Covalent functionalization of multi-walled carbon nanotubes with a gadolinium chelate for efficient T1-weighted magnetic resonance imaging

Article abstract of DOI:10.1002/adfm.201402234

Given the promise of carbon nanotubes (CNTs) for photothermal therapy, drug delivery, tissue engineering, and gene therapy, there is a need for non-invasive imaging methods to monitor CNT distribution and fate in the body. In this study, non-ionizing whole-body high field magnetic resonance imaging (MRI) is used to follow the distribution of water-dispersible non-toxic functionalized CNTs administrated intravenously to mice. Oxidized CNTs are endowed with positive MRI contrast properties by covalent functionalization with the chelating ligand diethylenetriaminepentaacetic dianhydride (DTPA), followed by chelation to Gd³⁺. The structural and magnetic properties, MR relaxivities, cellular uptake, and application for MRI cell imaging of Gd-CNTs in comparison to the precursor oxidized CNTs are evaluated. Despite the intrinsic T₂ contrast of oxidized CNTs internalized in macrophages, the anchoring of paramagnetic gadolinium onto the nanotube sidewall allows efficient T₁ contrast and MR signal enhancement, which is preserved after CNT internalization by cells. Hence, due to their high dispersibility, Gd-CNTs have the potential to produce positive contrast in vivo following injection into the bloodstream. The uptake of Gd-CNTs in the liver and spleen is assessed using MRI, while rapid renal clearance of extracellular Gd-CNTs is observed, confirming the evidences of other studies using different imaging modalities.

Full text of DOI:10.1002/adfm.201402234

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Covalent Functionalization of Multi-walled Carbon
Nanotubes with a Gadolinium Chelate for Efficient
T1-Weighted Magnetic Resonance Imaging
Iris Marangon, Cécilia Ménard-Moyon, Jelena Kolosnjaj-Tabi, Marie Lys Béoutis ,
Lénaic Lartigue, Damien Alloyeau, Elzbieta Pach, Belén Ballesteros, Gwennhael Autret,
Tsedev Ninjbadgar, Dermot F. Brougham, Alberto Bianco,* and Florence Gazeau*
1. Introduction
Given the promise of carbon nanotubes (CNTs) for photothermal therapy,
drug delivery, tissue engineering, and gene therapy, there is a need for
non-invasive imaging methods to monitor CNT distribution and fate in
the body. In this study, non-ionizing whole-body high field magnetic reso-
nance imaging (MRI) is used to follow the distribution of water-dispersible
non-toxic functionalized CNTs administrated intravenously to mice. Oxi-
dized CNTs are endowed with positive MRI contrast properties by covalent
functionalization with the chelating ligand diethylenetriaminepentaacetic
dianhydride (DTPA), followed by chelation to Gd3+. The structural and
magnetic properties, MR relaxivities, cellular uptake, and application for MRI
cell imaging of Gd-CNTs in comparison to the precursor oxidized CNTs are
evaluated. Despite the intrinsic T2 contrast of oxidized CNTs internalized in
macrophages, the anchoring of paramagnetic gadolinium onto the nanotube
sidewall allows efficient T1 contrast and MR signal enhancement, which is
preserved after CNT internalization by cells. Hence, due to their high dis-
persibility, Gd-CNTs have the potential to produce positive contrast in vivo
following injection into the bloodstream. The uptake of Gd-CNTs in the liver
and spleen is assessed using MRI, while rapid renal clearance of extracel-
lular Gd-CNTs is observed, confirming the evidences of other studies using
different imaging modalities.
Carbon nanotubes (CNTs) have great
promise for numerous technological as
well as biomedical applications, which
arise from their unique architecture and
their outstanding mechanical and elec-
tronic properties.[1] However, their toxicity
and fate in the organism is still a sub-
ject of debate. The lack of consensus on
a range of toxicology and biodistribution
issues regarding CNTs is due, on one part,
to the wide heterogeneity of CNTs used
in terms of length, diameter, number of
walls, purity (e.g., the presence of cata-
lysts), morphology, functionalization,
and aggregation state.[2] An additional
confounding factor is the limited range
of methods available to study CNTs, and
more generally carbon-based materials,
in biological environments.[3] As pristine
CNTs are water insoluble, a variety of
chemical strategies have been explored
to functionalize the nanotube surface
and render them dispersible in aqueous
Dr. D. Alloyeau
I. Marangon, Dr. J. Kolosnjaj-Tabi, M. L. Béoutis,
Dr. L. Lartigue, Dr. F. Gazeau
Laboratoire Matières et Systèmes Complexes
UMR 7057 CNRS/Université Paris Diderot
10 rue Alice Domon et Léonie Duquet
F-75205, Paris, Cedex 13, France
Laboratoire Matériaux et Phénomènes Quantiques
UMR 7162 CNRS/Université Paris, Diderot,
10 rue Alice Domon et Léonie Duquet
F-75205, Paris, Cedex 13, France
E. Pach, Dr. B. Ballesteros
ICN2 – Institut Catala de Nanociencia i Nanotecnologia
Campus UAB
E-mail: Florence.gazeau@univ-paris-diderot.fr
Dr. C. Ménard-Moyon, Dr. A. Bianco
08193, Bellaterra, (Barcelona), Spain
CNRS, Institut de Biologie Moléculaire et Cellulaire
Laboratoire d'Immunopathologie et Chimie Thérapeutique
UPR 3572, 67000, Strasbourg, France
Dr. T. Ninjbadgar
Faculty of Engineering
Shine Mongol Institute of Technology
Ulaanbaatar 13372, Mongolia
E-mail: a.bianco@ibmc-cnrs.unistra.fr
Dr. J. Kolosnjaj-Tabi, Dr. G. Autret
Inserm U970, Paris Cardiovascular Research Center-PARCC/
Université Paris-Descartes
56 rue Leblanc 75015, France
Dr. D. F. Brougham
National Institute for Cellular Biotechnology
School of Chemical Sciences
Dublin City University
DOI: 10.1002/adfm.201402234
Dublin 9, Ireland
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media.[4] The type of surface modification, for example, phys-
ical coating as opposed to covalent chemical functionalization,
has been shown to play a critical role in the interactions of
CNTs with cells, in their behavior in the blood compartment
and in their subsequent fate in vivo.[5] In particular, oxidation
and shortening of multi-walled carbon nanotubes (MWCNTs)
by acid treatment followed by chemical functionalization of
the hydrophobic carbon structure with hydrophilic amino
groups has led to a new generation of functionalized MWCNTs
with high dispersibility, low toxicity, high cellular uptake, and
favorable pharmacokinetics for medical use.[6] Although func-
tionalized MWCNTs have very promising applications, as they
can serve as multifunctional platforms for tumor targeting and
drug delivery,[7] therapeutic hyperthermia,[8] transfection of
siRNA,[9] and tissue engineering,[10] imaging of MWCNTs in
vivo remains a major challenge.
