Table Of ContentDellingeretal.JournalofCardiovascularMagneticResonance2013,15:7
http://www.jcmr-online.com/content/15/1/7
RESEARCH Open Access
Functionalization of gadolinium metallofullerenes
for detecting atherosclerotic plaque lesions by
cardiovascular magnetic resonance
Anthony Dellinger1,2, John Olson3, Kerry Link3, Stephen Vance2, Marinella G Sandros2, Jijin Yang4,
Zhiguo Zhou1* and Christopher L Kepley2*
Abstract
Background: The hallmark ofatherosclerosis is the accumulationof plaque invessel walls. This process is initiated
when monocytic cells differentiate intomacrophage foam cells under conditions withhighlevels ofatherogenic
lipoproteins. Vulnerable plaque can dislodge,enter theblood stream, and result in acute myocardial infarction and
stroke. Imaging techniques such as cardiovascular magnetic resonance (CMR) provides one strategyto identify
patients withplaque accumulation.
Methods: We synthesized an atherosclerotic-targeting contrast agent (ATCA) in which gadolinium (Gd)-containing
endohedrals were functionalized and formulatedinto liposomes withCD36 ligands intercalated into the lipid
bilayer. In vitroassayswere used to assess the specificity ofthe ATCA for foam cells. The ability of ATCA to detect
atherosclerotic plaque lesions in vivo was assessed using CMR.
Results: The ATCA was able to detectscavenger receptor (CD36)-expressing foam cellsin vitro and were specifically
internalized via the CD36 receptor as determined by focused ion beam/scanning electron microscopy
(FIB-SEM) and Western blotting analysis of CD36 receptor-specific signaling pathways.The ATCAexhibited
time-dependentaccumulationin atherosclerotic plaquelesions of ApoE −/− mice as determined using CMR. No
ATCA accumulation was observed invessels of wild type (C57/b6) controls. Non-targeted control compounds,
without theplaque-targeting moieties, were not taken upby foam cells in vitro and did not bind plaque in vivo.
Importantly, theATCAinjectionwas well tolerated, did notdemonstrate toxicity in vitroor in vivo,and no
accumulation was observed in the major organs.
Conclusions: The ATCAis specifically internalized by CD36 receptors on atherosclerotic plaque providing enhanced
visualization of lesions under physiological conditions. These ATCA may provide new tools for physicians to
non-invasively detect atherosclerotic disease.
Keywords: Atherosclerosis,Magnetic resonance imaging, Metallofullerenes,Contrast agent, Macrophage, CD36
Background neededtoidentifyincipientdisease,monitordiseasepro-
Atherosclerotic cardiovascular disease results in close to gression, and pinpoint factors that predict catastrophic
20 million deaths annually. A hallmark of the disease is ruptures.
the accumulation of plaque lesions in blood vessel walls At present, it is difficult for physicians to specifically
which can rupture and result in acute myocardial infarc- detect and quantify plaque lesion buildup in vessel walls.
tion and stroke. Clearly better diagnostic tools are Cardiovascular magnetic resonance (CMR) is one of sev-
eral techniques being investigated to identifyplaque bur-
*Correspondence:[email protected];[email protected] den in patients so that interventions can be conducted
1LunaInnovationsIncorporated,LunananoWorksDivision,521BridgeSt,
beforeruptureoccurs[1,2].Wehaveusednovelgadolin-
Danville,VA24541,USA
2JointSchoolofNanoscienceandNanoengineering2907ELeeSt, ium (Gd)-containing C80 endohedrals (Trimetaspheres™,
Greensboro,NC27401,USA TMS, Gd N@C ) [3] as a platform to develop new
3 80
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Dellingeretal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
Dellingeretal.JournalofCardiovascularMagneticResonance2013,15:7 Page2of12
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atherosclerotic targeting contrast agents (ATCA) for 100 μg/ml (five times the amount used for in vivo stud-
CMR. TheTMS-based molecules have 25 fold increased ies)for 10days.Nosignificantdifferences incellviability
relaxivity compared to other contrast agents, reduced wasobservedwith ATCA-treatedcellscompared tocon-
risk of metal toxicity, and can be customized to address trols(not shown)whichfurthersupportstheobservation
issues surrounding solubility, specificity, etc. [4]. TMS that derivatized fullerenes are not toxic in vitro [11,12].
