First soluble M @ C60 derivatives provide enhanced access to metallofullerenes and permit in vivo evaluation of Gd @ C60[C(COOH)2]10 as a MRI contrast agent

Article abstract of DOI:10.1021/ja0340984

M @ C₆₀ and related endohedral metallofullerenes comprise a significant portion of the metallofullerene yield in the traditional arc synthesis, but their chemistry and potential applications have been largely overlooked because of their sparse solubility. In this work, procedures are described to solublize Gd @ C₆₀ species for the first time by forming the derivative, Gd @ C₆₀[C(COOCH₂CH₃)₂]₁₀, and its hydrolyzed water-soluble form, Gd @ C₆₀[C(COOH)₂]₁₀. Imparting water solubility to Gd @ C₆₀ permits its evaluation as a magnetic resonance imaging (MRI) contrast agent. Relaxometry measurements for Gd @ C₆₀[C(COOH)₂]₁₀ reveal it to possess a relaxivity (4.6 mM^-1 s^-1 at 20 MHz and 40 °C) comparable to that of commercially available Gd(III) chelate-based MRI agents. An in vivo MRI biodistribution study in a rodent model reveals Gd @ C₆₀[C(COOH)₂]₁₀ to possess the first non-reticuloendothelial system (RES) localizing behavior for a water-soluble endohedral metallofullerene species, consistent with its lack of intermolecular aggregation in solution as determined by light-scattering measurements. This first derivatization and use of a M @ C₆₀ species suggests new potential for metallofullerene technologies by reducing reliance on the chromatographic purification procedures normally employed for the far less abundant M @ C₈₂ and related endohedrals. The recognition that water-soluble fullerene derivatives can be designed to avoid high levels of RES uptake is an important step toward fullerene-based pharmaceutical development.

Full text of DOI:10.1021/ja0340984

Published on Web 04/12/2003
First Soluble M@C60 Derivatives Provide Enhanced Access to
Metallofullerenes and Permit in Vivo Evaluation of
Gd@C60[C(COOH)2]10 as a MRI Contrast Agent
Robert D. Bolskar,*,† Angelo F. Benedetto,‡ Lars O. Husebo,‡ Roger E. Price,§
Edward F. Jackson,§ Sidney Wallace,§ Lon J. Wilson,‡ and J. Michael Alford†
Contribution from TDA Research Inc., 12345 West 52nd AVenue, Wheat Ridge, Colorado 80033,
the Department of Chemistry and the Center for Nanoscale Science and Technology, MS-60,
Rice UniVersity, Houston, Texas 77251-1892, and the MD Anderson Cancer Center, The
UniVersity of Texas, 1515 Holcombe BouleVard, Houston, Texas 77030-4009
Received January 9, 2003; E-mail: bolskar@tda.com
Abstract: M@C60 and related endohedral metallofullerenes comprise a significant portion of the metallo-
fullerene yield in the traditional arc synthesis, but their chemistry and potential applications have been
largely overlooked because of their sparse solubility. In this work, procedures are described to solublize
Gd@C60 species for the first time by forming the derivative, Gd@C60[C(COOCH2CH3)2]10, and its hydrolyzed
water-soluble form, Gd@C60[C(COOH)2]10. Imparting water solubility to Gd@C60 permits its evaluation as
a magnetic resonance imaging (MRI) contrast agent. Relaxometry measurements for Gd@C60[C(COOH)2]10
reveal it to possess a relaxivity (4.6 mM-1 s-1 at 20 MHz and 40 °C) comparable to that of commercially
available Gd(III) chelate-based MRI agents. An in vivo MRI biodistribution study in a rodent model reveals
Gd@C60[C(COOH)2]10 to possess the first non-reticuloendothelial system (RES) localizing behavior for a
water-soluble endohedral metallofullerene species, consistent with its lack of intermolecular aggregation
in solution as determined by light-scattering measurements. This first derivatization and use of a M@C60
species suggests new potential for metallofullerene technologies by reducing reliance on the chromato-
graphic purification procedures normally employed for the far less abundant M@C82 and related endohedrals.
The recognition that water-soluble fullerene derivatives can be designed to avoid high levels of RES uptake
is an important step toward fullerene-based pharmaceutical development.
Introduction
species have been largely overlooked, and they remain some-
what enigmatic. Small amounts of M@C60 species have been
Endohedral fullerenes consist of fullerene cages that encap-
sulate atoms, clusters, or small molecules.1,2 The prototypical
carbon arc synthesis produces mostly insoluble carbonaceous
material (ca. 80%) and soluble empty fullerenes (ca. 15%), while
the typical solvent-extractable metallofullerene yield is ca. 1
wt %.2,3 However, the total metallofullerene product can greatly
exceed this, particularly when M is a lanthanide element. For
example, previous measurements revealed that only 4% of
sublimable Gd endohedrals are of the soluble Gd@C82 variety,
with the balance of the material not soluble in solvents that
normally dissolve fullerenes (hydrocarbons, arenes and haloare-
nes, carbon disulfide, etc.).4 Because of this insolubility, M@C60
solubilized as electroreduced anions4 and by extraction with
chemically noninnocent amine bases such as aniline and
pyridine.5-7 In contrast, the routine solubility of the M@C82
class of metallofullerenes allows handling in a manner similar
to that of soluble C2n fullerenes, but their separation still requires
labor-intensive and expensive multistep HPLC purification,2,3
a limitation hampering advancement of metallofullerene-based
applications.
Here, we describe a method for utilizing the previously
unused, insoluble metallofullerene fractions. The more abundant
M@C60 fraction of endohedrals (consisting of mostly M@C60,
M@C70, and M@C74, and hereafter designated as “M@C60”)
is separated as a mixture from the soluble empty C2n and soluble
† TDA Research.
‡ Rice University.
§ MD Anderson.
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J. AM. CHEM. SOC. 2003, 125, 5471-5478
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A R T I C L E S
Bolskar et al.