Under controlled conditions imaging of CNTs in biological
environments can be achieved using the intrinsic properties
of the nanotubes, such as light absorption and scattering,[11]
acoustic or photoacoustic properties,[12] Raman signature[13]
or electron microscopy.[14] However, with current diagnostic
imaging modalities, exogenous labels (mainly fluorescent
probes[15] and radionuclides)[16] are usually required for efficient
non-invasive tracking of CNTs in living animals.[17]
their fate in vivo, as the organism must degrade inorganic
nanoparticles in addition to carbon materials, which are both
slow processes.[22] Alternative strategies such as nanoscale con-
finement of large quantities of Gd within ultra-short SWCNTs
through sidewall defects or at the opened ends (so called gado-
nanotubes),[23] synthesis of SWCNT-Gd oxide complexes by use
of Gd2O3 catalytic nanoparticles[24] or non-covalent adsorption
of Gd3+ chelates onto MWCNT surface[25] have been shown to
produce agents with very high r1 relaxivity (relaxation enhance-
ment per millimolar concentration of agent). However, non-
covalent approaches have the disadvantages of saturating the
loading capacity of CNTs or strongly reducing the possibility of
further functionalization. In addition, most of these Gd-CNT
complexes have unknown behavior in vivo, especially after
intravenous injection due their poor dispersibility. Only one
recent study described intravenous administration of a low
dose of SWCNTs, synthesized using gadolinium nanoparticles
as catalysts, however an in vivo MRI follow-up was not reported
and the fate of gadolinium nanoparticles is not known.[26] In
this context, our goal was to produce water-dispersible non-
toxic functionalized short MWCNTs suitable for targeting and
delivery of drugs[7a] with fully characterized MRI contrast prop-
erties. The critical point was to endow carbon nanotubes with
positive contrast properties without interfering with the nano-
tube structure and fate. We proposed a covalent grafting of
molecular paramagnetic species on the nanotubes.
For this purpose, oxidized MWCNTs were covalently func-
tionalized with the chelating molecule diethylenetriamine-
pentaacetic dianhydride (DTPA), followed by chelation to Gd3+.
As our approach is based on covalent bonding between CNTs
and DTPA, which has a high association constant for Gd3+, the
chelation of Gd3+ by DTPA/CNTs is strong, resulting in stable
Gd-CNT conjugates and thus reducing the risk of Gd3+ release.
The magnetic properties and magnetic resonance relaxivities
of the Gd-CNTs in comparison to oxidized CNTs showed the
paramagnetic contribution of Gd-chelates which led to positive
MR contrast. Remarkably these nanotubes were internalized by
cells without toxicity and the positive contrast effect was pre-
served following cell internalization. Finally, the potential for in
vivo monitoring of nanotube fate following intravenous admin-
istration of Gd-CNTs in mice was assessed demonstrating that
MRI can be used as suitable alternative imaging technique to
track CNTs in vivo.
Among non-invasive imaging methods, magnetic resonance
imaging (MRI) appears particularly advantageous because of its
excellent spatial resolution and soft-tissue contrast providing
simultaneously anatomic, functional and molecular informa-
tion. The main advantage of MRI over other modalities, such as
fluorescence or nuclear imaging, is its ability to penetrate deep
into tissue without employing ionizing radiation. In addition to
the enormous, and ongoing, growth in its application in rou-
tine clinical practice, MRI has also proven to be a method of
choice for molecular imaging, providing cell tracking and direct
information on molecular events through the use of smart con-
trast agents. To date there have been few reports on the use
of MRI for visualization of CNTs due to the poor MR contrast
generated by CNTs. Recently the magnetic properties of metal
impurities present in raw commercially available single-walled
CNTs (mainly iron and nickel catalysts) were exploited to inves-
3
tigate CNT distribution with hyperpolarized He and conven-
1
tional H MRI.[18] However, the intrinsic T2 and T2* contrast
that are exploited in this approach are highly dependent on the
quality of purification, on the type of CNTs, and indeed on the
aggregation propensity in physiological media;[19] all factors
which are not easily controllable. Alternatively, CNTs may be
coupled to exogenous contrast agents. These agents may allow
both, the T2 contrast and signal reduction, as for example is
the case of CNT conjugates with iron oxide superparamagnetic
nanoparticles[16b,20] or provide T1-based contrast and signal
enhancement when the tubes are conjugated to paramagnetic
gadolinium.[21] The CNT-contrast agent complexes might be
either made by loading the tube cavity or by functionalization of
their carbon backbone. For the goal of monitoring the behavior
of CNTs in vivo, it is important that any exogenous agent does
not influence either the CNT biodistribution, or their ability to
transport and deliver a therapeutic agent to an identified target.
In this regards both external coupling to or internal loading
of CNTs with iron oxide nanoparticles may partially modify
2. Results and Discussion
2.1. CNT Functionalization
MWCNTs with a diameter of 20–30 nm functionalized with a
DTPA derivative were prepared using a four step procedure
(Figure 1). Pristine purified MWCNTs were first oxidized in
acidic conditions to shorten the tubes to an average length
of ≈400 nm and to introduce COOH groups (Supporting
Information Figure S1).[27] The carboxylic acids were acti-
vated using oxalyl chloride. The corresponding acyl chloride
groups were then coupled to the DTPA derivative 2 bearing a
free amine function (Supporting Information Figure S2).[25,28]
The tert-butyl ester moieties were subsequently hydrolyzed
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1) Oxalyl chloride
Reflux
O
H2SO4 / HNO3
(3:1)
O
O
H
O
N
OH
N
24 h
Sonication
N
O
O
O
2)
O
N
O
O
O
O
N
1
3
O
O
O
O
N
O
O
O
H2N
O
N
O
O
2
THF (reflux)
60 h
TFA, H2O
20 h
Gd(III) perchlorate
HO
N
HO
OH
O
O
O
OH
O
H
H
H2O
24 h
N
N
N
Gd3+
N
O
N
O
O
O
N
N
O
O
OH
O
O
OH
4
5
OH
OH
OH
OH
Figure 1. Preparation of Gd-CNTs 5.
using trifluoroacetic acid (TFA). Finally, chelation of Gd3+ was
achieved by dispersing the DTPA/CNTs 4 in an aqueous solu-
tion of gadolinium(III) perchlorate, followed by dialysis against
water to remove free gadolinium. The usual approach to con-
jugation of molecules with DTPA involves the conversion of
one of the four carboxylic acids to an amide functionality by
the use of DTPA dianhydride. In addition to the disadvantage
of being non-selective, due to the concomitant formation of
diamide derivatives, this method results in conjugates with
lower chelating capacity for gadolinium.[29] In our case the
DTPA derivative 2 has the advantage of bearing five free COOH
groups available for chelation of gadolinium.
The Gd-CNTs were characterized by thermogravimetric
analysis (TGA), inductively coupled plasma atomic emission
spectrometry (ICP-AES), high resolution transmission elec-
tron microscopy (HR-TEM) and scanning transmission elec-
tron microscopy (STEM) coupled to energy-dispersive X-ray
spectroscopy (EDX). TGA in N2 of pristine MWCNTs, oxi-
dized MWCNTs (ox-CNTs) 1 and DTPA/CNTs 4 showed an
increasing weight loss, confirming the efficiency of the func-
tionalization (Figure 2A). The weight loss difference at 500 °C
between ox-CNTs 1 and DTPA/CNTs 4 provides an assessment
of the level of functionalization; the DTPA loading was esti-
mated 540 µmol per gram of CNTs. The level of functionaliza-
tion was also determined spectrophotometrically using xylenol
orange.[30] For this purpose, after chelation of DTPA/CNTs 4
with gadolinium(III) perchlorate and before filtration and dial-
ysis, the free Gd (i.e., not chelated to DTPA/CNTs) was titrated.