were functionalized with highly specific ligands for the Cells were treated with CD36-targeted or non-targeted
scavengerreceptorCD36foundonthesurfaceofmacro- controls atvarious concentrations for24hrs,centrifuged
phage foam cells inplaque lesions [5].Itisdemonstrated and the supernatant saved for Gd analysis. After a quick
that ATCA specifically bind to and are taken up within wash the cell pellet was disrupted by sonication and
foam cells in vitro and are able to detect lesions in supernatant and cell pellet subjected to ICP analysis or
plaque-susceptible mice in vivo (Apolipoprotein E defi- neutron bombardment (both methods gave similar
cient mice [ApoE −/−]). Thus, TMS can be targeted to results; not shown) to determine Gd concentration as
specific biomarkers in sufficient quantities to provide describedbelow.
enhanced imaging of atherosclerotic plaque under
physiological conditions. These results suggest that the CelluptakeoftheATCArevealedbyFIB-SEMcrossbeam
ATCA may be a new tool for detecting atherosclerotic workstation
plaque. In order to examine cell uptake of the ATCA we used a
Zeiss FIB-SEM crossbeam workstation. Foam cells were
Methods incubated with or without ATCA or non-targeted con-
ATCAsynthesis,functionalization,andcharacterization trol (20 μg/ml) for various times (16, 36, 72 hrs). As a
The method to synthesize the TMS uses an electric-arc control non-targeted ATCAwereincubated forthe same
process to encapsulate Gd within a carbonaceous cage, times. In addition, non-foam cells were incubated with
C [3], which was used as the starting material for the ATCA for 24 hrs. Cells were washed and fixed using 1%
80
ATCA. The molecular weight of TMS (1,446 Da) was glutaraldehyde solution and 0.1 M cacodylate buffer for
determined using matrix-assisted laser desorption/ 2.5 hrs at room temperature. Samples were mounted on
ionization (MALDI) with a time-of-flight (TOF) mass a microscope slide covered in aluminum foil and sub-
spectrometer. Elemental analysis determined that TMS jected to graded EtOH incubations (60%-100%/10 min
contains 10.15 ± 0.25 mM Gd which approximates the each) and stored in a desiccated environment until
value estimated from the molecular weight. No free Gd microscopicanalysis.
(Gd outside the carbon cage) was detected using an The mounted samples were put in the FIB-SEM cross-
Arsenazo III colorimetric test [6] and ICP analysis of li- beam workstation, while a focused ion beam (FIB) was
quid after dialysis of theTMS. Further, atomic force mi- used to cut the cells and expose inner structure of the
croscopy (AFM) measurement of the TMS showed the cell. Simultaneously, scanning electron microscope
majority of the nanoparticles are 1.1-1.3 nm in height (SEM) was used to image the cross section milled via
with some particles approaching 3.0 nm to 4.0 nm, sug- FIB, this is capable because both the ion beam and elec-
gesting minimalagglomerationand/oraggregation. tron beam can focus on the exactly same area. The FIB
The ATCA was developed as shown in Additional was operated at 30 kV, 20 pA and SEM was operated at
file 1 in which the TMS is functionalized with hydro- 1 kV to reduce potential beam charge and cell damage. A
philic and lipophilic groups [4] and incorporated into secondaryelectrondetectorwasusedtorevealmorphology
liposomes [7]. The resulting liposome-TMS were formu- and energy. Additionally, the energy and angle selective
lated with or without CD36 ligands intercalated within backscatteredelectron(EsB)detectorwasappliedtodetect
the liposome bilayer membrane [5,8]. The ATCA was ATCA existence and spatial distribution with nanometer
characterized asseeninAdditionalfile2. level resolution inside the cells, since heavy element Gd-
containing C endohedrals will express themselves much
80
Cellculture brighterinEsBimagesifTMSexists.