M@C2n metallofullerenes, and subsequently solubilized through
derivatization chemistry. The new derivatives of Gd@C60
described below, including Gd@C60[C(COOCH2CH3)2]10 and
Gd@C60[C(COOH)2]10, are the first highly soluble, air-stable
discrete molecules based on the more abundant M@C60 fraction
of metallofullerenes.
Derivatized fullerenes hold promise for medicinal applica-
tions.8,9 For example, C60 derivatives have been shown to inhibit
HIV protease,10 carboxylated C60 derivatives are potent anti-
oxidants and act as neuroprotectants in vivo,11 and endohedral
metallofullerenes offer potential in medicine as they present a
unique motif for metal ion “superchelation” based on entrapment
of metal ions in the fullerene interior space. In this regard,
diagnostic and therapeutic nuclear medicine are among the
proposed applications.8,12,13 Gd-containing metallofullerenes are
currently being pursued as a new generation of magnetic
resonance imaging (MRI) contrast agents8,14,15 because of their
high relaxivities and complete lack of Gd3+ ion release under
metabolic processes. Toward this goal, using a new water-
soluble Gd@C60[C(COOH)2]10 derivative, we demonstrate here
the first metallofullerene-based MRI contrast agent having a
favorable biodistribution similar to those of existing clinically
employed MRI contrast agents. The use of carboxyl functional
groups to water solubilize Gd-metallofullerenes has resolved
the earlier problem of excess reticuloendothelial system (RES)
uptake as seen for polydroxylated fullerene derivatives.
Figure 1. Positive-ion LD-TOF mass spectrum of the “Gd@C60 class” of
fullerenes. (Inset) Expansion of the 870-900 mass region, showing the
isotope patterns for Gd@C60 and the empty fullerene, C74.
of the M@C82 species are air sensitive. Higher yields of soluble
Sc3N@C80 and related endohedrals have been reported,17 but
their purification still relies on costly and time-consuming HPLC
separations of minor components of the arc process. For these
reasons, the relatively low availability of metallofullerenes has
hampered the advancement of metallofullerene-based applica-
tions.
We have pursued the chemistry of the more abundant M@C60
class as a step toward generating larger quantities of metallo-
fullerene-based materials. Here, the Gd@C60 class of endohedral
metallofullerenes was developed first because of our interest
in their potential as MRI contrast agents. Gd-containing
fullerenes were generated by the standard DC arc discharge of
Gd2O3-impregnated graphite rods, using cathode deposit “back-
burning” to maximize the total yield of fullerenes per arc run.
Sublimation at 750 °C and 1 mTorr separated the fullerenes
(including both soluble and insoluble empty fullerenes and
Gd@C2n endohedrals) from the nonfullerene carbon soot.4,18
Exploiting the insolubility of the M@C60 class, the soluble C2n
and Gd@C2n fullerenes were removed from the sublimate by
repeated o-dichlorobenzene washings using a Soxhlet extractor
operating at 40 Torr and 100 °C, until the washings were
colorless. The collection of the sublimate and the Soxhlet
extraction were performed anaerobically inside an argon-filled
glovebox due to the air sensitivity of some endohedral fullerene
materials.2,19
Reductive and oxidative treatments of mixed endohedral
fullerene materials can be used to separate fractions of fullerenes
having similar redox properties from other components with
differing redox properties.4,20 Here, a chemically oxidative
treatment was used to enrich the Gd@C60 content of the
insoluble material by solubilizing and removing several percent
of oxidizable Gd@C2n (2n g 72) and C74. The remaining
insoluble “Gd@C60 fraction” of metallofullerenes, the mass
spectrum of which is shown in Figure 1, is composed primarily
of Gd@C60 and Gd@C74, with smaller amounts of Gd@C70,
empty C74, and other minor Gd@C2n species with 2n > 70.
Only traces of C60, C70, etc. remain in this material. Over 500
Results and Discussion
M@C60 Species and Production of the Gd@C60 Fraction
Starting Material. M@C60 and related metallofullerenes are a
class of molecules completely insoluble in the usual fullerene
solvents. Their insolubility arises from intermolecular polym-
erization caused, at least in part, by their open-shell electronic
configuration and small HOMO-LUMO gaps.4 Largely because
of the M@C60-class insolubility, much of the previous work
with metallofullerenes has instead focused on the soluble
M@C82 class.2 Although this class also has open-shell electronic
configurations, the intermolecular association of certain M@C82
isomers is apparently much weaker because of significant
electron density localization of the unpaired electron inside the
fullerene cage.16 The reported processes for isolating these
soluble M@C82 species from the products of arc synthesis are
labor-intensive and expensive, relying on multistep HPLC
purification using costly specialty columns.2,3 In addition, some
(8) Wilson, L. J. Interface 1999, 8 (4, Winter), 24.
(9) Wilson, S. R. In Fullerenes Chemistry, Physics, and Technology; Kadish,
K. M., Ruoff, R. S., Eds.; John Wiley & Sons: New York, 2000; 437.
(10) Friedman, S. H.; DeCamp, D. L.; Sijbesma, R. P.; Srdanov, G.; Wudl, F.;
Kenyon, G. L. J. Am. Chem. Soc. 1993, 115, 6506.
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1997, 94, 9434.
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G. J.; Wilson, L. J. J. Am. Chem. Soc. 1996, 118, 8043. (b) Wilson, L. J.;
Cagle, D. W.; Thrash, T. P.; Kennel, S. J.; Mirzadeh, S.; Alford, J. M.;
Ehrhardt, G. J. Coord. Chem. ReV. 1999, 190-192, 199. (c) Thrash, T. P.;
Cagle, D. W.; Alford, J. M.; Wright, K.; Ehrhardt, G. J.; Mirzadeh, S.;
Wilson, L. J. Chem. Phys. Lett. 1999, 308, 329.
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Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5182.
(14) Zhang, S.; Sun, D.; Li, X.; Pei, F.; Liu, S. Fullerene Sci. Technol. 1997, 5,
1635.