The dye xylenol orange is used as complexometric indicator of
gadolinium as it shows a color change in the presence of free
Gd. This colorimetric test allowed us to determine that the
amount of bound gadolinium was 485 µmol per gram of CNTs
5, which is consistent with the value obtained from TGA. The
xylenol orange test was also performed to ensure that no free
Gd3+ was present in the Gd-CNT samples. Control reactions
were performed using the precursor CNTs (i.e., ox-CNTs 1). In
this case, we did not observe a significant complexation of Gd3+
ions by the carboxylic acid functions or the nanotube sidewall.
Finally, ICP/AES of Gd-CNTs 5 gave a more precise value of the
functionalization degree; 460 4 µmol of gadolinium per gram
of nanotubes. Overall, characterization of the CNT conjugates
by ICP/AES, TGA, and xylenol orange test is consistent and
demonstrate full chelation of Gd3+ by DTPA. TEM analysis of
Gd-CNTs 5 shows that conditions used for functionalization did
not alter the morphology and dispersion of the nanotubes (Sup-
porting Information Figure S1). The ultrastructure of Gd-CNTs
5, detailed by HR-TEM, revealed structural defects and disloca-
tions within the external graphene layers on which it is likely that
the functional groups and Gd-DTPA are anchored (Figure 2B).
Gd-CNTs were also characterized using high angle annular
dark field (HAADF)-STEM where the intensity in the image is
proportional to the atomic number of the elements present in
the sample. HAADF-STEM has been shown as a powerful tech-
nique to allow the direct visualization of heavy-element bearing
organic molecules down to the atomic scale.[31] In the HAADF-
STEM images of Gd-CNTs 5, bright dots correspond to the Gd
atoms (Figure 2C), while they did not appear in the HAADF-
STEM images of DTPA/CNTs 4. The presence of gadolinium
in Gd-CNTs 5 was confirmed by EDX (Figure 2D), while no Gd
was found in the case of DTPA/CNTs 4.
2.2. Magnetic Characterizations
Magnetic characterization confirmed the paramagnetic proper-
ties of Gd-CNTs 5 in comparison with the ox-CNT precursor
1. The field-dependent magnetization curve at 5 K (Figure 3A)
was fit using a Brillouin function characteristic of paramag-
netic materials, with a J angular momentum of 3.2. This gives
an effective magnetic moment of 6.9 µB, close to the theoretical
magnetic moment of Gd3+ (µeff = 7.94 µB, J = 7/2). However,
field-cooled (FC) and zero-field-cooled (ZFC) magnetization
measurements (using magnetic fields of 100 and 1000 Oe for
the former) revealed an additional ferromagnetic component
(Figure 3B) which is added to the paramagnetic contribution
arising from gadolinium. The FC and ZFC magnetization
curves were not strictly superimposable as expected for purely
paramagnetic behavior (inset in Figure 3B). Interestingly, the
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Figure 2. A) Thermogravimetric analyses of pristine MWCNTs, ox-CNTs 1, and DTPA/CNTs 4 performed in N2 atmosphere. B) Representative HR-TEM
images of Gd-CNTs 5 showing structural defects and dislocations (dotted circles) within the external graphene layers in which Gd-DTPA may anchor.
Inset shows the interlayer distance between graphene sheets C) HAADF/STEM images of DTPA/CNTs 4 (upper images) and Gd-CNTs 5 (lower images).
Groupings of Gd atoms might produce bright spots on HAADF due to the Z-contrast mechanism. D) Comparison of EDX spectrum of DTPA/CNTs 4
and Gd-CNTs 5 confirming the presence of gadolinium.
low temperature ferromagnetic component (with a blocking
temperature of about 65 K) was also displayed by the gado-
linium-free control nanotubes (ox-CNTs) (Figure 3C). This
suggests that the ferromagnetic contribution arises from
residual catalyst impurities (620 ppm of iron and 1283 ppm
of nickel), which are probably entrapped into the inner empty
cavity of CNTs and were not completely eliminated by the acid
treatment, for both ox-CNTs and Gd-CNTs. The chelation of
gadolinium into DTPA/CNTs gives rise to the stronger para-
magnetic contribution, demonstrated by the linear dependence
of magnetization with inverse temperature.
1H Larmor frequencies (from 0.01 to 200 MHz). Interestingly,
the resulting nuclear magnetic relaxation dispersion (NMRD)
profile of the Gd-CNT suspension is distinctly different from
that of non-conjugated Gd-DTPA, with r1 values enhanced by
a factor of two at low frequency (Figure 3E). The decrease of r1
at clinical fields above 10 MHz is less marked for the Gd-CNTs
in comparison to Gd-DTPA. However, the result is a three-
fold increase in r1 at 200 MHz (4.7 T) due to conjugation to
CNTs. This effect could be due to an enhancement of the inner-
sphere relaxivity mechanism by slowing down the tumbling
of Gd-chelates which exhibit fast exchange of the coordinated
water molecules. This characteristic effect has been achieved
before by binding Gd ions to objects of increasing size from
macromolecules to proteins or nanoparticles.[33] In our case,
the nearly one-dimensional structure of functionalized CNTs
together with their high dispersibility, highly hydrated surface
and high permeability to water may favor water exchange, while
increasing the correlation time for tumbling of the whole con-
jugate. Importantly the huge increase of low field relaxivity
demonstrated by non-covalent confinement of Gd-complexes or
gadolinium oxide into nanosystems permeable to water, such
as zeolites, apoferritin, silicon microparticles and single-walled
carbon nanotubes was not observed.[23a,34] This is consistent
with an external covalent coupling of water-accessible Gd-che-
lates at the surface of the oxidized nanotubes. It should also
be noted that we did not observe variations of relaxivity with
MRI contrast agents (CAs) act as enhancers of longitudinal
1
and transverse local H relaxation rates R1 = 1/T1 and R2 =
1/T2. The efficacy of a gadolinium-based CA is measured by
its relaxivities r1 and r2 defined as ri = (Ri – Ri0 )/[Gd], where i
= 1, 2 and Ri0 is the endogenous relaxation rate (Figure 3D).