Foam cells were generated using oxidized-low density
lipoproteins (oxLDL) from human plasma as described WesternblottingandquantificationofCD36-specific
previously [9] and verified using oil red-o (ORO) stain- phospho-signalingintermediates
ing [10]. The upregulation of CD36 expression in foam To confirm the specificity of CD36 ligand binding on
cells was confirmed using both PCR and FACs analysis the ATCA to the CD36 receptor and examine the
with CD36-specific antibodies. As a control, the non mechanisms underlying the interactions between the
CD36-expressing cell line 3 T3-F442A pre-adipocytes ATCA and foam cells, Western blotting was used as
were used to detect non-specific binding. TheU937 cells described previously [13]. Following ATCA challenge,
and foam cells were examined for toxicity using up to foam cells were lysed directly in boiling denaturing
Dellingeretal.JournalofCardiovascularMagneticResonance2013,15:7 Page3of12
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sample buffer consisting of tris-buffered saline with same reference ROI and then the contrast-to-noise ratio
triton-X-100 (0.5%) and protease inhibitors. Proteins (CNR) values were normalized to the pre-CA CNR
wereseparatedona10%gelsintris-glycineSDSrunning value. The quantification of signal intensities were tabu-
buffer. Western blotting and image quantification was lated and analyzed for all variables and expressed as
performed using the Odyssey Imaging System (LI-COR mean ± standard error (SE) from five separate animals
Biosciences). Band intensities were captured using the per group (Figure 1B and Figure 1C). The StatPlus
Odyssey scanner and bands quantified by measuring the (Analyst Soft, 2011) software was used to evaluate a
number ofpixelsineachband usingaboxdrawn for the two (group) × two (time) analysis of variance
same area of measurement for each separate blot. Band (ANOVA) with repeated measures was evaluated to
intensities were normalized for loading by dividing the analyze data. Tukey LSD post hoc tests were used to
number of pixels in each band with the housekeeping examine pair-wise differences. Significance was set at
bandintensity(β-actin) onthesame blot. p < 0.05.
Flowcytometry
InordertoconfirmtheupregulationofCD36expression Histology,immunohistochemistry,andGddetectionin
on foam cells, U937 cells (negative control) or PMA/ tissue
oxLDL-transformed cells (foam cells) were washed with The aorta of each animal was removed and fixed in
PBS/1% BSA, and blocked for 30 min at 4°C with a Tissue-Tek OCT (Miles, Elkhart, IN) and frozen with li-
1/500 dilution of normal human serum. The cells were quid nitrogen. The tissue was sectioned onto siloconized
washed and incubated with FITC-labeled anti-, CD36- slides and histopathology assessed using hematoxylin for
FITC, or FITC-labeled isotype control (BD Biosciences; assessing plaque accumulation [17,18]. To examine
10 μg/mL) for one hr. at 4°C. After three washes, cells CD36 and CD68 expression on macrophages in the
were resuspended in 400 μL of PBS. The mean intensity plaque lesions the tissue sections were fixed in Carnoys,
of fluorescence was determined for at least 10,000 cells embedded in parafilm, and tissue sections placed on
using a FACScan flow cytometer (BD Biosciences). All microscopic slides and probed with antibodies to CD36
experimentswereperformedinduplicate. and CD68 (both from Serotec) followed by horseradish-
conjugated secondary antibodies and developed using
CMRstudy AEC as described previously [19,20]. As a control non-
The lesions in arteries of ApoE −/− mice progress from specific, isotype-matched control antibodies (for CD36
fatty streaks to foam cell-containing plaque in a similar and CD68) were used. Images were captured using a
way as humans [14,15]. ApoE −/− mice (23 weeks; fe- camera-equipped Zeiss AXIO Observer. For tissue dis-
male) and non-diseased controls were anesthetized ini- tribution of Gd, organs (or ATCA sample used to inject
tially with isoflurane (3%) and oxygen (3 L/min) in an to determine total Gd counts) were weighed and placed
induction chamber and kept under constant sedation in pyrex tubes and combusted to ash at 900°C for 1 hr.
via a nose cone while monitoring vital statistics as The samples were again weighed and resuspended in
described [16]. Further details of the CMR are out- 1.0 ml of 12 M (37%) HCl. The Gd was measured
lined in Additional file 3. CMR was performed using ICP and percent Gd in tissue calculated based
blinded by personnel with no knowledge of targeted on the total amount of Gd in the ATCA sample
and non-targeted compounds. All animal studies used for injections.
