Controlled intracellular self-assembly of gadolinium nanoparticles as smart molecular MR contrast agents.

15 MB·English

by  Cao, Chun-Yan

Controlled intracellular self-assembly of
gadolinium nanoparticles as smart
SUBJECTAREAS: molecular MR contrast agents
PROTEASES
SELF-ASSEMBLY
Chun-YanCao1,Ying-YingShen2,Jian-DongWang3,LiLi2&Gao-LinLiang1
NANOPARTICLES
MAGNETICRESONANCE
IMAGING 1CASKeyLaboratoryofSoftMatterChemistry,DepartmentofChemistry,UniversityofScienceandTechnologyofChina,96Jinzhai
Road,Hefei,Anhui230026,China,2StateKeyLaboratoryofOncologyinSouthChina,ImagingDiagnosisandInterventional
Center,CancerCenter,SunYat-senUniversity,651DongfengRoadEast,Guangzhou510060,China,3LaboratoryofMolecular
Received PathologyandMolecularImaging,DepartmentofPathology,NanjingJinlingHospital,NanjingUniversitySchoolofMedicine,305
28August2012 ZhongShanDongLu,Nanjing210002,China.
Accepted
4December2012 Hereinwedevelopedanew‘‘smart’’Gd-basedMRcontrastagent(i.e.,1)whichissusceptivetofurin,a
proteaseoverexpressedintumor.Undertheactionoffurin,1condensestoformdimers(1-Ds)andthelatter
Published
self-assembleintogadoliniumnanparticles(Gd-NPs).Relaxivityof1-Dismorethan2foldsofthoseof1and
3January2013 magnevistat1.5 T,and1.4foldsofthatof1at3 T.Intracellularcondensationof1infurin-overexpressed
MDA-MB-468cellswasprovenwithdirecttwo-photonlasermicroscopy(TPLM)fluorescenceimagingof
thecellsincubatedwiththeeuropiumanalogof1(i.e.,2).IntracellularGd-NPsof1wereuncoveredand
characterizedforthefirsttime.MRIofMDA-MB-468tumorsshowedthat1hasenhancedMRcontrast
Correspondenceand
withinthetumorsthanthatofitsscrambledcontrol1-Scr.
requestsformaterials
shouldbeaddressedto
L.L.([email protected]. Molecular imaging (MI) is a growing research discipline aims at developing and testing novel tools,
edu.cn)orG.-L.L. reagents,andmethodstodetectuniqueinvivo‘‘biochemicalsignatures’’thatdifferentiateandchar-
acterizetissuesbeyondandbeforetheirgrossanatomicalfeaturesbecomingobvious1,2.Todate,imaging
([email protected])
modalitiesofMIthatmostcommonlyusedforextractingmolecularinformationarenuclear,MRI,andoptical
techniques3. Among them, MRI has become increasingly popular in experimental MI and clinical radiology
because it allows the interrogation of intact, opaque organisms in three dimensions at cellular resolution
(,10 mm)4. About 35% of all clinical MR scans utilize contrast agents (CAs). In proton MR, gadolinium
(Gd31)-basedT CAsareusedforreducingthespin-latticerelaxationtimesofnearbywater,increasingthesignal
1
fromtheseprotons,andmakingtheeffectedvoxelseem‘‘brighter’’inT -weightedimage.Superparamagnetic
1
ironoxidenanoparticle-basedT CAsareusedtoreducethespin-spinrelaxationtimeofwater,makea‘‘negative’’
2
contrasteffectinT */T -weightedimage5.Ingeneral,T -weightedsequencesprovideimagesofhigherresolution
2 2 1
andsignal-to-noiseratiothanT */T -weightedonesandarefreeofimageartifacts.However,duetothelow
2 2
sensitivityoftheCAs,highconcentration(0.1–0.6 mM)ofCAisalwaysrequiredforatypicalMRscanningand
this calls for the design of highly potent molecular CAs for success6,7. Common strategy for increasing the
longitudinalmolarrelaxivity(r )ofT CAistoprolongitsrotationalcorrelationtime(t i.e.,thetumblingtime
1 1 r
oftheCAinthewaterbulk).Toachievethisgoal,Gd-basedagentswithhighermolecularweightssuchasGd
functionalized polymer, peptide amphiphiles or viral caspid, dendrimer, liposomes, nanoparticles, micelles,
zeolites,fullerenes,carbonnanotubes,clays,andquantumdotswerepreparedandexplored8–12.Nevertheless,
these pre-made gadolinium complexes are facing the problem of cell membrane translocation and targeting,
besidesthedifficultyandreproducibilityoftheirfabrications13.Therefore,designof‘‘smart’’or‘‘activable’’MR
