40 60 80 100 120

(a) _DU145

NCTC1469

Concentration Gd ( M)

0 10 100 200

Ce

ll

Vi

a

bility

(%)

0 20 40 60 80 100 120

Concentration Gd ( M)

Cell

Via

bi

li

ty

(%

)

0 10 100 200 DU145

NCTC1469

(b)

[image:7.612.43.563.74.129.2] [image:7.612.348.528.431.718.2] [image:7.612.72.264.444.722.2]

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Fig.8.(a)3TT2MRimagesoftheliverofamousebeforeandafterinjectingan

aqueoussamplesolutionofBSA-PEGD-GNPsintoamousetailveinand(b)theplot ofsignalintensityinT2MRimagesasafunctionoftimeafterinjection(0indicates

“beforeinjection”).

wasfurthercarriedoutusinganaqueoussamplesolutionof BSA-PEGD-GNPs.AlthoughGNPsaregenerallyusedasT1MRIcontrast agents,theT2MRimageswereinvestigatedbecausether2value wasa lotlargerthanther1 value,due totheappreciable mag-netizationofultrasmallGNPs atroomtemperature[37].250␮L

(0.1mmolGd/kg)ofanaqueoussolutionofBSA-PEGD-GNPswas

injectedintoamousetailveinand3TT2 MRimagesoftheliver weretakenbeforeandafterinjectingtheaqueoussample solu-tion.AsshowninFig.8a,appreciablenegative(ordarker)contrast enhancementswereobservedinthemouseliverafterthe injec-tion,whichreturnedtoalmosttheoriginalcontrastafter24hdue likelytotheexcretionofBSA-PEGD-GNPs.Tomoreclearlyseethe timeevolutionofthecontrastchangeinT2MRimages,thesignal intensityinT2MRimageswasplottedasafunctionoftimeupto 24hinFig.8b.Thisplotclearlyshowsthatthenegativecontrast enhancementmaintainedupto91minafterinjectionbutreturned toalmostzeroabove91mindue likelytotheexcretionof BSA-PEGD-GNPs.Theseresultsclearlyindicatethatthesamplesolution functionedasaT2MRIcontrastagent.

4. Conclusions

Insummary,wesynthesizedPEGDcoatedultrasmallGd2O3NPs

(i.e.PEGD-GNPs)whichwerethenconjugatedtoBSAandC-BSA

through amidebonding (i.e. BSA-PEGD-GNPsand

C-BSA-PEGD-GNPs). We characterizedphysical and in vitro MRI properties,

andcytotoxicityofBSA-PEGD-GNPsand C-BSA-PEGD-GNPs,and

obtainedinvivoMRimagesusingBSA-PEGD-GNPs.

(1)BSA (Mn=66.5kD) could bind many ultrasmall PEGD-GNPs

(thecoredavg=2.0nm),showingthatBSAisagoodultrasmall

NP carrier.TheTGA showedthat ∼9 ultrasmall PEGD-GNPs

couldbeconjugatedtoeachBSA.However,C-BSAs(Mn<7kD) couldnotbindmanyultrasmallPEGD-GNPsduetoitsreduced

sizeand mass.Instead manyC-BSAs wereconjugatedtoan

ultrasmallPEGD-GNPlikepolymers.TheTGAshowedthat∼5

C-BSAswereconjugatedtoeachultrasmallPEGD-GNP.

(2)MR relaxivity measurements revealed that both

BSA-PEGD-GNPsandC-BSA-PEGD-GNPshadr1 andr2valueslargerthan thoseofmolecularGd-chelates.

(3)The3TT2MRimagesafterinjectinganaqueoussample solu-tionofBSA-PEGD-GNPsintothemousetailveinshowedclear

negativecontrastenhancements.

(4)Largerelaxivitiesand invivoT2 MR imagesprovethat

BSA-PEGD-GNPsandC-BSA-PEGD-GNPsarepotentialMRIcontrast

agents.

TheseresultssuggestthatbiologicalmoleculessuchasBSAcan beusedtoconjugatemanysurfacemodifiedultrasmallNPswhich canbeappliedtoavarietyofbiomedicalareassuchasMRIcontrast agentsstudiedinthiswork.

Acknowledgments

ThisstudywassupportedbytheBasicScienceResearch

Pro-gram(Grantno.2012R1A1B3004241toKSC,2011-0015353toYC,

and2013R1A1A4A03004511toGHL)andtheBasicResearch

Labo-ratory(BRL)program(Grantno.2013R1A4A1069507)throughthe

NationalResearchFoundationfundedbytheMinistryofEducation, Science,andTechnology,theR&DprogramofMKE/KEIT(Grantno. 10040393,developmentandcommercializationofmolecular diag-nostictechnologiesforlungcancerthroughclinicalvalidation),and theKNUResearchFund(2013).TheauthorswishtothanktheKorea BasicScienceInstitutefortheuseoftheirHVEMandXRD.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,athttp://dx.doi.org/10.1016/j.colsurfa.2014. 03.011.

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