Preparation of 14C-labeled multiwalled carbon nanotubes for biodistribution investigations

Article abstract of DOI:10.1021/ja906319z

(Figure Presented) A new method allowing the ¹⁴C-labeling of carboxylic acid functions of carbon nanotubes is described. The key step of the labeling process is a decarbonylation reaction that has been developed and optimized with the help of a screening method. The optimized process has been successfully applied to multiwalled carbon nanotubes (MWNTs), and the corresponding ¹⁴C-labeled nanotubes were used to investigate their in vivo behavior. Preliminary results obtained after i.v. contamination of rats revealed liver as the main target organ. Radiolabeling of NTs with a long-life radioactive nucleus like ¹⁴C, coupled to a highly sensitive autoradiographic method, that provides a unique detection threshold, will make it possible to determine for a long time period whether or not NTs remain in any organs after animal exposure.

Full text of DOI:10.1021/ja906319z

Published on Web 09/29/2009
14
Preparation of C-Labeled Multiwalled Carbon Nanotubes for Biodistribution
Investigations
Dominique Georgin, Bertrand Czarny, Magali Botquin, Martine Mayne-L’Hermite,
Mathieu Pinault, Brigitte Bouchet-Fabre, Marie Carriere, Jean-Luc Poncy, Quang Chau,
R e´ my Maximilien, Vincent Dive,* and Fr e´ d e´ ric Taran*
CEA, IBITECS, SCBM and SIMOPRO, CEA, IRAMIS, SPAM and SCM, Gif sur YVette, F-91191, France, and CEA,
IRCM, SREIT, Bruy e` res le Ch aˆ tel, F-91680, France
Received August 17, 2009; E-mail: frederic.taran@cea.fr; vincent.dive@cea.fr
Multiwalled carbon nanotubes (MWNTs) are currently produced
by the industry for multiple actual and future commercial applica-
tions, and consequently their potential health impact has been the
subject of intense research particularly over the past 5 years. One
of the main current concerns is related to the needle-like fiber shape
of carbon nanotubes (NTs) that might induce asbestos-like patho-
solvent, and the presence of additives (see Supporting Information)
that were rapidly screened thanks to the fluorescent properties of
3. From these screening experiments appeared Pd(PPh ) as the most
3 4
effective palladium source and CsF as a crucial additive (Table 1).
Table 1. Optimization of the Decarbonylation Reaction of Aroyl
Cyanides
1
genicity. Exploration of the in ViVo biodistribution and pharma-
cokinetics of MWNTs with approaches offering the highest
threshold in detection is therefore of prime importance. Although
several studies have been carried out on pristine single-walled
2
carbon nanotubes (SWNTs), information concerning the biodis-
tribution of nonmodified MWNTs is still lacking. Most current
research has been based on the anchoring of radiolabeled molecules
or radionuclide/chelate complexes on the NT surface. However,
a
entry
Pd catalyst
additive (1 equiv)
yields (%)
b
3
1
2
3
4
5
6
7
8
Pd(OAc)
Pd(OAc)
Pd(OAc)
2
2
2
-tertBu
3
P
-
-
6
2
22
23
5
0
0
3
31
11
65
99
b
-nBu P
3
such chemical modification might affect the in ViVo clearance of
b
3
-Ph P
-
b
NTs. In the present paper we describe a new method that allows
Pd(OAc) -DPPE
-
2
1
4
b
the C-labeling of purified MWNTs without modifying their
structure, as well as the results of a preliminary biodistribution study
in rats.
Pd(OAc)
Pd(OAc)
2
-(o-tolyl)
3
P
-
b
2 3
-Ph As
-
PdCl
Pd (dba)
Pd(PPh
Pd(PPh3)4
Pd(PPh
Pd(PPh3)4
2
(PPh
3
)
3 2
-
2
-
MWNTs are generally provided by companies as raw or as
purified NTs (low iron content). Many classical purification
methods, such as those involving acidic treatments, generate
9
)
3 4
-
10
11
12
TBAF
KF
CsF
3 4
)
4
carboxylic acid functions in MWNTs. Based on this finding, we
devised a radiolabeling approach, through a three-step process, that
a
Yields were determined by fluorescence measurements of product 3.
allows carbon isotope exchange of the CO
strategy is presented in Scheme 1 on 9-anthracen carboxylic acid
that was used as a model substrate for the development of the
2
H moiety. The designed
b
7
.5% mol of ligands were used.
1
We then investigated the preparation of 13C-labeled 9-anthracene
process. The anthracene moiety of this compound should mimic
the aromatic network of MWNTs and provides a fluorescent signal
that can be exploited.