CAsthatmodulatetheirMRproperties(e.g.,relaxivities)onsiteuponmoleculartargetinteractionwillovercome
theshortcomingsofMRIfrombottomup.Unfortunately,uptodate,onlyafewgadolinium-basedsmartMR
probeshavebeendeveloped,includingthoseresponsivetob-galactosidaseormyeloperoxidase4,14–17.Self-assem-
bly,aprevalentandimportantprocessinnature18,providesaneasyapproachtodesign‘‘smart’’MRprobes.In
brief,itisnotdifficultforasmallmolecularprobe(i.e.,buildingblockforself-assembly)toovercomethebarrierof
cellmembraneandbedeliveredtothetargetingsiteinsidecell.Atthetargetingsite,thebuildingblocks(i.e.,small
molecular probes) ‘‘smartly’’ start to self-assemble into nano/micro structures withhigher molecular weights
whicharecrucialforaT CA.Recently,Raoandco-workersdevelopedabiocompatiblecondensationreaction
1
between1,2-aminothiolgroupofcysteineandthecyanogroupof2-cyanobenzothiazole(CBT)whichcouldbe
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controlledbypH,reductionandproteaseatsuchlowaconcentration furinofferspeoplewithausefulhintofearlydevelopmentofcertain
asmicromolarforself-assemblingnanoparticleswithdiametersran- cancers.Thereisonebigadvantageforchemiststostudyfurinthatit
gingfrom8 nmto170 nminvitroandincells19.Usingthissystem, preferentially cleaves Arg-X-Lys/Arg-Arg#X motifs, where Arg is
Raoandco-workershavesuccessfullydevelopedthesmartMRCAs arginine,Lysislysine,Xcanbeanyaminoacidresidueand#indi-
of first generation which are susceptive to reducing agents (e.g., catesthecleavagesite24.
glutathione in cells) and have enhanced T relaxivity more than Inspiredbythese,asshowninFig.1,wedesignedAcetyl-Arg-Val-
1
100%20.Inspiredbythis,hereinwedesignedthesecondgeneration Arg-Arg-Cys(StBu)-Lys(Gd-DOTA)-CBT (1) for self-assembling
ofsmartmolecularMRICAswhicharenotonlyresponsivetointra- gadolinium nanoparticles (Gd-NPs) under the action of furin in
cellularglutathione(GSH)butalsocleavablebyintracellularprote- living tumor cells. In brief, 1 contains a RVRR peptide sequence
asefurinwhoisoverexpressedincancercells.UsingthissmartCA, forfurincleavageandcellmembranetranslocation,disulfidedCys
wesuccessfullyachievedenhancedMRIofMDA-MB-468tumorson forsupplyingthe1,2-aminothiolgroupforcondensation,Lyscon-
nudemiceundercommonclinicalfieldstrength(3Tesla). jugatedwithGd-DOTAforMRI.Afterenteringcells,thedisulfide
Thetrans-Golgiproteasefurinisakindofproteinconvertasethat bondoftheCysmotifof1isreducedbytheintracellularGSHand
playsimportantrolesinhomeostasis,andindiseasesrangingfrom subsequentlyitsRVRRmotifiscleavedbyfurinonthesiteofthis
Alzheimer’sdiseasetoanthraxandEbolafeverandcancer21.Several enzyme(i.e.,Golgibody),resultingintheactiveintermediate1-Core.
cancersupregulatefurin,includingnon-small-celllungcarcinomas, Two1-Corescondensequicklytoyieldtheamphiphilicdimer(i.e.,
squamous-cellcarcinomasoftheheadandneck,andglioblastomas22. 1-D)whichhasahydrophobicmacrocycliccoreforself-assembling
Moreover,theincreaseoffurinintumorscorrelateswithanincrease Gd-NPs via p-p stacking among each others. As-formed Gd-NPs
ofmembranetype1-matrixmetalloproteinase(MT1-MMP),oneof shouldgreatlyincreasethelocalconcentrationofGdinsidecellson
furin’ssubstrates.MT1-MMPactivatesextracellularpro-MMP2to onehand.Ontheotherhand,thehighermolecularweightofthe1-D
inducerapidtumorgrowthandmetastasis23.Thus,overexpressionof shouldhaverelaxivityenhancementcomparedwiththatof1atan
Figure1|Shematicillustrationofafurin-controlledcondensationandself-assemblyofGd-NPsincancercells.Afterenteringcancercells,thedisulfide
bondofprobe1isreducedbyGSHandtheRVRRpeptidesequenceiscleavedbyfurintoyieldtheactiveintermediate1-Core.Two1-Corescondenseto
yieldamphiphilicdimer1-Dwhichself-assemblesintoGd-NPsatornearthelocationsoffurinincells(i.e.,Golgibodies).