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Craft, J.; Hadju, E.; Bible, R.; Olmstead, M. M.; Maitra, K.; Fisher, A. J.;
Balch, A. L.; Dorn, H. C. Nature 1999, 401, 55.
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N.; Shinohara, H. Bioconjugate Chem. 2001, 12, 510.
(18) Diener, M. D.; Smith, C. A.; Veirs, K. D. Chem. Mater. 1997, 9, 1773.
(19) Hettich, R.; Lahamer, A.; Zhou, L.; Compton, R. Int. J. Mass. Spectrom.
1999, 182/183, 335.
(16) Kessler, B.; Bringer, A.; Cramm, S.; Schlebusch, C.; Eberhardt, W.; Suzuki,
S.; Achiba, Y.; Esch, F.; Barnaba, M.; Cocco, D. Phys. ReV. Lett. 1997,
79, 2289.
(20) Bolskar, R. D.; Alford, J. M. Chem. Commun. 2003, in press.
9
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In Vivo MRI Evaluation of Carboxylated Gd@C60
A R T I C L E S
mg of the Gd@C60 fraction is readily obtained from ca. 2.5 g
of starting sublimate, using this nonchromatographic process.
This is a considerably larger amount of material than can be
currently obtained by chromatographic separation of only the
soluble metallofullerenes, for example Gd@C82 or the various
soluble M3N@C2n species such as Sc3N@C80.2,17 While Gd@C60
has yet to be isolated as a pure material, it is the dominant
component of this class or fraction of fullerene molecules having
very similar properties (see Figure 1).
Gd@C60 Derivatization: Sythesis of Gd@C60[C(COOCH2-
CH3)2]10 and Gd@C60[C(COOH)2]10. Exohedral derivatization
chemistry of metallofullerenes has developed in a manner
analogous to that of the soluble C2n fullerenes, but more slowly,
due to the scarce amount of available metallofullerene starting
material. Akasaka and co-workers reported the first derivatiza-
tions of La@C82, Gd@C82, La2@C80, and Sc2@C84 with
disiliranes and digermanes.21 Suzuki and co-workers then
reported the reaction of La@C82 with substituted diazomethanes
to form methanofullerene derivatives.22 Gu and co-workers
reported generation of methanofullerene derivatives of Tb@C82
by Cu(I)-catalyzed addition of R-diazocarbonyl compounds.23
Additionally, several different groups have reported polyhy-
droxylation of the metallofullerene cages Ho@C82, Ho2@C82,
Pr@C82, and Gd@C82.8,13-15,24 However, these reported deriva-
tizations began with metallofullerenes already soluble in the
reaction medium, which in most cases was toluene or a similar
“standard” fullerene solvent. In contrast, the new process
described below is highly effective toward derivatizing insoluble
fullerenes, and it is this exohedral chemical modification which
now allows the endohedral fullerenes that previously went
unused as waste (including M@C60) to now be utilized.
Figure 2. Positive-ion MALDI-TOF mass spectra of the Gd@C60[C-
(COOCH2CH3)2]10 derivative product (S8 matrix): (a) low-resolution mass
spectrum; (b) high-resolution mass spectrum; the inset expands the 1650-
1700 mass region.
temperature with a ca. 15-fold excess of diethyl bromomalonate
and alkali metal hydride (NaH or KH), the Gd@C60-fraction
material is rapidly derivatized with multiple malonate ester
groups, which readily solubilize this otherwise intractable
material by breaking up the polymer into individually soluble
molecules. Hundreds of milligrams of a dark-brown, highly
organic-soluble and air-stable derivative can be obtained in only
minutes without heating.
The cycloaddition reaction widely used to add functionalities
across carbon-carbon double bonds of fullerenes is the base-
induced Michael addition of malonates first reported for C60
by Bingel25 and later expanded upon by Hirsch and co-
workers.26 These reaction conditions (hydrocarbon solvents such
as toluene and sodium hydride or 1,8-diazabicyclo[5.4.0]undec-
7-ene (DBU) as the base) are not optimal with metallofullerenes,
however. DBU use is not preferred with metallofullerenes
because, like basic nitrogen solvents (pyridine, aniline, di-
methylformamide, etc.),5-7 it adds readily to fullerene surfaces,27
a problem only exacerbated by the metallofullerenes’ electro-
negativity. For the Gd@C60-fraction metallofullerenes, we have
developed reaction conditions that derivatize and solubilize the
insoluble polymer material without requiring their prior dis-
solution. Using tetrahydrofuran (THF) as solvent at room
Mass spectral analysis of this product (Figure 2) reveals it to
be chiefly composed of Gd@C60[C(COOCH2CH3)2]x,with the
parent ion peak at x ) 10. Two laser-desorption mass spectra
taken under different conditions (see Experimental Section) of
this product are shown, Figure 2a with low-resolution conditions
and Figure 2b with higher-resolution conditions. Exohedral
fullerene derivatives are well-known to fragment in the gas phase
under laser desorption and other sampling conditions, generating
mass spectral peaks for derivatives having fewer adduct groups
per fullerene sphere than are present for the parent compound.
Figure 2a shows, despite low resolution, a preponderance of x
) 9 and 10 for the parent ion peaks, and while Figure 2b does
show x ) 10 for the parent ion peak, it also shows a greater
intensity for the lower-mass adduct peaks. Also visible in Figure
2b is the Gd isotope pattern for the derivatives x ) 5 and lower
(see inset) that provides unambiguous evidence for the deriva-
tization of the Gd@C60 molecule. The Gd isotope pattern is
not readily discernible for the higher-mass adduct peaks, most
likely because of the high degree of fragmentation operating
with the parent ion and higher adducts. Very minor peaks for
other derivatized fullerene cages (e.g., Gd@C70, Gd@C74, etc.;
see Figure 1) are also present. The differences between the two
spectra in Figure 2 underscore the difficulty in analyzing
fullerene derivatives by mass spectroscopy, but the data serves
(21) (a) Akasaka, T.; Kato, T.; Kobayashi, K.; Nagase, S.; Yamamoto, K.;
Funasaka, H.; Takahashi, T. Nature 1995, 374, 600. (b) Akasaka, T.;
Nagase, S.; Kobayashi, K.; Suzuki, T.; Kato, T.; Kikuchi, K.; Achiba, Y.;
Yamamoto, K.; Funasaka, H.; Takahashi, T. Angew. Chem., Intl. Ed. Engl.