were approved by Wake Forest School of Medicine
Animal Care and Use Committee. Toxicologyevaluation
ApoE −/− were injected i.v. with PBS or 1,000 μg/100 μl
Signalenhancementmeasurement (10 times more than optimized for imaging studies) of
Images acquired after the ATCA injection were exam- ATCA. Mice (four/group) were sacrificed at days two,
ined for areas of hyper-intensity in the aorta wall seven, and 14 and alanine aminotranferease (ALT) and
(selected at random). As a comparison whole aortas aspartate aminotransferase (AST) levels evaluated in
were analyzed as described in Additional file 3. Regions serum.TheALTandASTaretransaminaseenzymesthat
appearing hyper-intense after ATCA injection were leak out into the general circulation when the liver is
manually traced using ImageJ (nih.org). The contrast to injured. In separate experiments, ATCA were injected as
noise ratio was calculated using a reference region of above, tissue harvested at day seven, and subjected to
interest (ROI) next to the plaque lesion or of the whole neutron bombardment or ICP for Gd quantification. An
aorta. The same regions were traced in the pre-ATCA aliquot of the ATCA (not injected) was measured
injection image and in all the post-ATCA time point separately to determine the percentage cleared from
images. The contrast to noise was calculated using the the mice.
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Figure1(Seelegendonnextpage)
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(Seefigureonpreviouspage.)
Figure1ATCAcandetectinflammatoryplaqueinvivo.ApoE−/−mice(23wks;n=5/grp)wereinjectedi.v.(100μg/100μl)withATCA(top
andmiddle)ornon-targetedcontrol(bottom)andimagesacquiredattheindicatedtimes.Bar=1.0mm.Arrowsindicatecontrastagentbinding
totheplaque.A.Representativeimagesfromtwo(ATCA)orone(control)differentmiceinjectedwithATCA.B,C.Quantificationofsignal
enhancementofATCA.InB,thebrightestvoxels(approximately>10spots/section)intheaortawallfrom5separateanimalsweremeasured
excludingartifact.TheratiooftheSignalIntensity(SI)ofthebrightestvoxelintheaortawalltotheSIofthe(non-affected)myocardiumforeach
timepointwascalculated.InC,theratioofSIofthewholeregionliningtheaortawalltotheSIofthe(non-affected)myocardiumwasmeasured
andtheaveragesignalintensitywasrecordedfor5separateslicesfromeachtime-point.TheSI-enhanced/SI-myoratiofromeachtimepointto
itsprescanSI-enhanced/SI-myoratiowerenormalized(prescanvalues=1).Dataisexpressedasmeanof5CMRslices±SD(n=5;animals/grp;
*indicatessignificance(p<0.05).D.PlaquelesionsexpressmacrophagesandCD36receptors.AortaswerestainedwithH&E(left),ratanti-mouse
CD36(middle),ratanti-mouseCD68(bottom)ornon-specificratIgG(nsIgG).Peroxidase-conjugatedanti-ratAbswereaddedanddeveloped
usingAEC.ThebrownstainingrepresentsCD36-orCD68-positivecellslocatedintheplaquelesions.Resultsarerepresentativeofatleastthree
separateaortas.
Results [22],wehypothesized that this may be a way toincorpor-
Plaquetargetingcontrastagentsareselectivelytakenup ate the ATCA contrast agent into foam cells in sufficient
byfoamcellsthroughCD36receptor enough quantities that CMR could be performed. Foam
Atherosclerotic plaque have higher numbers of macro- cells were induced using PMA/oxLDL and the upregula-
phage foam cells that express CD36 scavenger receptors tion of CD36 expression was confirmed using FACs ana-
ontheircellsurface[21].GiventhattheCD36activelyup- lysis (Figure 2A). As seen in Figure 2B, ATCA had a
take extracellular lipids into their cytoplasmic membrane significant uptake into the CD36-expressing foam cells.
A CD36 surface expression
U937+CD36-FITC
Foam cells
nsIgG-FITC
Foam
cells+
CD36-
FITC
B ATCA Control
600 600
2% 98% 42% 58% 3% 97% 2% 98%
500 500
e
pl 400 400
m
a
M/s 300 300
P
D 200 200
100 100
0 0
P S P S P S P S
Non-foam Foam cells Non-foam Foam cells
cells cells
Figure2A.UpregulationofCD36receptorsonfoamcells.U937cells(induplicate)weretreatedasdescribedinMethods,washedand
incubatedwithantibodiestoCD36oranisotypematchedcontrol.Thegraphshowstheaveragemeanfluorescentintensity(MFI)ofthesignal
(±SD)andisrepresentativeofthreeseparateexperiments.B.TestingATCAforfoamcellbinding.U937cells(non-foam)wereconvertedinto
foamcells(foam)usingoxLDLandPMAasdescribed.CellswerewashedandATCA(10μg/ml)addedovernightinaCO incubator.Thenextday
2
cellswerewashedwithmediaandthesupernatants(S)andpellets(P)subjectedtoICPforGddetection.ThepercentageofGdinthepelletand
supernatantwascalculatedbasedonthetotalamountaddedtothecells.