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identicalGdconcentration,presumablyowingtoanincreasedrota- Furin-controlledcondensationof1andself-assemblyofGd-NPs,
tional correlation time. In this work, we demonstrated the furin- andnanocharacterizations.Totestourhypothesis,weused1forin
controlledcondensationof1andself-assemblyofGd-NPsinvitro, vitrostudy.AsshowninFig.3a,after17 hincubationof1at100 mM
measuredtheenhancedrelaxivitiesofthecondensationproductof1 and30uCwith1 nmol/Uoffurin,wedirectlyinjectedtheincubation
(i.e.,1-D),directlyvisualizedtheintracellularcondensationof2with mixture into a HPLC system and collected the peaks for matrix-
TPLMcellimaging,andcharacterizedtheGd-NPsof1insidecells assisted laser desorption/ionization (MALDI) mass spectroscopic
forthefirsttime.WealsosuccessfullyimagedMDA-MB-468tumors analysis. Interestingly, peaks onHPLC tracesat retention times of
on nude mice with this second generation of smart molecular CA 38.2 min(1-D-1,19.8%),39.5 min(1-D-2,21.2%),40.5 min(1-D-2,
(i.e.,1). 20.0%), and 42.8 min (1-D-4, 13.5%) share an identical molecular
weightandwereidentifiedasthecondensationproductsof1(i.e.,1-
D,SupplementaryFig.S1).OwingtothepresenceofL-lysine,these
Results fourpeaksprobablyrepresentthefourdiastereoisomersof1-Dthat
arise from two different ring-closing orientations during the
Synthesis.Webeganthestudywiththesynthesisandpreparation
condensation between L-cysteine motif and cyano group of 125.
of five compounds: 1 and 1-Scr, 2 and 2-Scr, and 1-D (Fig. 2 &
Thesefourdimersaccountfor74.5%oftheenzymaticproductsof
Supplementary information). The synthesis for these five com-
1 in total. UV-Vis spectrum at 500–700 nm of the above reaction
pounds is simple and straightforward (Supplementary infor-
mixture showedan obvious increase ofabsorption compared with
mation). Briefly, the Ac-Arg-Val-Arg-Arg-Cys(StBu)-Lys-OH
that of the solution without furin, suggesting the formation of
(A) peptide sequence with protection groups was synthesized
nanostrcutures (Supplementary Fig. S2). Directly taking the above
with Solid Phase Peptide Synthesis (SPPS), then coupled with
dispersion for scanning electron microscope (SEM) and trans-
CBT, purified with high performance liquid chromatography
mission electron microscope (TEM) observation, we uncovered
(HPLC) to yield B. Deprotection of B yields C after HPLC
the 3-dimensional and 2-dimensional depositions of the Gd-NPs
purification. Coupling of C with DOTA(OtBu) yields D and
3 of 1 (Fig. 3b&c). The Gd-NPs have uniform spherical shapes and
subsequent deprotection of D with trifluoroacetic acid (TFA)
anaveragediameterof57.1611.9 nm.
yields E. At pH value of 6–7, 10 equiv. of GdCl ?6H O chelates
3 2
with E at room temperature (RT) for 3 h yields 1 after HPLC Measurement of longitudinal molar relaxivity (r ). The MR
1
purification. Synthesis of 1-Scr is similar to that of 1 with A contrast properties of 1, 1-Scr, and 1-D were evaluated in vitro
used for the synthesis of 1 being replaced by peptide sequence together with commercial CA Gd-DTPA (Magnevist) as control
Ac-Arg-Lys-Arg-Cys(StBu)-Arg-Val-OH (F). Syntheses of 2 and using phosphate buffered phantoms (pH 7.4, 0.2 M). T -weighted
1
2-Scraresimilartothoseof1and1-ScrwithGdCl3?6H2Ousedat MRphantomimagingoftheseCAswasconductedonboth1.5Tesla
the last steps for the syntheses of 1 or 1-Scr being replaced with (T)and3TMRIscanners(SupplementaryFig.S3–12).Plotsofsignal
EuCl ?6H Orespectively.Followingtheliterature,wesynthesized intensityversusinversiontimegivetheT relaxationtimesofeach
3 2 1
NH -Cys(SEt)-Lys(Gd-DOTA)-CBT (K)20. Reduction of K with CAsatcertainconcentrations.Wecalculatedthelongitudinalmolar
2
4 equiv. of tris(2-carboxyethyl)phosphine (TCEP) yields 1-D relaxivity(r )ofeachCAaccordingtotheequationr 5gR /[CA],
1 1 1
after HPLC purification. wheretherelaxationrateR is1/T .AsshowninFig.4a,at1.5 Tand
1 1
Figure2|Chemicalstructuresofthefivedesignedprobes. 1,1-Scr,and1-DareGd-basedT MRCAs.1issusceptivetofurin,while1-Disthe
1
condensationproductof1afterfurincleavage.1-Scristhescrambledcontrolprobeof1.2istheEuanalogof1forTPLMcellimaging.2-ScristheEu
analogof1-Scr.
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Figure3|Characterizationsoffurin-controlledcondensationandself-assemblyofGd-NPsof1invitro. (a)Upper,HPLCtraceof1inwater;lower,
HPLCtraceoftheincubationmixtureof1at100mMafterincubationwith1nmol/Uoffurinat30uCfor17h.(b)SEMand(c)TEMimagesoftheGd-
NPsof1intheaboveincubationmixture.