1995, 34, 2139. (c) Akasaka, T.; Nagase, S.; Kobayashi, K.; Suzuki, T.;
Kato, T.; Yamamoto, K.; Funasaka, H.; Takahashi, T. Chem. Commun.
1995, 1343.
(22) Suzuki, T.; Maruyama, Y.; Kato, T.; Akasaka, T.; Kobayashi, K.; Nagase,
S.; Yamamoto, K.; Funasaka, H.; Takahashi, T. J. Am. Chem. Soc. 1995,
117, 9606.
(23) Feng, L.; Zhang, X.; Yu, Z.; Wang, J.; Gu, Z. Chem. Mater. 2002, 14,
4021
(24) Sun, D.; Huang, H.; Yang, S.; Liu, Z.; Liu, S. Chem. Mater. 1999, 11,
374.
(25) Bingel, C. Chem. Ber. 1993, 126, 1957.
(26) Hirsch, A.; Lamparth, I.; Karfunkel, H. R. Angew. Chem., Int. Ed. 1994,
33, 437.
(27) Skiebe, A.; Hirsch, A.; Klos, H.; Gotschy, B. Chem. Phys. Lett. 1994, 220,
138.
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A R T I C L E S
Bolskar et al.
pounds,14 while Wilson et al. reported r1 ) 20 mM-1 s-1 for
Gd@C82(OH)x (at 0.47 T and 40 °C).8 More recently, Shinohara
and co-workers reported Gd@C82(OH)x (x ≈ 40) with an r1
value of 67 mM-1 s-1 (at 0.47 T and 25 °C) and r1 ) 81 mM-1
s-1 (at 1.0 T and 25 °C).15 These r1 values are all higher than
the relaxivities of clinically used Gd(III) chelates and demon-
strate that Gd metallofullerene compounds can serve as potent
T1 relaxation agents for water protons.32
In this work, the measured r1 relaxivity in water for Gd@C60-
[C(COOH)2]10 is 4.6 mM-1 s-1 (at 20 MHz, 40 °C, and based
on Gd content by ICP analysis). This is comparable to the best
[GdIII(chelate)] MRI contrast agents in the marketplace today
such as ProHance ([Gd(HP-DO3A)(H2O)]; r1 ) 3.6 mM-1 s-1
)
and Magnevist ([Gd(DTPA)(H2O)]2-; r1 ) 4.3 mM-1 s-1) un-
der similar conditions.30 For comparison, La@C60[C(COOH)2]x
was prepared in a manner analogous to that for Gd@C60-
[C(COOH)2]10. This La-containing metallofullerene should be
similar in electronic structure to its Gd analogue, as both contain
trivalent endohedral lanthanide metals (resulting in one unpaired
electron on the fullerene cage); however, with its d0 configu-
ration, La3+ has no metal-centered unpaired f electrons. The r1
relaxivity of La@C60[C(COOH)2]x in water was determined to
be less than 1 mM-1 s-1 (ca. 0.3 mM-1 s-1 at 20 MHz and 40
°C with x ≈ 10).
Figure 3. Ball-and-stick depiction of Gd@C60[C(COOH)2]10, illustrating
a possible arrangement of 10 C(COOH)2 addends on a single C60 cage (light
blue, C; red, O; white, H; dark blue, Gd).
to establish the identity of the Gd@C60[C(COOCH2CH3)2]10
derivative material.
This ester derivative was readily converted into the water-
soluble Gd@C60[C(COOH)2]10 carboxylate acid using the
method reported by Hirsch for the conversion of C2n[C(COOCH2-
CH3)2]x to the corresponding C2n[C(COOH)2]x species.28 Figure
3 illustrates a likely structural isomer, consistent with the
addition pattern proposed for the deca-addition of 1,8-naphthyne
to C60.29
These results raise some important points and generate
questions about the nature of metallofullerene-induced relaxivity
in water. While the magnitude of the relaxivity for Gd@C60-
[C(COOH)2]10 is nearly the same as for the [GdIII(chelate)]
compounds, the relaxation mechanisms must differ, since water
molecules have no direct access to a Gd3+ ion inside the
fullerene carbon cage. Most plausible is an outer-sphere
relaxation mechanism with water molecules hydrogen-bonded
to the water-solubilizing groups on the fullerene surface, with
the unpaired f electrons of the encaged Gd3+ ion magnetically
coupled to the unpaired electron in the fullerene-centered
molecular orbital, which itself transfers some spin density to
the substituents. This explanation is consistent with the large
drop-off in relaxivity seen in going from the carboxylated
Gd@C60 compound (seven f electrons) to the La@C60 analogue
(zero f electrons).
The first derivatization of Gd@C60 reported here is significant
for several reasons. It provides a solution to the long-standing
problem of how to exploit the polymerized M@C60 species that,
while more abundant than soluble M@C82 metallofullerenes,
previously went unused. Using more polar solvents such as
tetrahydrofuran (instead of nonpolar hydrocarbons such as
toluene) allows the incipient malonate carbanion to derivatize
the solid Gd@C60 surface in an apparently heterogeneous solid-/
solution-phase reaction. The reaction proceeds very rapidly to
the deca-addition stage without heating (unlike the traditional
Bingel and Hirsch conditions) with the exohedral derivatization
breaking up the intermolecular polymerization of Gd@C60.
Finally, starting from the Gd@C60-containing arc product, the
process is easily scalable to produce hundreds of milligrams of
the water-soluble, air-stable Gd@C60[C(COOH)2]10 product over
several days.