Gd-DTPA (Magnevist, Bayer Schering Pharma) is one of the
most common clinically approved CAs used in the majority of
−1
clinical procedure.[32] It has r1 value of 4.1 s−1 mM and r2 of
4.6 s−1 mM−1 at an imaging field of 1.5 T (or 60 MHz 1H Larmor
frequency). A relaxivity ratio r2/r1 of close to 1 ensures that the
CA acts as a good local signal enhancer (a positive CA), par-
ticularly in T1-weighted sequences. Here, the MR properties of
Gd-CNTs are compared to those of a Gd-DTPA chelate we syn-
thesized (Supporting Information Figure S3). The longitudinal
relaxivities, r1, were measured at 25 °C over a large range of
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Figure 3. A) Magnetization curve of Gd-CNTs 5 at 5K. The solid line indicates the fit using a Brillouin function. B) FC and ZFC temperature dependent
magnetization of Gd-CNTs 5 registered at two different magnetic fields: 1000 Oe (black diamonds) and 100 Oe (gray diamonds). The shoulder at low
temperature (<70 K) indicates the presence of a ferromagnetic component. The inset shows the magnetization as a function of inverse temperature. A
purely paramagnetic sample would display a linear increase with inverse temperature. C) FC and ZFC magnetization curves (magnetic field 100 Oe) for
the ox-CNTs 1. D) 1H magnetization longitudinal relaxation rate (1/T1) recorded at 25 °C as a function of Gd concentration for Gd-CNTs 5 dispersed in
water at 0.47 T (20 MHz) and in agarose gel at 0.47 T (20 MHz) and 4.7 T (200 MHz). The linear slope yields the relaxivity r1 of Gd-CNTs. E) NMRD
profiles (r1 as a function of 1H Larmor frequency) recorded at 25 °C for Gd-CNTs 5 in comparison to Gd-DTPA 8.
CNT concentration, as previously reported.[25] All relaxation
rates showed linear variation with CNT and Gd concentration
with identical r1 relaxivities whether CNTs have been dispersed
in water or further immobilized in agarose gel (Figure 3D).
All relaxation rates showed linear variation with CNT and Gd
concentration, with identical r1 values obtained irrespective of
whether the Gd-CNTs were dispersed in water or immobilized
in agarose gel (Figure 3D). The former observation confirms
both the remarkably high dispersibility of the functionalized
CNTs and their colloidal stability in water suspension over the
concentration range studied. The latter suggests that further
increases in the overall tumbling correlation time (in agarose)
have no effect, and so this is no longer the r1-limiting process.
Once bound to the CNT it seems that the timescale for local
dynamics about the chelate determines the relaxivity.
CA is enhanced for Gd-CNTs on increasing the field, in com-
parison to Gd-DTPA. Therefore, for the in vitro and in vivo
evaluation of Gd-CNTs, an imaging field of 4.7 T was used.
2.3. Cellular Uptake and MRI Cell Imaging
As intrinsic MR contrast highly depends on tissue structure
and water dynamics, the ability of a CA to modify the 1H signal
is strongly affected by the local environment and by CA organi-
zation in this environment. During their odyssey through the
organism, CNTs are likely internalized by cells, and most prob-
ably by cells of the reticulo-endothelial system (resident mac-
rophages or circulating phagocytic cells). However, the magnetic
properties of MRI contrast agents are deeply affected by cellular
internalization and intracellular compartmentalization. For
instance it is well-known that there is a drastic reduction of the
longitudinal relaxivity of iron oxide nanoparticles following cel-
lular uptake.[35] Similar effects were observed for gadolinium
oxide nanoparticles depending on the internalized dose[36]
and for a macromolecular Gd(III)-based probe[37] depending
on its intracellular localization.[38] This so-called intracellular
“quenching” effect tends to enhance r2/r1 ratio, in such manner
Transverse relaxivities r2 were measured in the frequency
range between 20 MHz (0.47 T) and 200 MHz (4.7 T). The values
−1
of r2 of Gd-CNTs and Gd-DTPA decreased from 13.0 s−1 mM
and 5.40 at 20 MHz to 5.22 s−1 mM and 4.07 s−1 mM at 200
MHz, respectively. This change corresponds to an evolution of
the r2/r1 ratio from 1.8 to 0.7 for Gd-CNTs and from 1.42 to 1.
40 for Gd-DTPA when the imaging field is increased from 0.47
to 4.7 T. Hence, in terms of r2/r1 ratio, the efficacy as a positive
−1
−1
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Figure 4. A) Optical micrographs of RAW 264.7 mouse macrophages after 20 h incubation with Gd-CNTs 5 at concentrations of 10, 20, 50 and
100 µg/mL. B) Electron micrographs of RAW cells incubated with 20 µg/mL of Gd-CNTs 5 for 20 h. Isolated Gd-CNTs and bundles of Gd-CNTs can be
observed inside membrane-bound endosomes.
that the generation of positive contrast by labeled cells becomes
challenging. Regarding CNTs, it is not clear how r1 could be
modified by cell confinement and how intrinsic properties of
precursor ox-CNTs could affect transverse and longitudinal
relaxation in that environment. It was reported that, despite a
very high r1 value in suspension, gadolinium-loaded SWCNTs
failed to induce positive contrast when internalized by murine
macrophages and were subsequently detected with a negative
signal on T2 or T2*-weighted sequence.[21c] Likewise, CNTs that
power of high throughput flow cytometry with imaging of each
analyzed cell.[11b,39] As illustrated in Figure 5A, Gd-CNT-labeled
cells exhibit dark spots on bright field images that were not
observed in non-labeled cells. These dark spots colocalized with
high intensity spots on the dark field image, corresponding to
areas of CNT accumulation that strongly absorb and scatter the
light. The biparametric dot plot representing the mean inten-
sity on dark field versus the mean pixel intensity on bright
field (Figure 5B) clearly shows that both parameters increase
(in absolute value) with increasing concentration of CNTs in
the incubation medium. This indicates a dose dependent CNT
uptake for the entire cell population, which tends to saturate
at the highest concentration. Similar results were obtained for
both Gd-CNTs and ox-CNTs.
Subsequently the MR contrast properties of cells labeled with
increasing concentration of Gd-CNTs or ox-CNTs and dispersed
in agarose gels mimicking the cell density of tissue (7 × 106
cells in 70 µL of gel) were examined. MR images were acquired
using a spin echo sequence, while varying the echo time TE or
repetition time TR in order to determine T1 and T2 values and
the optimal conditions for T1- or T2-weighting. Representative
T1-weighted images in Figure 6A show a dose-dependent signal
increase in the macrophages labeled with Gd-CNTs in compar-
ison to the non-labeled cells or to the cells labeled with ox-CNTs.
The signal increase was nearly twofold for the highest Gd-CNT
concentration with respect to non-labeled cells. Conversely, in
T2-weighted images, the macrophages labeled with Gd-CNTs
display decreased MR signal intensity with increasing CNT con-
centration (Figure 6A). Hence, the Gd-CNTs act as a bimodal
contrast agent for cell imaging, generating positive contrast
under T1-weighted and negative contrast under T2-weighted
imaging conditions. By comparison, no signal change was
1
were free of any gadolinium label[18b] were found to induce H
signal suppression in mouse liver. These confusing results call
for a thorough examination of MR properties depending on cel-
lular uptake and on the intrinsic magnetic properties of CNTs.