RT,ther sweredeterminedtobe6.00 s21mM21for1,7.42 s21mM21 thanthoseat1.5 T.Allthedataoftheirrelaxivitiesat1.5 Tor3 T
1
for 1-Scr, and 13.24 s21mM21 for 1-D respectively. Relaxivities of aresummarizedinTable1.
Magnevist at this condition were determined to be 5.39 s21mM21,
whichagreewellwiththosereportedinliterature26.Therelaxivityof Direct imaging furin-controlled intracellular condensation of 2
1-D at 1.5 T (13.24 s21mM21) is 2-fold more than that of 1, and withTPLM.AstheEu-analogof1,2hasallthenecessariesforfurin-
comparabletothoseprotein-boundGd-DOTAanaloguesreported controlled intracellular condensation. Luminescence of europium
by Caravan et al27. Measurement of the relaxivities of abovemen- enablesthisintracellularprocessof2visibleunderaTPLM.Before
tioned MR CAs at 3 T is shown in Fig. 4b. Similar to those at applying 2 or 2-Scr for TPLM cellular imaging, we tested the
1.5 T, 1-D has the highest relaxivity and that of Magnevist is the expression level of furin in human breast cancer cell line MDA-
lowest. In general, relaxivities of these MR CAs at 3 T are lower MB-468 with western blot and immunofluorescence staining.
Figure4|T relaxivitymeasurementsof1,1-Scrand1-D.Spin-lattice1/T relaxationratesof1,1-Scr,and1-Datdifferentconcentrationsinphosphate
1 1
buffer(pH7.4,0.2M)at1.5T(a)and3T(b),comparedtothecommerciallyavailableMRCA(Magnevist).Relaxivityratesr wereobtainedby
1
comparingthemeasured(symbols)andtheoretical(lines)values.
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furin while very weak signal of furin was detected in LoVo cells.
Table1|T relaxivities(r ,s21mM21)ofcontrastagentsstudiedat
1 1 QuantificationofthewesternblotsignalswithimageJ(NIH,USA)
RTanddifferentfieldstrengths
indicatedthatfurininMDA-MB-468cellhasanexpressionlevelof
96.2%ofGAPDHwhilethatinLoVois24.5%ofGAPDH(4-fold,
MRfieldstrength[T] 1 1-D Change[%] 1-Scr Magnevist
p50.008)(Fig.5b).HighexpressionoffurininMDA-MB-468cells
1.5 6.00 13.24 121 7.42 5.39 wasalsoconfirmedwithimmunofluorescencestainingoffurinusing
3 5.48 7.68 40 7.00 4.10
rhodamine-labelledsecondaryantibody(Fig.5c).Anoverlayofthe
fluorescence staining of furin with that of nucleus staining (4’,6-
HumancoloncarcinomaLoVocellswerechosenascontrolcelllines diamidino-2-phenylindole, DAPI staining) clearly shows that the
for western blot study because they are reported to be furin- locations of furin (i.e., the Golgi bodies) as reported (Fig. 5d)21.
deficient28. The protein cell lysates prepared from these two cell TPLMimagingofMDA-MB-468cellsincubatedwith2at100 mM
lines were analyzed with western blot. Using glycolytic enzyme for8 hshowsstrongfluorescencesignalssimilartothoseinFig.5c
glyceraldehydesphosphatedehydrogenase(GAPDH)ascontrol,as (Fig. 5e & Supplementary Movie S1), suggesting 2 was under the
showninFig.5a,MDA-MB-468cellsrevealedapositivesignalfor action of furin and trapped at/near the locations of furin.
Figure5|ExpressionoffurininMDA-MB-468cellsandtwo-photonlasermicroscopyimagesofMDA-MB-468cellsincubatedwith2or2-Scr.
Westernblotanalysis(a)andquantification(b)offurininMDA-MB-468cellsandLoVocells.FurinwashighlyexpressedinMDA-MB-468cells(96.2%
ofGAPDH)whileinLoVocellsitwaslessexpressed(24.5%ofGAPDH,4-fold,p50.008).(c,d)ImmunofluorescencestainingofMDA-MB-468cells
withrhodamine-labeledantibodyagainstfurin:DsRedchannel(red,furin)(c);mergedimagewithDAPI(blue,nucleus)(d).Scalebar:20mm.(e,f)
TPLMimages(lex5725nm,lem5565–636nm)ofMDA-MB-468cellsincubatedwith2(e)or2-scr(f)at100mMfor8handthenrinsedandfixed
priortoimaging.Scalebar:20mm.