Another issue raised by the relaxivity results for Gd@C60-
[C(COOH)2]10 is why the relaxivities of Gd@C82(OH)x com-
pounds are up to an order of magnitude higher. Also unexplained
is the significant variation in the r1 relaxivity values reported
for the various Gd@C82(OH)x preparations. A contributing factor
to the former may be the closer proximity of proton-water
exchange to the paramagnetic fullerene surface for the poly-
hydroxyl derivatives. A possible explanation for the latter is
that different syntheses of the polyhydroxyl fullerene compounds
give somewhat different materials both in number and disposi-
tion of the hydroxyl groups. Both of these issues are challenging
questions to unravel, since definitive spectroscopic and structural
characterization of polyhydroxyl fullerene compounds is difficult
to achieve. While the elevated relaxivities of the polyhydroxyl
materials remain incompletely explained by the above consid-
erations, a likely explanation does emerge if intermolecular
aggregation of water-soluble fullerene compounds is considered.
Magnetic Relaxivity Measurements. Measuring the r1
“relaxivity′ of a water-soluble paramagnetic compound is a
quantitative way to compare its efficacy in relaxing solvent water
protons (shortening T1 or the longitudinal relaxation time) to
that of other paramagnetic ions and their complexes.30,31 Several
different groups have identified water-solubilized polyhydroxyl
Gd metallofullerene compounds as potential MRI contrast
agents, with each reporting different r1 values. Zhang et al.
measured r1 ) 47 mM-1 s-1 (at 9.4 T) for a mixed sample of
empty fullerene and Gd metallofullerene polyhydroxyl com-
(28) (a) Lamparth, I.; Hirsch, A. Chem. Commun. 1994, 1727. (b) Lamparth,
I.; Schick, G.; Hirsch, A. Liebigs Ann./Recl. 1997, 253.
(29) Hoke, S. H., II; Molstad, J.; Yang, S.-S.; Carlson, D.; Kahr, B. J. Org.
Chem. 1994, 59, 3230.
(30) (a) Lauffer, R. B. Chem. ReV. 1987, 87, 901. (b) Caravan, P.; Ellison, J. J.;
McMurry, T. J.; Lauffer, R. B. Chem. ReV. 1999, 99, 2293.
(31) To´th, E.; Helm, L.; Merbach, A. In The Chemistry of Contrast Agents in
Medical Magnetic Resonance Imaging; Merbach, A., To´th, E., Eds.; John
Wiley & Sons: Chichester, 2001; pp 45-120.
(32) As relaxivity measurements are dependent on temperature and magnetic
field, it is most meaningful to compare r1 values obtained under the same
conditions.
9
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In Vivo MRI Evaluation of Carboxylated Gd@C60
A R T I C L E S
Assessing Intermolecular Aggregation. Intermolecularly
aggregated MRI contrast agents are known to exhibit increased
rotational correlation times, which results in enhanced relax-
ivities relative to nonaggregated agents.31,33 Thus, the propen-
sity of water-soluble fullerene derivatives toward intermolecu-
lar aggregation or spontaneous clustering in aqueous solution
would have consequences for interpreting their measured
relaxivities. Indeed, previous independent laser-light scattering,
small-angle neutron scattering, and small-angle X-ray scattering
measurements on several water-soluble fullerene derivatives
provide complimentary experimental evidence for aggregation.
Guldi and co-workers have found evidence of aggregation for
water-soluble C60 derivatives, including C60(OH)18 and C60-
[C(COOH)2].34 Pulse radiolysis and triplet lifetime measure-
ments on the C60 monoadduct, C60[C(COOH)2], suggested
aggregation in aqueous solution.34a,c Pulse radiolysis and optical
absorption spectroscopy with C60(C4H10N+) also revealed ag-
gregation for this monoadduct.34b,c Guldi concluded that covalent
attachment of only one addend to the C60 surface was insuf-
ficient to prevent hydrophobic attraction and aggregation of these
derivatives. Bensasson et al. reported that lower singlet oxygen
quantum yields for C60[C(COOH)2]n derivatives (with n ) 2-6)
in aqueous solution as compared to those for the corresponding
ethyl esters in toluene was indicative of clustering of the acids
in water.35
Several aggregation mechanisms may contribute to the
clustering of these water-soluble fullerene derivatives, including
both intermolecular hydrogen bonding and hydrophobic
fullerene-fullerene attraction with the degree of aggregation
varying with concentration. There may be a minimum level of
derivatization needed for sufficient solubility and minimization
of hydrophobic interactions, the extent of which is dependent
on the fullerene cage as well as the type of derivative group.
Because of the above considerations, we have performed a
qualitative assessment of aggregation of water-solubilized
Gd@C60 compounds by dynamic light scattering (DLS). DLS
measurements comparing polyhydroxylated fullerene com-
pounds such as Gd@C60(OH)x and Gd@C60[C(COOH)2]10 under
the same conditions revealed that the polyhydroxylated ful-
lerenes form aggregates in excess of 100 nm in diameter, while
Gd@C60[C(COOH)2]10 displayed no aggregation down to the
instrumental detection limit (∼10 nm). This order-of-magnitude
difference in aggregation propensity correlates with the elevated
r1 values seen for the polyhydroxyl compounds in comparison
to that for Gd@C60[C(COOH)2]10. Large clusters of slowly
tumbling polyhydroxyl fullerenes will have higher relative
relaxivities than comparable molecules that are not intermo-
lecularly aggregated. The decamethano Gd@C60 compound
(with its 20 carboxyl groups) lends itself to less intermolecular
aggregation than the polyhydroxylated fullerenes because of its
highly charged surface. Also, the steric disposition of the 10
derivative groups (Figure 3) protects the underivatized fullerene
surface(s) of Gd@C60[C(COOH)2]10 from hydrophobic-induced
aggregation. Thus, the structure and identity of the derivative
groups on the Gd@C60[C(COOH)2]10 inhibits intermolecular
aggregation that dominates the solution structure of polyhy-
droxyl fullerene derivatives. Comparative in vivo biodistribu-
tion studies of Gd@C60[C(COOH)2]10 and M@C82(OH)x species
also support this aggregation hypothesis, as discussed below.