12
carboxylic acid from its C-analogue 1 according to Scheme 1 and
using the above optimized decarbonylation reaction. Compound 4
was obtained in three steps with a reasonable global yield (52%)
and an excellent isotopic enrichment (98%). The formation of aroyl
cyanide 2 was the limiting step in terms of efficiency (58% yield)
although the decarbonylation and hydrolysis steps proceeded with
almost quantitative yields. This validated labeling process was then
applied to MWNTs produced in our institute by an aerosol-assisted
Scheme 1. Proposed Labeling Strategy
6
CVD process as previously described. The iron catalyst was
removed, and the MWNTs were shortened by acidic treatments
(average length and diameter: 10 µm and 40 nm, respectively). Each
step of the labeling process was followed by careful repeated
washing to eliminate noncovalent radioactivity. Radioactive count-
ing was used to estimate the efficiency of the labeling process
(Scheme 2). After the first step (i.e., formation of R-keto nitrile), a
specific activity of 0.99 MBq/mg was found. A control experiment
The first step of the process involves the formation of 14C-labeled
14
aroyl cyanides from a classical reaction of Cu CN with aroyl
chlorides. The second step corresponds to a palladium-catalyzed
1
4
decarbonylation of the aroyl cyanides into the corresponding C-
labeled nitriles. This particular reaction has been only sporadically
5
described and therefore represents the key step of the process that
requires optimization efforts. Hydrolysis of the so-formed labeled
nitriles into carboxylic acids should then afford 14C-MWNTs that
are identical to the MWNT starting material. To study the
decarbonylation of aroyl cyanides, we carried out a series of more
than 100 reaction conditions by varying the palladium source, the
2
carried out without SOCl , therefore avoiding the formation aroyl
chlorides, afforded nonradioactive MWNTs. Step 2 was carried out
under the optimized conditions presented in Table 1 followed by
specific basic treatments to convert the remaining labeled R-keto
nitriles into cold carboxylic acids. A control experiment (without
1
4658 9 J. AM. CHEM. SOC. 2009, 131, 14658–14659
10.1021/ja906319z CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
the Pd catalyst) proved that the elimination of nonreacted R-keto
nitriles was efficient. At the end of this process a 2-fold lower
specific activity was obtained suggesting a 50% yield in the
decarbonylation reaction. Finally, alkaline hydrolysis afforded the
14
desired C-MWNTs with a specific activity of 0.45 MBq/mg
corresponding to a 14C/ C ratio of 3/1000. These results have been
confirmed by surface nitrile measurements using X-ray photoelec-
tron microscopy (XPS) conducted on samples issued from each
step of the process (see Supporting Information). Further charac-
terization of the 14C-MWNTs by transmission electron microscopy
12
(
TEM) showed no significant change in their size and shape after
Figure 2. Tissue radioimaging in liver (A), lungs (D, D′′), spleen (B),
the labeling process.
14
kidney (C), and optical microscopy in lungs (D′) of C-MWNT 24 h
postinjection.
Scheme 2. 14C-Labelled MWNT Preparation
laboratory proved that the presented method is general and can be
successfully applied to a panel of commercially available SWNTs,
Hipco, and MWNTs (see Supporting Information). Since many
2
reported molecular anchorings use the CO H functions present in
NTs, the present method offers an interesting way to obtain
radiolabeled starting nanomaterials that can be further functionalized
for desired properties. Radiolabeling with a long-life radioactive
1
4
nucleus like C will make it possible to critically assess for long
time periods (6 months) the biopersistence of NTs in any organs
after animal exposure, as well as the possible crossing of the
pulmonary barrier by NTs after inhalation, a critical safety concern
for humans in the working place.
These 14C-labeled MWNTs were used for preliminary in ViVo
biodistribution studies in rats. C-MWNTs in rat serum (1 mg/
14
mL) were sonicated until the formation of a stable suspension. TEM
analysis of this suspension showed that the 14C-MWNTs were
Acknowledgment. This work was supported by a program of
interdisciplinary research called “Nanoscience” developed by the
CEA. The authors thank Dr. P. Jegou for help in XPS analysis.
shortened by this protocol (Figure 1A and B) but without loss of
radiolabeling according to controls. Three groups of rats (n ) 6)
were intravenously injected with 0.35 mg of 14C-MWNTs (0.15
MBq) and sacrificed 1, 7, and 14 days later. Blood and urine, as
well as organs, were collected after sacrifice. The quantitative
determination of the radioactivity in fluids and tissue sections were
performed with a radioimager (Figure 1C).
Supporting Information Available: Complete analytical data of
anthracene derivatives and screening results. Detailed experimental
procedures of NT labeling including characterization (XPS, TEM, etc.)
data and in vivo experiments. This material is available free of charge
via the Internet at http://pubs.acs.org.