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Interestingly,TPLMimagingofMDA-MB-468cellsincubatedwith injections of 1 (1st injection: 0.15 mmol/kg at 0 min; 2nd injection:
2-Scr at same condition only exhibits uniform, weak fluorescence 0.15 mmol/kgat50 min)(Fig.7a&bandSupplementaryFig.S16).In
signals(Fig.5f). theprecontrastimages(i.e.,0 min),therewaslittleintrinsiccontrast
between the implanted MDA-MB-468 tumors and surrounding
Self-assembly of Gd-NPs of 1 in MDA-MB-468 cells. After muscle. At 50 min following the administration of the 1st dose of
validation of the intracellular condensation of 2 upon furin 1, significantly increased enhancement was observed within the
cleavageinMDA-MB-468cells,weusedelectronmicroscope(EM) MDA-MB-468 tumors (49.1% increase of grey value compared to
to localize the Gd-NPs self-assembled from the condensation thatat0 min).By90 min(i.e.,40 minafterthe2nddoseofinjection),
products of 1 incubated with the cells. Cells treated with 1-Scr slightlyenhancedsignaltothatat50 mincouldbeobserved(53.9%
were studied in parallel because 1-Scr is inactive to furin. Before increaseofgreyvaluecomparedtothatat0 min),andby240 min
EM observation, furin protein expression in the cells was the signal within the tumors remained obvious (20.5% increase of
quantified with western blot after 8 h incubation of the cells with greyvaluecomparedtothatat0 min,SupplementaryFig.S16).To
theprobes.Unexpectedly,asshowninFig.6a,after8 hincubation assessspecificityof1,controlexperimentswereperformedbytwoi.v.
with1 at 100 mM, furin protein expression in MDA-MB-468 cells injectionsof1-ScrintomicewithsubcutaneousMDA-MB-468cell
exhibits an obvious decrease compared with that in the cells xenograftsattheexactlysamedosesandtimepointstothoseof1.In
untreated.Quantificationofthewesternblotsignalsindicatedthat these animals, enhancement in contrast signal within tumors was
furinproteinincellstreatedwith1hasanexpressionlevelof41.7%of also observed. However, at each of the time points studied, the
GAPDHwhilethatincellsuntreatedis93.9%ofGAPDH(2.3-fold, signalwasclearlylowerthanthatofmiceinjectedwith1(Fig.7a).
p50.001)(Fig.6b).Incontrast,cellstreatedwith1-Scrdidnotshow QuantitativeanalysisoftheMRimagesispresentedinFig.7b.These
decreased expression level of furin protein, compared with that in resultsindicatedthat1isobviouslybetterthan1-ScrasaMRCAfor
cellsuntreated(SupplementaryFig.S13).After8 hincubationwith1 imagingMDA-MB-468tumors,probablythat1issusceptivetofurin
or 1-Scr, the cells were fixed with 2.5% glutaraldehyde and thin while1-Scrisnot.SincefurinproteinlevelsweredecreasedinMDA-
sections of cells were cut and mounted on copper grids for EM MB-468cellsinvitrotreatedwith1(Fig.6a&b),wealsoquantified
observation.ForMDA-MB-468cellsthattreatedwith1,largearea furin protein expression in MDA-MB-468 tumors in vivo with
of clustered Gd-NPs at/near the sites of Golgi bodies were clearly westernblotafterMRI.AsshowninFig.7c,after240 minofMRI,
observed (Fig. 6c). High magnification of the EM image indicated expression level of furin in MDA-MB-468 tumors treated with 1
thattheseintracellularGd-NPsof1haveanaveragediameterof24.0 exhibited an obvious decrease compared with that in control
6 2.3 nm (Fig. 6d), much smaller than those formed in vitro groups. In contrast, expression of furin in tumors treated with 1-
(Fig. 3b). In contrast, there are no Gd-NPs presented in MDA- Scr did not show obvious change compared with that in control
MB-468 cells treated with or without 1-Scr (Supplementary Fig. groups. Quantification of the western blot signals indicated that
S14&15). furin in tumors of mice injected with 1 has an expression level of
54.8%ofGAPDHwhilethatintumorsofmiceuntreatedis90.9%of
MRI of MDA-MB-468 tumors with 1. Having shown that 1 GAPDH (1.7-fold, p 5 0.002) (Fig. 7d). Furin in tumors of mice
selectively condenses and self-assembles into Gd-NPs in furin- injected with 1-Scr has an expression level of 106.3% of GAPDH,
overexpressed MDA-MB-468 cells, coronal MR images of mice noobviousdifferencefromthatintumorsofmiceuntreated(p5
with subcutaneous MDA-MB-468 cell xenografts were acquired 0.08). To directly observe furin expression levels, after 240 min of
precontrast and at various times after the first intravenous (i.v.) MRI,thetumorswereexcisedforimmunofluorescencestainingand
Figure6|ExpressionoffurininMDA-MB-468cellsbeforeandafterincubationwith1,andelectronmicroscopyimagesofthecellsafter8h
incubationwith1. Westernblotanalysis(a)andquantification(b)offurinexpressionlevelsinMDA-MB-468cellsbeforeandafterincubationwith1at
100mMfor8h.Expressionoffurinincellstreatedwith1hasanobviousdecrease(41.7%ofGAPDH),comparedwiththatincellsuntreated(93.9%of
GAPDH,2.3-fold,p50.001).Low(c)andhigh(d)magnificationElectronmicroscopicimagesofMDA-MB-468cellsafterincubationwith1at100mM
for8h.LargeareaofclusteredGd-NPsof1werefoundat/nearGolgibodies.Scalebarinc:2mm.Scalebarind:400nm.