In Vivo MRI Measurements and Biodistribution Data for
Gd@C60[C(COOH)2]10. To study the biodistribution of Gd@C60-
[C(COOH)2]10 in vivo, an MR imaging experiment with a rodent
model was performed at an approximate dosage of 35 mg/kg,
which was well tolerated by the animal. The MR imaging
experiment (see Experimental Section) revealed that Gd@C60-
[C(COOH)2]10 behaved much like commercially available Gd
chelate-based MRI contrast agents (e.g., Prohance and Magne-
vist) in this animal model.39 Typical biodistribution results are
shown in Figures 4 and 5. The Gd@C60[C(COOH)2]10 agent
moved rapidly to the kidneys, with only minimal uptake into
the liver as illustrated graphically in Figure 4, which compares
the relative MRI enhancement proffered by the metallofullerene
derivative in the kidney vs that in the liver in the minutes after
injection. Figure 5 shows examples of the MR images obtained,
comparing the MRI intensity of one kidney before contrast agent
injection and again 16 min after injection. The lighter color of
the kidney in the latter image results from the proton relaxation
induced by the paramagnetic Gd@C60[C(COOH)2]10 contrast
agent. The agent began undergoing excretion via the bladder
within 1 h of injection. This biodistribution behavior is in
striking contrast to that seen previously with polyhydroxylated
fullerene derivatives.
Furthermore, dynamic light-scattering measurements on the
polyhydroxyl C60 compound C60(OH)18 showed evidence for
aggregates at high solute concentrations (up to 39 mM).34d
A
small-angle X-ray scattering study by Chiang and co-workers
measured C60(OH)18 aggregates in aqueous solution of 20 Å
Rg (Rg ) radius of gyration) at 0.7 mM, with the aggregates
doubling in size to 40 Å Rg at 50 mM.36 They also found water-
soluble C60[(CH2)4SO3Na]6 to have 19 Å Rg across the range
of concentrations from 0.4 to 26 mM in aqueous solution.36
Dynamic light-scattering measurements on a highly water-
soluble dendro-C60 monoadduct derivative (having a second-
generation bis(polyamide) malonate dendrimer with 18 carbox-
ylate groups) by Brettreich and Hirsch revealed clusters of at
least two different size ranges. Clusters with average hydrody-
namic radii of ca. 10 and 38 nm were seen at pH ) 8, with a
decrease in size to 5 nm for the smaller clusters at pH ) 11.37
Chu, Sawamura, Nakamura, and co-workers recently reported
that water-soluble Ph5C60K forms vesicles having a hydrody-
namic radius of 17 nm as detected by laser-light scattering.38
These different examples reveal that intermolecular aggregation
of water-soluble fullerene derivatives occurs with a range of
different derivative groups having a variety of dispositions on
fullerene cages.
(33) Fatin-Rouge, N.; Toth, E.; Perret, D.; Backer, R. H.; Merbach, A. E.; Bunzli,
J.-C. G. J. Am. Chem. Soc. 2000, 122, 10810.
(34) (a) Guldi, D. M.; Hungerbu¨hler, H.; Asmus, K.-D. J. Phys. Chem. 1995,
99, 13487. (b) Guldi, D. M.; Hungerbu¨hler, H.; Asmus, K.-D. J. Phys.
Chem. A 1997, 101, 1783. (c) Guldi, D. M. J. Phys. Chem. A 1997, 101,
3895. (d) Mohan, H.; Palit, D. K.; Mittal, J. P.; Chiang, L. Y.; Asmus,
K.-D.; Guldi, D. M. J. Chem. Soc., Faraday Trans. 1998, 94, 359.
(35) Bensasson, R. V.; Berberan-Santos, M. N.; Brettreich, M.; Frederiksen, J.;
Go¨ttinger, H.; Hirsch, A.; Land, E. J.; Leach, S.; McGarvey, D. J.;
Scho¨nberger, H.; Schro¨der, C. Phys. Chem. Chem. Phys. 2001, 3, 4679.
(36) (a) Jeng, U.-S.; Lin, T.-L.; Chang, T. S.; Lee, H.-Y.; Hsu, C.-H.; Hsieh,
Y.-W.; Canteenwala, T.; Chiang, L. Y. Prog. Colloid Polym. Sci. 2001,
118, 232. (b) Jeng, U.-S.; Lin, T.-L.; Tsao, C.-S.; Lee, C.-H.; Canteenwala,
T.; Wang, L. Y.; Chiang, L. Y.; Han, C. C. J. Phys. Chem. B 1999, 103,
1059.
(38) Zhou, S.; Burger, C.; Chu, B.; Sawamura, M.; Nagahama, N.; Toganoh,
M.; Hackler, U. E.; Isobe, H.; Nakamura, E. Science 2001, 291, 1944.
(39) Wedeking, P.; Eaton, S.; Covell, D. G.; Nair, S.; Tweedle, M. F.; Eckelman,
W. C. Magn. Res. Imag. 1990, 8, 567.
(37) Brettreich, M.; Hirsch, A. Tetrahedron Lett. 1998, 39, 2731.
9
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A R T I C L E S
Bolskar et al.
polyhydroxyl derivatization of the fullerene cage, while the
differences among these compounds (different cage sizes,
different endohedral metals or lack thereof, different electronic
structures, etc.) apparently do not significantly affect the
biodistribution. It also appears that differing degrees of hy-
droxylation do not produce a major impact on the observed
biodistributions.