In this work, RAW 264.7 mouse macrophages were used as
a cell model to study Gd-CNT uptake in vitro. The cells were
incubated for 24 h with Gd-CNTs in the concentration range
of 0–100 µg/mL. Cell morphology and adherence on culture
flasks was not affected by CNT labeling as shown by optical
microscopy (Figure 4A). Cellular metabolic activity, assessed
by the Alamar blue test, revealed a slight decrease in compar-
ison to non-labelled cells at the highest concentration tested
(100 µg/mL) (see Supporting Information Figure S4). TEM
confirmed the intracellular uptake of CNTs; indeed elongated
hollow structures were easily identifiable within the intracel-
lular organelles (Figure 4B). Most nanotubes were assembled
as large bundles within vesicular membranes, only a small
fraction could be observed isolated within the cytoplasm. This
suggests an active process of cellular internalization, involving
membrane trafficking and dynamic compartimentalization
within lysosomal storage organelles. The dose dependent CNT
uptake by macrophages was assessed by imaging flow cytom-
etry (ImageStream).[11b] This technique combines the statistical
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Figure 5. Imaging flow cytometry analysis of Gd-CNT uptake by RAW 264.7 macrophages. A) Representative bright field and corresponding dark field
images of cells (among 10 000) acquired with ImageStream for different Gd-CNT incubation conditions. Black spots on bright field images corre-
sponding to bright spots on dark field images are indicative of CNT uptake. B) Relative quantification of Gd-CNT uptake on biparametric dot plot of
dark field intensity versus mean pixel intensity on bright field image.
observed for macrophages labeled with ox-CNTs. The quanti-
tative determination of relaxation rates (1/Ti expressed in s−1,
i = 1,2) reveals a linear increase of 1/T1 with Gd-CNT concen-
tration in macrophages, whereas no variation was observed
after labeling with control ox-CNTs (Figure 6B). This shows that
Gd-DTPA chelates covalently coupled to CNTs can still be effec-
tive as T1-contrast agents when internalized in cells. Regarding
the transverse relaxation, the analysis is more complex. In com-
parison to cell-free agarose, the high density of cells induces a
3-fold increase in transverse relaxation time 1/T2 (Figure 6B).
This observation is consistent with relaxation dynamics in
tissue, which is accelerated in comparison to water.[40] More
surprisingly, 1/T2 also increased with the concentration of
CNTs in cells, regardless of whether they were labeled with Gd-
CNTs or ox-CNTs. The T2 effect appears as an intrinsic char-
acteristic of CNTs, not of the complexed gadolinium probe. In
summary covalent grafting of Gd-DTPA induces an efficient
intracellular T1 reduction that depends on CNT cellular uptake.
Despite the associated T2 reduction in labeled cells arising from
the nanotubes, positive cell contrast due to the paramagnetic
label can be observed when appropriate T1-weighting is applied.
A T1-weighted sequence (fast spin echo) was applied since it
offered the best contrast-to-noise ratio after Gd-CNT admin-
istration. Following the injection of the low dose of Gd-CNTs,
significant signal changes in liver, spleen, kidneys and bladder
were not observed. However a five-fold increase in Gd-CNT
dose (250 µg corresponding to a gadolinium dose of 5.75 µmol/
kg body weight) yielded a clear signal enhancement in liver,
spleen and to a much larger extent in the bladder, where signal
enhancement in excess of +250% was obtained (Figure 7).
While the contrast enhancement decreased after 2 h in the
bladder (Figure 7C), it was slightly increased over time in liver
reaching a value of +27% after 320 min (Figure 7AB). In spleen
the signal reached a maximum after two hours (+16%) and
then decreased to close to its initial value (+3%). Histological
specimens of hepatic and splenic tissues confirmed the pres-
ence of nanotubes with a spotty homogenous distribution in
liver and in the red pulp periphery of the spleen (Figure 8 and
Supporting Information Figures S5,S6).
Unambiguous identification of carbon nanotubes in the
intracellular compartments of Kupffer cells in liver was pro-
vided by electron energy loss spectroscopy spectrum (EELS).
This technique can differentiate graphitic sp2 signal from CNTs
and amorphous carbon signal either from the endogenous
cellular background or from carbon membranes present on
TEM grids (Figure 9).[41] Moreover, EDX spectroscopy also con-
firmed the presence of gadolinium co-localizing with carbon
nanotubes within membrane-bound intracellular compart-
ments that are presumably lysosomes, in both liver and spleen
(Figure 9). Thus, the increased MR signal in these organs is
unambiguously related to the rapid uptake of CNTs by the
2.4. In Vivo Imaging
The next step was to evaluate the use of Gd-CNTs in vivo to
monitor the organ biodistribution of functionalized nanotubes.
Gd-CNTs were injected intravenously to mice at two different
doses (50 µg and 250 µg corresponding to 2.5 and 12.5 mg/kg,
respectively) and monitored over 5 hours following injection.
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Figure 6. A) T1-weighted and T2-weighted fast spin echo MR images of compacted cells mimicking tissue (70 µL) labeled with different concentrations
(10, 20, 50 and 100 µg/mL) of Gd-CNTs 5 or ox-CNTs 1. For comparison, images of agarose gel alone (agar), water alone or non-labeled cells (cells)
are also displayed. TR/TE are respectively 445/12 ms for T1-weighted sequence and 4500/95 ms for T2-weighted sequence. B) Longitudinal, 1/T1, and
transverse, 1/T2, relaxation rates of compacted cells for different labeling conditions (NM: not measurable).
reticuloendothelial system, as confirmed by histology and TEM.
The massive and transient signal enhancement in bladder also
suggests that a significant fraction of Gd-CNTs is eliminated
by renal clearance. Both histology and TEM confirmed MRI
results showing that Gd-CNTs do not accumulate in the kidneys
(as only one isolated aggregate was observed in histological
sections), which is consistent with effective elimination in the
urine (Supporting Information Figure S7). Overall, our find-
ings are consistent with the recent SPECT/CT imaging study
on functionalized CNTs radiolabeled with 111Indium-DTPA.
Different distribution profiles have been reported with a bal-
ance of renal clearance versus RES accumulation depending on
the degree of chemical functionalization of the nanotubes.[16a]
Individual functionalized CNTs with outer diameter lower than
the glomerular filtration cut-off have been shown to escape
the glomerular kidney filter to be excreted in urines in sev-
eral studies.[42] In contrast, most of the largest CNTs or CNTs
forming bundles in vivo are uptaken in liver, spleen and lung.[5]
Therefore, full dispersion (or individualization) of CNTs which
is favored by chemical functionalization appears to be a pivotal
factor determining their excretion profile. In the present study,
a single bolus injection of a relatively high dose of Gd-CNTs
(250 µg versus 50 µg and 10 µg in two recent studies)[16a,26] was
used with no acute or sub-acute effect on the animal behavior
apparent (until day 8 post-injection) and with no signs of tissue
damage, inflammatory response and cellular toxicity that could
be noticed on histological and TEM images in liver, spleen,
kidney and lung. This clearly indicates that the dispersion of
the functionalized nanotubes in physiological saline medium at
the concentration of 5 mg/mL was appropriate for intravenous
administration. Moreover, unlike previous studies reporting
accumulation of CNTs in the lungs after their systemic admin-
istration,[5] the lung histological specimens show very few
nanotubes despite the high dose injected (Supporting Informa-
tion Figure S7). The lack of lung accumulation is a further indi-
cation of the good dispersibility of Gd-CNTs which persists in
blood circulation. The ultrastructural examination of Gd-CNTs
in liver and spleen also revealed that well individualized CNTs
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Figure 7. A) MRI contrast enhancement in liver, spleen, kidneys and bladder following intravenous injection of Gd-CNT 5 (5 mg/mL, 100 µL) in com-
parison to the signal prior injection. Standard deviations are deduced from three independent measurements. B,C) Example of MR slices showing liver
(L), spleen (S) and kidney (K) (B) or bladder (B) (C) at different-time points before and after injection of Gd-CNTs. A fast spin echo RARE T1-weighted
sequence was used with TR/TE = 445/12 ms.
could be sequestered into intracellular compartment while
keeping their native hollow and elongated structure (Figure 8).