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The control compound for ATCA without the CD36 tar- compoundscanbedetectedinsidehumanfoamcells.The
geting moiety ligands (control) was not taken up within ATCAisshowntobemorescatteredinsidethecellatearl-
thecellssuggestingtheCD36receptorwasresponsiblefor ier time points (B,D) and begins to aggregate with lesser
the uptakeof the compoundswithin the cells.Indatanot amounts inside the cell at later time points (F) suggesting
shown, there was no uptake in non-foam cell monocytes cell excretion. The non-targeted control was not detected
ornon-monocytic,tissuecells(3T3). inside the foam cells (G, H) further suggesting the specifi-
We next used a FIB-SEM - to follow the ATCA in- cityoftheATCAforfoamcells.
side foam cells and determine the length of time it To definitively establish the specificity of ATCA bind-
remains. Figure3A-3Hdemonstratesthatfullerene-based ing to the CD36 receptor we used Western blotting and
Area of cut
Gd inside cells
A B
1 µm
3 µm
C D
1 µm 1 µm
E F
1 µm
2 µm
H
G
2 µm
1 µm
Figure3A-H.ATCAaretakenupbyfoamcells.FoamcellswereincubatedwithATCA(10μg/ml;A-F)ornon-targetedcontrol(G,H)for16
(A,B),48(C,D)or72(E,F)hrs,washed,fixed,andsubjectedtoFIB-SEM.Leftcolumns(A,C,E,G)showsthemorphologyimagestakenbysecondary
electrondetectorafterfocusedionbeamcutandtherightcolumn(B,D,F,H)showsthecorrespondingmaterialcontrastimagestakenbyenergy
andangleselectivebackscatteredelectron(EsB)detectorforthesameregion.Thefoamcell-targetedATCAareclearlyrevealedinsidethecells
butnotinthenon-targetedcontrols.Inparallel,analiquotofcellswereusedtomeasurecellviabilitybytrypanblueexclusionandrevealedno
celldeathinducedbyATCAadditioncomparedtonon-treatedcontrols(notshown).
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quantification of CD36-associated signaling molecules ATCAdetectatheroscleroticplaqueinvivo
induced by ATCA treatment. Previous studies have We used ATCA to visualize atherosclerotic disease in
shown that Erk, Lyn, and JNK2 are activated by the ApoE −/− mice [14]. As seen in Figure 1A (top and mid-
binding of oxLDL to CD36 receptors on macrophages dle), mice injected i.v. with the ATCA had a striking
[23,24]. When foam cells were challenged with ATCA enhanced T1 image of the plaque attached in the mouse
there was a dose (Figure 4A) and time (Figure 4B) aorta that could not be seen prior to injection. Of note is
dependent activation of these signaling molecules. the observation that the imaging agent accumulates over
This provides definitive evidence that ATCA specific- time, suggesting it circulates through the blood for
ally target foam cells through CD36 receptors. periods long enough for biomarker targeting to occur
Figure4A,B.ATCAactivateCD36-specificsignalingintermediates.FoamcellswereincubatedwithorwithoutvaryingconcentrationsofATCA
(A;doseresponse)for10minorwith10μg/mlATCA(B;timecourse)forvaryingtimes.CellswerelysedandWesternblottedusingphospho-specific
antibodies.Bandintensitieswerequantifiedandthenumbersrepresentthefoldincreaseofeachbandcomparedtothehousekeepinggeneβ-actin
whichdidnotdemonstrateanychangeuponATCAchallenge.Resultsarerepresentativeofthreeseparateexperiments.