The RES uptake of polyhydroxyl fullerene compounds has
important implications for the development of pharmaceuticals
based on water-soluble fullerenes. Shinohara and co-workers
have noted that polyhydroxyl fullerene compounds induce
spontaneous aggregation of erythrocytes when in contact with
blood and that addition of mannosyl groups to a polyhydroxyl
C
60 surface diminishes this effect.41 It is not yet clear if in vivo
RES uptake stems from intermolecular aggregation causing
larger particles to be targeted by the RES or if the RES uptake
results from polyhydroxyl fullerene-induced aggregation of
erythrocytes (or other blood components/proteins), which are
then targeted by the RES. A combination of the two actions
seems plausible, but one must note that the intermolecular
clustering as measured by light-scattering techniques and as
inferred by relaxivity measurements does not require the
presence of blood or blood components to be manifested. For
fullerene-based pharmaceuticals to be successful, sufficient water
solubility without significant RES uptake is required, and the
new Gd@C60[C(COOH)2]10 species of this report demonstrates
that this goal can be realized for a fullerene-based material.
Figure 4. Representative in vivo MRI intensity-derived biodistribution data
showing the Gd@C60[C(COOH)2]10 signal enhancement within the first 5
min of administration, revealing rapid renal uptake with a minimum of liver
uptake (red circles, kidney; blue squares, liver).
Conclusion
For some years now empty fullerenes, mostly C60 and C70,
have been readily available.42 Many applications have been
proposed, and after more than a decade of research, their use
in electronics43 and medicine8-11 now seems imminent. En-
dohedral metallofullerenes are also expected to contribute to
these areas, but their development has been hampered by their
low availability.
This contribution reports new methodology whereby highly
soluble Gd@C60 molecular species have been obtained for the
first time by a nonchromatographic procedure. This procedure
increases the efficient use of metallofullerene products from the
traditional arc synthesis by accessing previously unused M@C60-
fraction metallofullerenes. The derivatization procedure solu-
bilizes individual Gd@C60 molecules via multiple cycloaddi-
tion of bromomalonates, the utility of which has been demon-
strated by the production of several hundred milligrams of
Gd@C60[C(COOCH2CH3)2]10 and Gd@C60[C(COOH)2]10.
The potential of Gd@C60[C(COOH)2]10 as a new MRI
contrast agent has also been evaluated by in vivo MRI
biodistribution measurements, relaxometry, and dynamic light
scattering. Biodistribution results comparing the M@C2n(OH)x
species with Gd@C60[C(COOH)2]10 complement the light-
scattering and relaxivity measurements, revealing aggregation
for the polyhydroxyl fullerene derivatives but not for the
carboxylated fullerene derivative. The emerging pattern is one
showing that aggregated polyhydroxyl fullerene derivatives
concentrate in the organs of the RES with little or very slow
Figure 5. Representative in vivo rodent MR images focusing on a cross
section containing a portion of one kidney. (a) Baseline image without
contrast agent; (b) image of the same cross section 16 min after administra-
tion of Gd@C60[C(COOH)2]10 with increased signal intensity in the kidney.
Several complimentary studies on the biodistribution of the
polyhydroxylated compounds 166Hon@C82(OH)x (n ) 1, 2; x
≈ 16), Gd@C82(OH)x (x ≈ 40) and 99mTc-labeled C60(OH)x have
recently revealed high uptake levels of these compounds by the
reticuloendothelial system (RES). The radiotracer study con-
ducted by Cagle et al. with 166Hon@C82(OH)x (n ) 1, 2; x ≈
16) showed significant RES uptake in mice, including concen-
tration in liver and bone.13 The MR imaging and biodistribution
study performed by Shinohara and co-workers with Gd@C82-
(OH)x (x ≈ 40) reported similar results.15 Qingnuan et al. have
recently published a radiotracer study with the (nonendohedral)
polyhydroxyl C60 derivative 99mTc-C60(OH)x.40 The biodistri-
bution results in mice and rabbits also showed significant uptake
of the polyhydroxyl fullerene by the kidneys, bone, spleen, and
liver. The common feature in all of these studies is the
(41) Kato, H.; Yashiro, A.; Mizuno, A.; Nishida, Y.; Kobayashi, K.; Shinohara,
H. Bioorg. Med. Chem. Lett. 2001, 11, 2935.
(42) Kra¨tschmer, W.; Lamb, L. D.; Fostiropoulos, K.; Huffman, D. R. Nature
1990, 347, 354.
(40) Qingnuan, L.; Yan, X.; Xiaodong, Z.; Ruili, L.; Qieqie, D.; Xiaoguang,
S.; Shaoliang, C.; Wenxin, L. Nuclear Med. Biol. 2002, 29, 707.
(43) Hinokuma, K.; Ata, M. Chem. Phys. Lett. 2001, 341, 442.
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In Vivo MRI Evaluation of Carboxylated Gd@C60
A R T I C L E S
further treated with aluminum trichloride in o-dichlorobenzene to
deplete the amount of C74.4,20 The solids were collected by filtration,
rinsed with dichloromethane and hexane, and then dried under vacuum.
The resulting material is composed of the Gd@C60-dominated fraction
of fullerenes, including chiefly Gd@C60 with smaller amounts of
Gd@C70, Gd@C74, other higher Gd@C2n and C74 (with negligible
amounts of C60 and C70).
clearance, while Gd@C60[C(COOH)2]10 does not. In fact, this
carboxylated metallofullerene derivative is the first fullerene
compound proven to exhibit a favorable (non-RES localizing)
biodistribution. The development of a nonaggregating and non-
RES-targeting fullerene derivative is a significant finding that
should contribute to the development of water-soluble fullerene-
and metallofullerene-based pharmaceuticals. It now seems
reasonable that a variety of fullerene-based materials can be
developed for a range of both extra- and intercellular drug
delivery.44 The present report also suggests that metallo-
fullerenes, as derivatized M@C60 species, are now more likely
to play a role in these developments for medicine and other
nanotechnologies as well.