A similar intracellular distribution of CNTs was still observed
8 days after injection. This suggests that individual as well as
CNT clusters were internalized in the first hours post-injection.
practice (5.75 µmol/kg versus 100 µmol/kg body weight), the
high longitudinal relaxivity of Gd-CNT conjugates enabled their
detection with T1-weighting in mice using a 4.7 T scanner. In
contrast to other strategies using nanoparticles to enhance MR
contrast of carbon materials, the present approach exploits par-
amagnetic molecular chelates that are widely used in the clinics
and minimally interfere with the structure, dispersibility, func-
tionalization, loading capacity and degradability of CNTs. The
approach to material design described here could open new
routes to highly dispersible nanomaterials functionalizable
with significant payloads for drug delivery, tumor targeting and
photothermal therapy or vaccination. The second aspect is the
retention of T1-weighting properties for MRI following cellular
uptake, which may provide possibilities for study the biodistri-
bution of functionalized CNTs and for image guided delivery.
3. Conclusion
In conclusion, we covalently functionalized oxidized MWCNTs
with DTPA for chelation of Gd3+ and the Gd-CNTs were
administrated intravenously to mice. The CNT uptake in liver
and spleen could be successfully assessed using in vivo MRI
owing to the preserved capacity of Gd-CNTs to generate signal
enhancement after cellular internalization. In addition, the
rapid clearance into the urines of extracellular functionalized
CNTs confirmed previous studies using other different imaging
modalities.[43] Overall, this study represents the first success
in monitoring the CNT distribution with functionalized tubes
used to generate a T1 positive MR contrast after intravenous
injection. This work shows that combining the properties of
Gd3+ and CNTs allows performing MRI in vivo. MRI has a
high resolution compared to other imaging techniques and can
thus be used as an alternative imaging method to track CNTs.
Despite the fact that the amount of gadolinium chelates injected
with nanotubes is far lower the gadolinium dose in clinical
4. Experimental Section
4.1. General Information
MWCNTs (20–30 nm diameter, 0.5–2 µm length, 95% purity; batch
1240XH) were purchased from Nanostructured and Amorphous
Materials. When stated, suspensions of CNTs were sonicated in a
water bath (Transsonics Digitals Elma, 20 W, 40 kHz). All reagents and
solvents were purchased from different commercial suppliers and used
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Figure 8. Histological (left) and transmission electron microscopy (TEM) (right) micrographs of A) liver and B) spleen 6 h after injection of Gd-CNTs
5. A) Note the homogenous distribution of Gd-CNT aggregates (spots) within the liver (histological slices were stained with Nuclear Fast Red, Mag
×20). TEM shows that Gd-CNTs are mainly localized in endosomes within Kupffer cells (N-h: nuclei of hepatocytes, N-K: nucleus of the Kupffer cell,
asterisks: glycogen, white arrowhead: endosome containing nanotubes and its magnified view in the inset. White arrows point to nanotubes). B) Black
arrows point to Gd-CNT aggregates (spots on histology (left)) within the spleen (Nuclear Fast Red and Pearls staining, which reveals endogenous iron
storage ferritin (spots), Mag ×10). Aggregates are mainly localized in the periphery or, in case of smaller aggregates, in the center of the white pulp.
TEM images show Gd-CNTs localized in endosomes of phagocytic cells (N: nuclei, red square: endosome containing nanotubes and its magnified
view on the right).
as received. In particular, xylenol orange tetrasodium salt was purchased
from Alfa Aesar. Thermogravimetric analyses were performed by using
a TGA Q500 TA instrument with a ramp of 10 °C min−1 under N2 using
the brominated compound 9 (6.54 g, 0.019 mol) and DIEA (3.87 mL,
0.022 mol) in acetonitrile (32 mL) was added a solution of H-Lys(Z)-
OtBu·HCl (2.78 g, 0.0074 mol) in a phosphate buffer (Na2HPO4: 1.1 g/
KH2PO4: 0.137 g, pH 8) (36 mL). The reaction mixture was vigorously
stirred at 40 °C for 3 days. Water and ethyl acetate were added and the two
phases were separated. The organic phase was washed with water (twice)
and brine, dried over Na2SO4, filtered and evaporated under vacuum. The
crude was purified by chromatography on silica gel using ethyl acetate/
cyclohexane from 20/80 to 60/40 as eluant. 3.53 g of compound 10
1
a flow rate of 60 mL min−1. H and 13C NMR spectra were recorded on
Bruker DPX 300 instrument. The peak values were obtained as ppm (δ)
and referenced to the solvent. The resonance multiplicity is indicated
as s (singlet), br s (broad singlet), and m (multiplet). LC/MS analyses
were performed on ThermoFisher Finnigan 6 LCQ Advantage Max.
TLC was performed on aluminum sheets coated with silica gel 60 F254
(Merck, Darmstadt). Chromatographic purification was done with Merck
silica gel (Kiesegel 60, 40–60 µm, 230–400 mesh ASTM). Dialysis was
performed using Spectra/Por dialysis membrane, MWCO: 12–14 000 Da.
1
was obtained as yellow oil (yield: 54%). The H NMR spectrum was in
agreement with literature.[28] LC-MS (ESI): m/z 879.6 [M + H]+.
6-Amino-2-{bis-[2-(bis-tert-butoxycarbonylmethyl-amino)-ethyl]-amino}-
hexanoic acid tert-butyl ester 2: To a solution of compound 10 (1.77 g,
2.0 mmol) in methanol (20 mL) was added a catalytic amount of
Pd/C. The mixture was stirred at room temperature under a hydrogen
atmosphere for 17 h. The suspension was filtered over celite and the
filtrate was evaporated under vacuum to give compound 2 as colorless
oil (1.33 g, 85%) with a satisfactory purity. The 1H and 13C NMR spectra
were in agreement with literature (Anelli, Bioconjugate Chem. 1999,
137). LC-MS (ESI): m/z 745.5 [M + H]+.