References
(
1) Poland, C. A.; Duffin, R.; Kinloch, I.; Maynard, A.; Wallace, W. A. H.;
Seaton, A.; Stone, V.; Brown, S.; MacNee, W.; Donaldson, K. Nat.
Nanotechnol. 2008, 3, 423–429.
(
2) (a) Cherukuri, P.; Gannon, C. J.; Leeuw, T. K.; Schmidt, H. K.; Smalley,
R. E.; Curley, S. A.; Weisman, R. B. Proc. Natl. Acad. Sci. U.S.A. 2006,
1
03, 18882–18886. (b) Leeuw, T. K.; Reith, R. M.; Simonette, R. A.; Harden,
M. E.; Cherukuri, P.; Tsyboulski, D. A.; Beckingham, K. M.; Weisman,
R. B. Nano Lett. 2007, 7, 2650–2654.
(
3) (a) Deng, X.; Jia, G.; Wang, H.; Sun, H.; Wang, X.; Yang, S.; Wang, S.;
Wang, T.; Liu, Y. Carbon 2007, 45, 1419–1424. (b) Wang, H.; Wang, J.;
Deng, X.; Sun, H.; Shi, Z.; Gu, Z.; Liu, Y.; Zhaoc, Y. J. Nanosci.
Nanotechnol. 2004, 4, 1019–1024. (c) Singh, R.; Pantarotto, D.; Lacerda,
L.; Pastorin, G.; Klumpp, C.; Prato, M.; Bianco, A.; Kostarelos, K. Proc.
Natl. Acad. Sci. U.S.A. 2006, 103, 3357–3362. (d) Liu, Z.; Cai, W.; He, L.;
Nakayama, N.; Chen, K.; Sun, X.; Chen, X.; Dai, H. Nat. Nanotechnol.
Figure 1. (A) Length characterization, (B) TEM picture, and (C) biodis-
tribution of 14C-MWNT after i.v. injection.
The 14C-MWNTs were rapidly cleared from blood and distributed
into organs, with a prominent liver uptake. Lungs, spleen, and
kidneys were the other targeted organs; no radioactivity was
detected in brain, heart, bones, stomach, and muscle. Given the
2
007, 2, 47–52.
(
4) See for example: (a) Liu, J.; Rinzler, A. G.; Dai, H.; Hafner, J. H.; Bradley,
R. K.; Boul, P. J.; Lu, A.; Iverson, T.; Shelimov, K.; Huffman, C. B.;
Rodriguez-Macias, F.; Shon, Y.-S.; Lee, T. R.; Colbert, D. T.; Smalley, R. E.
Science 1998, 280, 1253–1256. (b) Dillon, A. C.; Gennett, T.; Jones, K. M.;
Alleman, J. L.; Parilla, P. A.; Heben, M. J. AdV. Mater. 1999, 11, 1354–
1358. (c) Duesberg, G. S.; Blau, W.; Byrne, H. J.; Muster, J.; Burghard,
M.; Roth, S. Synth. Met. 1999, 103, 2484–2485. (d) Holzinger, M.; Hirsch,
A.; Bernier, P.; Duesberg, G. S.; Burghard, M. Appl. Phys. A 2000, 70, 599–
602. (e) Duesberg, G. S.; Muster, J.; Krstic, V.; Burghard, M.; Roth, S.
Appl. Phys. A 1998, 67, 117–119.
1
4
C-labeling level reported in this study and the threshold of the
14
2
radioimager used for the C detection (0.02 cpm/mm ), up to 10
pg of 14C-MWNTs can be detected. A focus on the 14C-MWNT
distribution within liver and lungs revealed radioactive hot spots
of several µm that were shown to correspond to dark cluster by
(
5) (a) Murahashi, S.-I.; Naota, T.; Nakajima, N. J. Org. Chem. 1986, 51, 898–
901. (b) Brown, R. F. C.; Eastwood, F. W.; Kissler, B. E. Tetrahedron Lett.
1988, 29, 6861–6864. (c) Brown, R. F. C.; Eastwood, F. W.; Kissler, B. E.
Aust. J. Chem. 1989, 42, 1435–1445.
optical microscopy analysis (Figure 2). This observation suggests
that 14C-MWNTs accumulate in the liver and lungs under ag-
gregates, leading to slow elimination from these organs.
In summary, we report a convenient method for the 14C-labeling
of purified MWNTs. Recent investigations conducted in our
(6) Pinault, M.; Pichot, V.; Khodja, H.; Launois, P.; Reynaud, C.; Mayne-
L’Hermite, M. Nano Lett. 2005, 5, 2394–2398.
JA906319Z
J. AM. CHEM. SOC. 9 VOL. 131, NO. 41, 2009 14659