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(Supplemental Video). Control compounds without the based molecular imaging nanomaterials and standard
CD36 ligands demonstrated no accumulation in the vessel CMR techniques. Our rationale for developing ATCA
wallsofApoE−/−mice(Figure1A-bottom).Quantification was driven by the need for new ways to diagnose blood
oftheimageintensityofROI’sappearingafterATCAinjec- vessel plaque burden with existing technologies such as
tion (Figure 1B) demonstrates accumulation of the com- CMR. It is well established that atherosclerotic plaque
poundsoccursafter30min;oneandtwohourtime-points express high levels of CD36 receptors for which ligands
demonstrated the optimal imaging time. When measuring have already been identified. Importantly, these recep-
the entire aorta the normalized mean intensity was very tors normally and actively internalize the ligands
similar to what was observed in the bright spot ROI’s (oxLDL); a prerequisite for accumulation of sufficient
(Figure 1C). This quantification clearly demonstrates the levels so that enhanced contrast can be observed. It is
ATCA significantly increases the contrast representing shown that this strategy was successful in identifying
plaquelesions. new ATCA which may provide new tools for physicians
Separate experiments were performed with non- todetectplaquebuildup inpatients.
diseased animals. In these experiments the ATCA and Magnetic resonance imaging is one of many techni-
controlwereinjectedandthedescendingaortaimagedat ques used to assess atherosclerotic plaque lesions in
thesametimepoints.Asseeninthebottompanelswild- patients [25,26]. We functionalized theTMSthrough the
type C57 mice did not demonstrate any signal enhance- addition of CD36 ligands which were hypothesized to
ment in the descending aorta further demonstrating the home to macrophage foam cells found in atherosclerotic
specificity of the ATCA for plaque detection. These plaque [27-30]. Indeed, many studies have used this
results suggest the ATCA specifically detect disease- strategy of targeting diagnostics to macrophages through
induced plaque lesions that are not expressed in normal scavenger receptors as well as other macrophage-
vessel walls. Histochemical evaluation of each animal expressedbiomarkersthroughseveraltechniques includ-
revealed that plaque lesions were present in the diseased ing CMR [27,31-41]. For example, the Fayad group
animals(Figure1D;left)withtheaccumulationofCD36- investigated the ability of macrophage scavenger
and CD68-positive cells on the anterior portion of the receptor-targeted immunomicelles to assess macro-
plaquelesions(Figure1D;middle/bottom). phages in atherosclerotic plaque using CMR in vivo. The
targeted micelles increased thesignalintensity ofathero-
sclerotic aortas 79% in ApoE−/− mice compared with
Trackingtheinvivofateandtoxicityofplaque-targeting
only 34% using untargeted controls [27]. Others have
contrastagents
targeted macrophages through the use of Lyp-1 peptides
In order to determine the fate of the ATCA we per-
attached to a fluorescent dye [34]. Thus, targeting
formed whole body scans after i.v. injection as described
macrophages in atherosclerotic lesions is a valid strategy
above. In allexperiments theinjectionwaswell tolerated
for developing diagnostics capable of detecting and
in each group of mice with no noticeable adverse reac-
quantifying plaque lesions. The advantages of using the
tions observed. As determined using CMR, there was no
CD36 receptor for targeting CMR contrast agents to
accumulation of any of the compounds in any major
plaque is its well established connection in atheroscler-
organs (e.g. liver and kidneys) at the same time points
osis [28,30], its high expression on plaque foam cells
measured intheApoE−/−mice(Figure5A).Toconfirm
[28], and its ability to be internalized upon ligand bind-
these resultswe performedICP analysis onthe organs of
ing [42] which allows the accumulation of the ATCA
these animals after seven days post injection. As seen in
ligand even under physiological flow conditions as was
Table 1 Gd was only detected in the liver and kidneys
shown in Figure 1. The key limitation presently is the
demonstrating that approximately 85% of the ATCA is
lack of a specific biomarker (or biomarkers) for plaque
excretedfromthebodyatthistime-point.
lesions that will not cross-react with other sites in the
In separate experiments in which high concentra-
body. An even more difficult challenge is identifying
tions of ATCA were injected, there was no significant
vulnerableplaquebiomarkers.
increaseinserumactivityofALTandASTbetweenthe
We used the Gd-containing TMS as they provide a
untreated and ATCA-treated animals indicating no
unique starting material for developing biomarker-
livertoxicity(Figure5B).Thus,theATCAdonotaffect
targetingdiagnostics[43].TheTMSoffersseveraladvan-
liver function and are mostly cleared from the body
tages over current Gd-based CMR contrast agents for
withinfivedaysafterinjection.