4.2. Synthesis of DTPA Ligand 2
[(2-Bromo-ethyl)-tert-butoxycarbonylmethyl-amino]-acetic acid tert-butyl
ester 9: To a solution of tert-butylbromoacetate (9.96 mL, 0.067 mol) and
N,N-diisopropylethylamine (DIEA) (19.5 mL, 0.11 mol) in DMF (25 mL)
was added dropwise a solution of 2-bromoethylamine hydrobromide
(4.61 g, 0.022 mol) in DMF (20 mL). The reaction mixture was stirred
at room temperature for 1 day. Water and ethyl acetate were added and
the two phases were separated. The organic phase was washed with
water (3 times) and brine, dried over Na2SO4, filtered and evaporated
under vacuum. The crude was purified by chromatography on silica gel
using ethyl acetate/cyclohexane from 3:97 to 7/93 as eluant. 3.73 g of
compound 9 was obtained as colorless oil (yield: 47%). The 1H NMR
spectrum was in agreement with literature.[44]
4.3. Synthesis of Gd-DTPA Ligand 8
6-Acetylamino-2-{[2-(bis-tert-butoxycarbonylmethyl-amino)-ethyl]-[2-(tert-
butoxycarbonylmethyl-isopropoxycarbonylmethyl-amino)-ethyl]-amino}-
hexanoic acid tert-butyl ester 6: To a solution of compound 2 (166 mg,
0.23 mmol) in methanol (2.3 mL) was added acetic anhydride (24 µL,
0.25 mmol), and the mixture was stirred at room temperature. The
reaction was monitored by TLC. After 7 h, the reaction mixture was
evaporated under vacuum to give 171 mg of compound 6 as a yellow
6-Benzyloxycarbonylamino-2-{bis-[2-(bis-tert-butoxycarbonylmethyl-
amino)-ethyl]-amino}-hexanoic acid tert-butyl ester 10: To a solution of
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Figure 9. A) TEM of liver 6 h after injection of Gd-CNTs. B) EELS analysis on carbon K-edge can differentiate between Gd-CNTs and the carbon rich
cellular background. The strength of the first sharp peak (285 eV) due to the 1s to 2π* can be related to the predominance of sp2 hybridizations.
Therefore, the strong initial peak unambiguously reveals the presence of carbon nanotubes in liver. This characteristic signal is easily differentiated
from the carbon rich cellular background which shows an amorphous carbon-like K-edge. C) EDX spectrum confirms the presence of Gd on CNTs
internalized by liver Kupffer cells.
1
oil (yield: 98%) with a satisfactory purity. H NMR (300 MHz, CDCl3): δ
4.4. Functionalization of MWCNTs
6.05 (br s, 1 H), 3.41 (s, 8 H), 3.31–3.16 (m, 3 H), 2.84–2.58 (m, 8 H),
1.91 (s, 3 H), 1.64–1.22 (m, 51 H). 13C NMR (75 MHz, CDCl3): 172.9,
170.8, 170.2, 81.0, 80.8, 63.9, 56.0, 53.7, 50.2, 39.4, 29.5, 29.0, 28.4, 28.3,
23.6, 23.4. LC/MS (ESI): m/z 787.3 [M + H]+.
ox-CNTs 1: A suspension of pristine MWCNTs (1 g) in a mixture of
sulfuric acid 98% (108 mL) and nitric acid 65% (36 mL) was sonicated in
a water bath for 24 h. The temperature was kept below 35 °C. Deionized
water was then carefully added and the CNTs were filtered (OmniporeTM
membrane filtration, 0.45 µm), re-suspended in water, filtered again until
pH became neutral and dried under vacuum.
Functionalized CNTs 3: A suspension of ox-CNTs 1 (21 mg) in oxalyl
chloride (8.5 mL) was sonicated in a water bath for 30 min and then
heated at reflux for 24 h. After evaporation under vacuum, the acyl
chloride-CNTs were dispersed in a solution of DTPA ligand 2 (237 mg) in
dry THF (10 mL). The mixture was sonicated for 2 min and then heated
at reflux for 60 h. The suspension was filtered over a PTFE membrane
(0.1 µm, Omnipore, Millipore). The solid recovered on the filter was
dispersed in DMF (100 mL), sonicated for 3 min in a water bath, and
filtered over a PTFE membrane (0.1 µm). This sequence was repeated
with DMF, methanol (twice), and dichloromethane. The resulting solid
was dried under vacuum.
6-Acetylamino-2-{bis-[2-(bis-carboxymethyl-amino)-ethyl]-amino}-
hexanoic acid 7: A solution of compound 6 (39 mg, 0.050 mmol) in
TFA (0.9 mL) and water (0.1 mL) was stirred at room temperature for
1 day. The reaction was monitored by LC/MS. The reaction mixture was
precipitated in diethyl ether and filtered. The solid was dissolved in water
and the solution was lyophilized to give 17 mg of compound 7 as a beige
solid (yield: 68%) with a satisfactory purity. 1H NMR (300 MHz, DMSO):
δ 3.35 (s, 8 H), 3.41–3.28 (m, 3 H), 3.10–2.98 (m, 8 H), 1.90–1.81 (m,
2 H), 1.79 (s, 3 H), 1.52–1.31 (m, 4 H). 13C NMR (75 MHz, DMSO):
172.6, 171.4, 169.1, 63.9, 54.4, 50.6, 50.3, 38.3, 29.1, 27.1, 23.6, 22.6. LC/
MS (ESI): m/z 507.3 [M + H]+.
Gd-DTPA Ligand 8: To a solution of gadolinium(III) perchlorate (Strem
Chemicals, 50% in water) (14.9 µmol) in water (9.4 mL) was added
DTPA ligand 7 (9.4 mg). The mixture was stirred at room temperature
for 3 days. An aliquot of the solution was titrated with xylenol orange to
check that there was no more free Gd. The solution was lyophilized to
obtain Gd-DTPA ligand 8.
DTPA/CNTs 4: A suspension of the functionalized CNTs 3 (21 mg)
in TFA (1.8 mL) and water (200 µL) was sonicated in a water bath for
3 min and stirred at room temperature for 2 h. A solution of TFA (1.8 mL)
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and water (200 µL) was added and the mixture was further stirred for
18 h. After evaporation under vacuum, diethyl ether was added and
the mixture was evaporated under vacuum. The residue was dispersed
in water and dialyzed against water for 2 days. The suspension was
lyophilized to obtain DTPA/CNTs 4.
Gd-CNTs 5: A suspension of DTPA/CNTs 4 (8.3 mg) in gadolinium(III)
perchlorate 50% aqueous solution (4.04 µmol, 8.3 mL) was sonicated
for 30 min and stirred at rt for 24 h. An aliquot of the suspension was
centrifuged. The supernatant was titrated with xylenol orange to assess
the amount of free Gd. The reaction mixture was then dialyzed against
water for 2 days. The suspension was lyophilized to obtain Gd-CNTs 5.
100 µg/mL for 20 h at 37 °C. Cell metabolic activity was assessed by the
Alamar blue test in 48-well plates seeded with cells the day before labeling.
The labeled macrophages were washed twice in PBS and were incubated
with 10% Alamar Blue in culture medium for 2 h. The fluorescence in cell
medium due to the reduction of resazurin (oxidized form) to resorufin
by cell activity was quantified on a FLUOstar OPTIMA microplate reader
(excitation 550 nm, emission 590 nm) and normalized to the fluorescence
signal of the control non-labeled cells. The conditions were run in
quadruplicate. For TEM observation, cells were trypsinized, centrifuged at
1200 rpm for 5 min, washed in 0.1 M cacodylate buffer and fixed with 2.5%
glutaraldehyde in cacodylate buffer at 4 °C for 30 min. Cells were then
post-fixed with osmium tetraoxide 1% and passed through uranyl acetate.
Samples were then dehydrated in an ethanol series (30–100%) and
embedded in epoxy medium (EPON 812; Shell Chemical, San Francisco,
California). Thin sections (70 nm) were collected onto 200 mesh cooper
grids, and counterstained with lead citrate before examination with
Zeiss EM902 electron microscope operated at 80 kV (MIMA2- UR1196
Génomique et Physiologie de la Lactation, INRA, Plateau de Microscopie
Electronique 78352, Jouy en Josas, France).