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Figure7|InvivoimagingMDA-MB-468tumorswith1. (a)RepresentativecoronalMRimagesofmicewithsubcutaneouslyxenograftedMDA-MB-
468tumorsat0min,50min,and90minaftertwointravenousinjectionsof1(upper)or1-Scr(lower)viatailveins(1stinjection:0.15mmol/kgat
0min;2ndinjection:0.15mmol/kgat50min).Tumorsareindicatedbyarrows.(b)QuantitativeanalysisofgreyvaluesoftumorMRimagesatvarious
time.(c)Westernblotanalysisand(d)quantificationoffurinexpressioninMDA-MB-468tumorsonmiceinjectedwith1,with/without1-Scrafter
240minofMRI.Furinproteinintumorsofmiceinjectedwith1hasadecreasedexpressionlevelof54.8%ofGAPDH,comparedwiththatintumorson
miceuntreated(90.9%ofGAPDH,1.7-fold,p50.002).Expressionoffurinproteinintumorsofmiceinjectedwith1-Scrdoesnotshowobviouschange
(106.3%ofGAPDH),comparedwiththatintumorsonmiceuntreated(p50.08).(e)Immunofluorescencestainingimagesoftumorsonmiceuntreated
(Control),injectedwith1or1-Scr.FurinisstainedredandnucleusesarestainedbluewithDAPI.Scalebar:20mm.
imaging. The results are shown in Fig. 7e. Unlike those of mice treatedwith1or1-Scrdidnotshowpathologicchangescompared
injected with or without 1-Scr which show connective red withthoseofmiceuntreated,suggestingthatthedosesof1or1-Scr
fluorescence signal (i.e., staining of furin) among the nucleuses hereininjectedforMRIdidnotresultintoxicitytothemicewithin
(DAPI staining), tumor sections of mice injected with 1 exhibited thetimewindowofimaging(SupplementaryFig.S17).
disconnected,attenuatedredsignal,whichalsoindicatesthedown
regulation of furin protein level by the probe. Although the furin Discussion
proteinitselfhaslowerexpressioninmiceinjectedwith1,ICP-MS 1-Scrisanisomerof1butwithascrambledpeptidesequencewhich
analysisindicatedthatat240 minafterthefirstinjectionof1,tumors could not be cleaved by furin. Compound 2 is the europium (Eu)
onmicehaveanaverageGdcontentof0.12 mg/g,2.6-foldhighof analog of 1. We designed 2 to evaluate the cell permeability and
thatoftumorstreatedwith1-Scr(0.046 mg/g)(SupplementaryTable demonstratetheintracellularcondensationof1bydirectlyimaging
S1),suggestingthat1isaverypotentprobeforimagingMDA-MB- the intracellular behavior of 2 with TPLM because 2 has lumin-
468 tumors in vivo. Other organs (lung, liver, spleen, and kidney) escence emissions at 594 nm and 616 nm when excited with two-
except brain in mice treated with 1, all exhibit higher contents of photonexcitationat725 nm(SupplementaryFig.S18).Inparallel,
Gd than those treated with 1-Scr (Supplementary Table S1). we synthesized 2-Scr to mimic the intracellular behavior of 1-Scr
Hematoxylin and eosin (HE) staining of the tissue slices of mice with TPLM imaging. Since the majorities of the condensation
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productsof1uponfurincleavageare1-Ds,wealsosynthesized1-D Compared with that of its scrambled control probe 1-Scr, 1
forinvitrostudy. showed enhanced MR contrast within MDA-MB-468 tumors.
Invitrorelaxivitymeasurementresultsindicatedthatboth1and Immunofluorescencestainingofthetumorsindicatedthatitisfurin
1-ScrhaverelaxivitieshigherthanthatofMagnevist.Thisprobably to trap 1 in tumors. Encouraged by these exciting results above,
duestothatthemolecularweightof1or1-Scr(1645Da)ishigher we envisioned that more ‘‘smart’’ probes based onother proteases
thanthatofMagnevist(662Da).Interestingly,although1-Dhasa overexpressedintumorscouldalsobeinventedfortumorimaging,
molecularweight(1860Da)closetothatof1,itsr is2-foldmore usingthisversatilecondensationplatformorother‘‘clickchemistry’’
1
higherthanthatof1,near2-foldofthatof1-Scrandnear3-foldof techniques31.