use as targeted diagnostics. First, the toxic Gd (in cage)
is separated from active targeting moieties (outside cage)
Discussion by the carbon shell. Adding targeting ligands/moieties to
Theresults presented here demonstrate that atheroscler- current contrast agents may affect the ability of Gd to
otic plaque can be detected in vivo using targeted, Gd- becomefree ofthe compound. Ofcourse,furtherstudies
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A
ATCA
lungs
liver
control
kidney
Pre 30 min 60 min 120 min
B Day 2 Day 7
Untreated Treated Untreated Treated
n Activity U/L n Activity U/L n Activity U/L n Activity U/L
Aspartate Amino Transferase (AST) 4 27.5 (±9.2) 4 21.7 (±8.6) 4 20.5 (±3.2) 4 19.7 (±8.6)
Alanine Amino Transferase (ALT) 4 64.2 (±11.5) 4 52.7 (±7.7) 4 68.7 (±11.5) 4 62.9 (±7.7)
n= Number of Mice evaulated in Duplicates
Figure5A.Plaque-targetingcontrastagentsdonotaccumulateinmajororgans.Arepresentativeplaque-targetingcontrastagentand
non-targetedcontrolwasinjectedasaboveandthemouseplacedsupineforwholebodyscan.Resultsarerepresentativeoffourseparatemice.
B.C.ATCAdoesnotaccumulateinliverorinducetoxicity.Miceweretreatedwithorwithout1000μg/100μlofATCAviatailveininjection.Two
andsixdayslatertheserumwasusedforALTandASTlevels.Dataispresentedasanaverageoffour(untreated)orfour(treated)mice±SD.No
increaseinactivitywasobservedbetweentheuntreatedandtreatedsamples.ActivityismeasurebyUnits/Lobtainedusinglinearregression
fromastandardcurve.
are needed for these molecular entities but we and chelates [43]. Targeted imaging agents require strong
others have not observed any toxicity of empty cage or signals by which to report the presence of an agent at a
TMS (Figure 5) using several in vitro and in vivo assays particular location. Third, the fullerene cage can be tar-
[12,44,45]. Second, the TMS are more sensitive than geted to disease biomarkers or encapsulated into lipo-
existing contrast agents with three Gd/molecule com- somes/micelles without compromising release of Gd
pared to one Gd/molecule in commercially available into the body; a strategy already being used for targeting
glioblastoma [46]. The chemistry for the addition of dif-
ferent moieties to the cage offers many possibilities for
Table1TotalpercentGdintissue
not only biomarker targeting of plaque accumulation
Day7(n=4)
butalsodetectionof biomarkersassociatedwithremoval
Average SD
of lesions (e.g. peptidoglycans) so that new tools for
Liver 9% ±6%
evaluative diagnosisofthisdisease can bedeveloped.
Heart ND ND The specificity of the ATCA for targeting foam cells
Spleen ND ND through CD36 is demonstrated three separate ways.
Kidney 3% ±2% First, ICP analysis of Gd within foam cells was con-
firmed in Figure 2B demonstrating that the CD36
Lung ND ND
ligand-decorated ATCA were significantly taken up
TotalGdintissuewasvaluatecomparedtototalGdinjectedND-none
detected. within foam cells while the non-targeted ATCA were
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not. Second, we used FIB-SEM; a powerful technology seven days as demonstrated above. Similarly, the TMS
that can be used to cut the cell open and detect nano- were recently shown to have a favorable toxicity profile
materials, to further show the targeted ATCA is specific- in vitro and in vivo [12]. Since it is anticipated that a
ally internalized within foam cells were levels begin to patient will only be given a single injection before
diminish after 72 hrs. This suggests the cells remain CMR is performed, these results are encouraging for
viableandcancontinuetoexocytose asanormalmacro- future studies in humans. However, more studies are
phage would. Third, we show the targeted ATCA, and clearly needed to examine the long-term effects of
not the non-targeted, specifically activate signal trans- fullerenes on biological systems. Efforts are focused
duction intermediates in the CD36 receptor pathway in- on systematically identifying any acute and long-
cluding the ligand binding-dependent phosphorylation term toxicity issues with ATCA.