Imaging Flow Cytometry: For imaging flow cytometry analysis, labeled
macrophages were trypsinized, fixed with 4% formaldehyde in PBS and
centrifuged to obtain a pellet of about 106 cells in 50 µL. Cell images
were acquired using the ImageStreamX multispectral imaging flow
cytometer (Amnis Corporation), collecting 10,000 events per sample
at 40× magnification. Dark field images were acquired using a 785 nm
laser. Cell images were analyzed using IDEAS image-analysis software
(Amnis). Gating on bivariate plot of aspect ratio versus cell area was first
used to isolate a population of single cells. Cells within the focal plane
were further selected using a two-dimensional plot of image contrast
versus root mean squared (RMS) gradient. Mean pixel feature on bright
field images versus intensity feature on dark field images was used as
a biparametric dot plot to discriminate cells with respect to their CNT
uptake.
4.5. TEM and Magnetic Characterizations of MWCNTs
Aberration-corrected High Resolution Transmission Electron Microscopy
(HR-TEM), Energy-dispersive X-Ray Spectroscopy (EDX), and High Angle
Annular Dark Field Scanning Transmission Electron Microscopy (HAADF)-
STEM: HR-TEM investigations were performed with the newly developed
JEOL ARM 200 F microscope operating at 80 kV. This microscope
is equipped together with a CEOS aberration corrector, a cold field
emission gun, a Gatan GIF quantum ER and a JEOL EDX Diode.[45]
HAADF-STEM images and EDX spectra were acquired on a FEI Tecnai
G2 F20 HRTEM operated at 200 kV and equipped with an EDAX super
ultra-thin window (SUTW) X-ray detector. Samples were deposited on
lacey carbon Cu TEM grids (Agar).
Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES)
Analysis: Determination of gadolinium, iron and nickel content in CNTs
was performed by ICP-AES analysis (Agilent 7500ce). The samples were
digested in concentrated HNO3 (3:1, v/v) solution and diluted with
ultrapure water for the analysis.
Magnetization Measurements: Magnetic measurements were carried
out on a Quantum Design MPMS-5S SQUID Magnetometer. Field-
dependent magnetization curves were measured at 5K as function of
the external field in the range 0–5 Tesla. Field-cooled (FC) and zero-
field-cooled (ZFC) temperature-dependant magnetization was measured
using a magnetic field of 100 Oe or 1000 Oe and the sample was cooled
with the same magnetic field.
4.7. Animal Experiments and MRI Protocol
Animal experiments were conducted in line with French Agriculture
Ministry guidelines. During the MRI protocol the animals were
anesthetized with 2% isofluorane (Aerrane, Baxter, Maurepas, France)
supplied in an air mixture, while their body temperature was kept
constant by circulating thermostated warm water. Twelve eight-
NMRD and Relaxivity Measurements: The frequency dependence of
1
the H relaxation for the aqueous nanotube suspensions was recorded
over the frequency range 0.01–20 MHz using a Spinmaster FFC-
2000 Fast Field Cycling NMR Relaxometer (Stelar SRL, Mede, Italy).
T1 measurements were performed as a function of external field, B0,
with standard pulse sequences incorporating B0 field excursions.
The temperature of the samples was maintained at 25 1 °C using a
thermostated airflow system. All of the 1H magnetisation recovery
curves were singly exponential within experimental error and the random
errors in fitting T1 were always less than 1%. T2 was measured using
the CPMG pulse sequence at 20, 40, and 60 MHz using the Stelar
spectrometer and a reconditioned Bruker WP80 electromagnet. T2 was
measured using the CPMG pulse sequence at 20, 40 and 60 MHz.
Images of CNT liquid suspensions, agarose gel (0.3%) and cell samples
(7 × 106 cells in 70 µL agarose) were obtained on a Bruker Bio-Spec
47/40 USR scanner operating at 4.7 T (200MHz) using fast spin echo
sequences with variable echo time TE and repetition time TR. T1 and T2
were deduced from monoexponential fit of MR signal as function of TR
and TE, respectively, using the ParaVision software.
weeks-old female C57/Bl6 mice (mean weight 20.5
1 g) (Janvier,
France) were used in this study. The mice were imaged by the whole
body MRI 20 min before injection and 10, 75, 120, 320 min after
injection of Gd-CNTs in the retro-orbital sinus at a dose of 1 or 5 mg/mL
suspended in a 100 µL physiological saline medium. Three animals
were analyzed at each time point. MRI was performed on a Biospec
47/40 USR (40-cm bore actively shielded 4.7 Tesla magnet) scanner
interfaced to ParaVision software (both provided from Bruker Biospin
GmbH, Rheinstetten, Germany). The whole body imaging protocol
was performed with a volume transmission/reception RF coil for mice
(Bruker), using a fast spin echo (Turbo RARE) sequence with a TE of 12
ms and a TR of 445 ms (T1-weighted sequence), flip angle of 180°, 30
averages, in-plane resolution of 150 µm, slice thickness of 500 nm, for a
total of seven slices. At the end of the experimental protocol, mice were
sacrificed with a lethal injection of sodium pentobarbital. Livers, spleens,
lungs and kidneys were excised and prepared for histological and TEM
characterizations. Image processing and analysis were made with the
open source software OsiriX (3.9.2. version). The percentage of contrast
4.6. Cell Culture, Cell Labeling, Cell Metabolic Activity, and Cell
Preparation for TEM
CNRpost-injection-CNRpre-injection
CNRpre-injection
enhancement was defined as
,
(
)
× 100
The murine macrophage RAW 264.7 cells were maintained as
monolayer cultures in RPMI (Roswell Park Memorial Institute) medium
supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin at
37 °C and 5% of CO2. For cell labeling, cells were incubated with complete
RPMI medium supplemented with CNTs at concentration of 10, 20, 50,
with the contrast-to-noise ratio (CNR) measured in different organs
of interest at the different investigation time points. CNR was defined
SNRROI-SNRref
SNRref
Mean Signal
SDNoise
(
)
as
with
, where "ref" denotes the
SNR=
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water tube reference and SD is the standard deviation of the noise in the
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Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author. TEM of ox- and Gd-CNTs, HAADF-STEM images of
ox-CNTs, synthesis of DTPA ligand 2, HAADF-STEM images and EDX
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I.M. and C.M.-M. contributed equally to this work. The authors thank
C. Péchoux and S. Chat for electron microscopy, C. Fabbro for TGA
measurements, O. Clément and L. Pidial for the access to the Small
Animal Imaging Platform Paris-Descartes PARCC-HEGP and to the
animal facility, N. Bogetto and the platform Imagoseine (University Paris
Diderot, Institut Jacques Monod) for imaging flow cytometry. This work
was supported by ANR P2N (NANOTHER project 2010-NANO-008–398
04) and by the Region Ile-de-France (convention SESAME E1845 for
the JEOL ARM 200F electron microscope recently installed at the Paris
Diderot University). E.P., B.B., C.M.-M., and A.B. acknowledge the EU
FP7-ITN Marie-Curie Network programme RADDEL (290023). E.P. is
enrolled in the UAB PhD program.
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