thatofMagnevist.Thismightbeascribedtothatthehydrophobic
macrocyclicringof1-Dincreasesitsrotationalcorrelationtime(t)
r Methods
inaqueoussolution,suggestingthat1isa‘‘smart’’MRcontrastagent
Generalmethods.AllthestartingmaterialswereobtainedfromAdamasorSangon
susceptivetofurin. Biotech.Commerciallyavailablereagentswereusedwithoutfurtherpurification,
Since2and2-Scraretheeuropiumanalogsof1and1-Scrrespect- unlessnotedotherwise.Allotherchemicalswerereagentgradeorbetter.Furinwas
ively,theirintracellularbehaviorshouldrepresentthatof1or1-Scr purchasedfromBiolabs(2,000UmL21);oneunit(U)isdefinedastheamountoffurin
thatreleases1pmolofmethylcoumarinamide(MCA)fromthefluorogenicpeptide
accordingly. For 2-Scr, it is the diastereoisomer of 2. Therefore, it
BOC-RVRR-AMC(Bachem)inoneminuteat30uC.1HNMRspectrawereobtained
should have similar cell permeability to that of 2, proven by the ona300MHzBrukerAV300.MALDI-TOF/TOFmassspectrawereobtainedona
TPLM imaging of 2 and 2-Scr on furin-deficient LoVo cells time-of-flightUltrflexIImassspectrometer(BrukerDaltonics),HPLCanalyseswere
(Supplementary Fig. S19). The difference between the two TPLM performedonanAgilent1200HPLCsystemequippedwithaG1322Apumpandin-
linediodearrayUVdetectorusingaYMC-PackODS-AMcolumnwithCHOH
images(i.e.,Fig.5e&f)suggeststhat2couldbeintracellularlycleaved 3
(0.1%ofTFA)andwater(0.1%ofTFA)astheeluent.SEMimageswereobtainedon
byfurinandcondenseatthesitesoffurinwhile2-Scrcouldnotbe JEOL-JSM-6700Felectronmicroscopeatanacceleratingvoltageof5.0KV.TEM
trappedinsidethecellsbecause2-Scrisnotsusceptibletofurin.To imageswereobtainedonaJEOL2010electronmicroscope,operatingat100KV.The
our best of knowledge, caspases and cathepsins are also able to cryo-driedsampleswerepreparedasfollowing:acoppergridcoatedwithcarbonwas
dippedintothesuspensionsolventandplacedintoavial,whichwasplungedinto
hydrolyzeapeptidesubstratetoyieldaN-terminalcysteinemotif.
liquidnitrogenuntilnobubbleswereapparent.Thenwaterwasremovedfromthe
However,theyhavetheirownspecificsubstratesforcleavageinstead
frozenspecimenbyafreeze-drier.ICP-AESmeasurementswereconductedonan
ofRVRR(e.g.,DEVDforcaspase-3andZVKMforcathepsinB)29,30. ICP-96BmachineequippedwithaPGS-2atomicemissionspectrometer(Zeiss).ICP-
Therefore,thespecificityof1(or2)tofurinforcondensationshould MSmeasurementswereconductedonanXSeries2machine(ThermoFisher
be much higher than those to other proteases. Interestingly, the Scientific).
intracellularGd-NPsobservedinsidethecellsafterincubationwith
Cellculture.MDA-MB-468humanbreastadenocarcinomaepithelialcellsand
1aremuchsmallerthanthoseobtainedviainvitroincubationof1
HumancoloncarcinomaLoVocellswereculturedinDulbecco’smodifiedeagle
withfurin(24.0 nmvs.57.1 nm).Wesuspectedthatthismightdue medium(GIBCO)supplementedwith10%fetalbovineserum(FBS,GIBCO).
tohighviscosityofcytosolandtheintracellularfibrousnetworksof
thecellshinderingthesmallGd-NPsfromfurtheraggregation. Twophotonlasermicroscopy.MDA-MB-468orLoVocellswereculturedonthe
PreliminaryresultsofinvivoimagingMDA-MB-468tumorswith glassslide,incubatedwith2or2-scrat100mMfor8h,washedwithphosphate
bufferedsaline(PBS)forthreetimes,fixedwith4%paraformaldehydeatRTfor
1wereobtained.MRIresultsindicatethat1isobviouslybetterthan
30min,washedwithPBSafurtherthreetimesandoncewithdistilledwater.Thenthe
1-ScrforMDA-MB-468tumorimagingeveninthemicewithlower cellsweremountedwith50%glycerolandimagedunderaZeiss710confocallaser-
expressionoffurinprotein,whichfurthersuggeststhat1isapower- scanningmicroscopeequippedwithaCoherentMrux1titanium:sapphiremode-
fulprobe.ThebigdifferenceofGdcontentsintheorgans(lung,liver, lockedlaser.TheexcitationwavelengthforTPLMwas725nm(23362.5nm5
725nm).A565–636nmbandpassfilterwasusedforcellimaging.