ofErk,Lyn,andJnk.Thus,theATCAspecificallydetects
and is taken up by macrophage foam cells though the Conclusions
CD36 receptor. This study describes the synthesis and testing of a new
The use of ApoE −/− mice to study CMR contrast class of ATCA based on a novel metallofullerene nano-
agents has been described extensively [14,15]. We dem- material.Invitroscreeningwasusedtoselecthighrelax-
onstrate that the ATCA specifically recognize athero- ivity compounds that were taken up by foam cells via
sclerotic plaque in diseased animals. No contrast was the scavenger receptor CD36. The CD36 compounds
observed with non-targeted controls or in ATCA-trea- were shown to target inflammatory lesions in ApoE −/−
ted, non-diseased animals. The signal enhancement mice while the same compounds without the CD36
occurs in a time-dependent fashion suggesting the foam ligands did not. These and other similar studies dis-
cells are actively internalizing the ATCA via the CD36 cussed above suggest the rationale for targeting macro-
scavenger receptor. While it has been reported that the phage foam cell may provide physicians with new tools
lesion burden can vary among animals it was demon- for non-invasively diagnosing and quantifying otherwise
strated that each animal tested had high levels of plaque undetectable plaque lesions. These results also suggest
lesions containing macrophages and CD36 receptors as the TMS may serve as a platform to attach other bio-
identified usinghistochemistry andimmunohistochemis- markerstotargetandimagedisease-specificbiomarkers.
try (Figure 1D). While each animal was verified to con-
tain inflammation one limitation of this study was the Additional files
small numbers of animals used. We are in the process of
verifying these results in higher-order mammals (rab-
Additionalfile1:DesignandsynthesisofATCA.
bits). The high power 7 Tscanner used warrants further
Additionalfile2:A.DLS,zetapotential,andrelaxivityofliposome
validation of the ATCA in a more clinically relevant controlandATCA.
scanner. However, the TMS have been studied in lower Additionalfile3:MRIanalysis.
field magnets and demonstrate similar relaxivity and
sensitivity [47,48]. Competinginterests
Investigation of biocompatibility and biodistribution of Allauthorshavedeclarednocompetinginterests.
theTMS-based protein cages in vivo is critical for future
Authors’contributions
clinical translation. To this end, the studies examining AD:substantialcontributionstocollection,analysis,andinterpretationof
the toxicity of fullerenes on human systems are still data,anddrafting/revisionofthemanuscript.JO:substantialcontributionsto
acquisitionandinterpretationofMRIcontrastdata.KL:substantial
emerging and the subject of debate [44,49]. Several
contributionstoacquisitionandinterpretationofMRIcontrastdata.SV:
in vivo publications suggest that (in general) fullerenes providedsubstantialcontributionstointerpretationandcharacterizationof
show no overt signs of toxicity up to 2000 mg/kg [50,51] TMS.MS:providedsubstantialcontributionstointerpretationand
characterizationofTMS.JY:substantialcontributionstoacquisitionand
and have been shown to extend the lives (11%) and im-
interpretationofmicroscopicdata.ZZ:substantialcontributionstodesign
prove cognitivefunction ofmice chronically treated with andsynthesisoftheatherosclerotic-targetingcontrastagent,andrevisionof
these compounds [52]. More recently, rats given fuller- themanuscript.CK:substantialcontributionstodesignofstudy,
interpretationofdata,anddrafting/revisionofmanuscript.Allauthorsread
enes in olive oil lived over 90% longer than control mice
andapprovedthefinalmanuscript.
[53]. We demonstrated that a single dose of ATCA,
given at a 100 fold concentration higher than that Acknowledgements
needed for plaque imaging in vivo, caused no overt signs ThisworkwassupportedinpartbyWakeForest’sCenterforBiomolecular
Imaging.CKacknowledgesNIHGrantsR01GM083274,R21ES015696and
of toxicity, does not accumulate in the liver, and does
R43HL087578.
not induce liver damage. These results are consistent
with the observation that TMS injected i.v. into mice is Authordetails
1LunaInnovationsIncorporated,LunananoWorksDivision,521BridgeSt,
passed through the kidney and into the urine (data not
Danville,VA24541,USA.2JointSchoolofNanoscienceandNanoengineering,
shown).TheATCAismostlyclearedfromthebodyafter 2907ELeeSt,Greensboro,NC27401,USA.3CenterforBiomolecularImaging,