spleen,andkidney)exceptbrainbetweenthemicetreatedwith1and
1-Scrat240 min(SupplementaryTableS1)mightbeascribedtothe
Electronmicroscopicimaging.MDA-MB-468cellswereincubatedwith1or1-scrat
structuraldifferencebetween1and1-Scr.Detailedly,cleavageofthe 100mMfor8h,washedforthreetimeswithphosphate-bufferedsaline(PBS),fixed
amidebondbetweentheCysandArgmotifsof1bytheproteinase with2.5%glutaraldehydeatRTfor30min.Thecellswerethendetachedfromculture
(furinorotherproteinases)intheseorgansresultsincondensation dishes,centrifuged(300rpm,15min)andwashedwithPBSforafurtherthreetimes,
andthenstainedwith1%OsO indouble-distilledwaterfor1.5h.Thenthecellswere
reactionandformationoftheGd-NPswhichtraptheagentinthe 4
dehydratedinethanolandembeddedinEpon.Thinsections(80nm)werecutand
tissues/organs.Incontrast,eventheamidebondbetweentheCysand mountedoncoppergrids,stainedwithsaturatedsolutionofuranylacetateandlead
Arg motifs of 1-Scr is cleaved by the proteinase resulting in the citrateforelectronmicroscopeobservation.
condensationandformationofnanoparticles,theGd-DOTAmotif
of1-Scrisexcludedfromneitherthecondensationreactionnorthe InvitroandinvivoMRI.TheinvitrophantomMRexperimentswereperformedon
a1.5T(Simens,Magnetom-essenza)and3T(Simens,Trio-Tim)scanners,usinga
nanoparticle formation thereafter. Thus, it is conceivable that the
headRFcoil.Thescanningprocedurebeganwithalocalizerandthenconsistedofa
Gd-DOTAmotifcleavedfrom1-Scrshouldhaveasmallermolecular seriesofinversion-preparedfastspinechoimages,identicalinallaspects(TR
weight and be excreted from the body within a very short time 1740ms,TE13,BW140kHz,percentphasefieldofview50,slicethickness3mm,
window(theplasmahalf-lifeforGd-DOTAis90 mininpatientwith matrix1363136,NEX1)exceptfortheinversiontime(TI)whichwasvariedas
follows:1500,1200,1000,800,500,400,200,150,100,and75ms.Signalintensity(SI)
normalrenalfunction).HEstainingresultsindicatethattheMRCAs
versusTIrelationshipswerefittothefollowingexponentialT decaymodelbynon-
designedinourworkarebiocompatible,suggestingthat1couldbe linearleastsquaresregression:SI(TI)5A1*exp(2TI/T)1S1I(0).Relaxationrates
1
likelydevelopedforclinicaltrialinthenearfuture. (R)weredeterminedas1/T.Longitudinalmolarrelaxivities(r,unitsofs21mM21)
1 1 1
Insummary,takingadvantageofabiocompatiblecondensation, werecalculatedastheslopeofR1vs[CA]afterthedeterminationoftrueGd
concentrationofeachsamplebytheICP-AESorICP-MSmeasurement.Theinvivo
wehavesuccessfullydevelopedthesecondgenerationofnewsmart
MRimagingofMDA-MB-468tumorxenograftednudemicewasperformedon3T
Gd-basedMRcontrastagent(i.e.,1)forimaginginvitroandinvivo. scanner(Simens,TrioTim),usingheadRFcoil.Femalemiceof3–4weeksoldwere
1 is susceptive to furin, a protease overexpressed in tumor. Upon providedbySunYat-senUniversityLaboratoryAnimalCenter(Guangzhou,China).
furincleavage,1condensestoformamphiphilicdimer(1-D)andthe MDA-MB-468tumorlesionswereestablishedbysubcutaneousdorsalflankinjection
of43107tumorcellsin100mLPBSforeachmouse.Visibletumorswerenormally
latter self-assembles into Gd-NPs thereafter. Relaxivity of 1-D is
observed2–4weeksafterinjection.Thetumor-xenograftedmicewerethensubjected
morethan2-foldofthatof1.Usingtheeuropiumanalogof1(i.e., randomlyintotwogroupsfortailveininjectionsof1or1-scr(1stinjection:
2),wedirectlyimagedtheintracellularcondensationof2infurin- 0.15mmol/kgat0min;2ndinjection:0.15mmol/kgat50min).Theimageswere
overexpressedMDA-MB-468cells.Byincubatingthecellswith1for takenatatimesequencefrom0minto240minusingT1-weightedMRacquisition
sequencewiththefollowingparameters:TR2000ms,TE70,BW289kHz,percent
8 h,forthefirsttimetoourbestofknowledge,weuncoveredand
phasefieldofview60,slicethickness2mm,matrix1443144,NEX1.Theintensity
characterized the intracellular Gd-NPs self-assembled from the
ofMRsignalintumorforeachtestwasdeterminedbystandardregion-of-interest
condensationproductsofthissmallmoleculeuptakenbythecells. measurementwithImageJ.
SCIENTIFICREPORTS |3:1024|DOI:10.1038/srep01024 8
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