Derivatization of Gd@C60: Synthesis of Gd@C60[C(COOCH2-
CH3)2]10. In a typical synthesis of Gd@C60[C(COOCH2CH3)2]10
(conducted in the glovebox), a suspension of Gd@C60 (307 mg, 0.350
mmol) and KH (210 mg, 5.25 mmol) (NaH is an acceptable substitute)
was prepared in THF (20 mL) with vigorous stirring (15 min). With
continued stirring, diethyl bromomalonate (1.255 g, 5.25 mmol) in THF
(∼1 mL) is added dropwise to the mixture. Vigorous bubbling is
immediately observed (evolution of H2(g)), and a dark-brown solution
color develops within minutes. The mixture was stirred (30 min) after
which the dark-brown soluble derivative was separated from excess
alkali hydride and small amounts of unreacted fullerene material by
filtration (0.45 µm PTFE filter). The product was isolated by THF
removal under reduced pressure, rinsed with hexanes, and dried under
reduced pressure (yield 331 mg, 41%). FTIR, KBr matrix: C-H
Experimental Section
General. Gd(NO3)3‚6H2O (99.9%) was purchased from Strem
Chemicals and used as received. All other chemicals were purchased
from Sigma-Aldrich and used as received. Solvents were distilled and
dried under inert atmosphere according to standard procedures, except
for carbon disulfide, which was used as received. Inert atmosphere
manipulations were conducted inside a Vacuum Atmospheres glovebox
under argon (O2, H2O < 5 ppm). Mass spectrometry was performed
with two separate instruments having different resolutions; higher-
resolution spectra were obtained with a Bruker BiflexTM III MALDI-
TOF MS (N2 laser, λ ) 337 nm), and lower-resolution spectra were
obtained with a custom-built laser-desorption combination linear and
reflectron time-of-flight mass spectrometer (Nd:YAG laser, λ ) 355
nm). A sulfur matrix deposited from a carbon disulfide solution was
used for derivative mass spectra when indicated. Fourier transform
infrared spectroscopy was conducted with a Nicolet Magna-IR 550
FTIR spectrometer.
aliphatic stretch, 2980, 2927, 2855 cm-1; CdO stretch, 1743 cm-1
.
Conversion of Gd@C60[C(COOCH2CH3)2]10 to Gd@C60[C-
(COOH)2]10. Conversion of Gd@C60[C(COOCH2CH3)2]10 to the water-
soluble carboxylate salt Gd@C60[C(COOH)2]10 was accomplished by
reflux in toluene with NaH followed by a methanol quench, according
to the method reported by Lamparth and Hirsch for the conversion of
C2n[C(COOCH2CH3)2]x to C2n[C(COOH)2]x.28 Lamparth and Hirsch
speculate that this transformation takes place via trace nucleophilic OH-
formed by reaction NaH with trace water in the methanol or by
hydrogenolysis of the O-Et bonds.28b The aqueous-soluble product was
converted to the free acid by passage over an acid-form ion-exchange
chromatography column (without Gd loss). Next, the solution pH was
adjusted to 7.0 with NaOH, and the product was dried under reduced
pressure at room temperature. FTIR, KBr matrix: O-H, 3425 cm-1
(v br); CdO asymmetric stretch, 1743 cm-1; CdO symmetric stretch,
Arc Fullerene Production of Mixed C2n/Gd@C2n. Gd2O3 impreg-
nated graphite rods (0.25 in. diameter Poco Graphite, 40% porosity)
doped to a level of ca. 1% Gd were produced according to published
procedures.12a The graphite rods were first evacuated (∼1 Torr) and
then soaked in a saturated absolute ethanolic solution of Gd(NO3)3‚
6H2O for 30 min. The solution-saturated rods were air-dried and then
heated in a quartz furnace at ca. 850 °C under vacuum for 3 h,
converting the metal nitrate to the oxide. Gd-containing fullerene soot
was generated by the standard direct current (DC) arc-discharge of
Gd2O3-impregnated 6 in. graphite rods using a custom-built arc
apparatus, operating at 150 Torr of helium. Cathode deposit “back-
burning” was employed to maximize the yield of fullerenes and
metallofullerenes. “Back-burning” (reverse-polarity arcing) consists of
periodically briefly reversing the arc polarity so as to arc the solid
deposits of material formed on the cathode (relative to the original
polarity). Anaerobic sublimation of the raw arc-produced soot4,18 onto
an isolated, water-cooled coldfinger inside the arc chamber at 750 °C
separated the fullerenes (a mixture of soluble and insoluble empty
fullerenes and Gd@C2n endohedral metallofullerenes) from the non-
fullerene carbon soot. Approximately 2.5 g of sublimed fullerenes (the
“sublimate”) per 10 rod arc run was obtained.
1146 cm-1
.
Relaxivity Measurements. Single-point r1 relaxivity measurements
(expressed by the relationship (1/T1)obsd ) (1/T1)d + r1[solute]) in
aqueous solution were conducted using an IBM PC/20 MiniSpec
Relaxometer operating at 40 °C and a fixed field of 0.47 T (20 MHz).
All relaxivity data on Gd metallofullerene compounds calculated the
r1 values in terms of Gd content, which was independently determined
by ICP-AES.
Dynamic Light-Scattering Measurements. DLS measurements
were performed using a Coulter N4 Plus Dynamic Light Scattering
instrument (detection angle 90°) with a lower detection limit of ca. 10
nm in diameter; samples that did not scatter light to a significant degree
were judged not to contain particles of sufficient size. DLS measure-
ments in aqueous solution at pH ≈ 7 comparing C60(OH)x, Gd@C2n-
(OH)x, and Gd@C60[C(COOH)2]10 species demonstrated aggregation
of the polyhydroxylated compounds (having aggregates in excess of
100 nm diameter), but no such aggregation was observed for the
polycarboxylated species.
In Vivo MRI Measurements. To study the MRI contrast and
biodistribution behavior of Gd@C60[C(COOH)2]10 in vivo, an experi-
ment was performed using a Fischer 344 female rat (Sasco, Wilmington,
MA) weighing 200-220 g and housed at the Department of Veterinary
Medicine and Surgery, University of Texas, M. D. Anderson Cancer
Center (Houston, TX) with all procedures conforming to institutional
guidelines for animal welfare. The rat was injected via the tail vein
with 1 mL of a 3 mM solution of Gd@C60[C(COOH)2]10 in saline at
pH ) 7.4. The dosage was approximately 35 mg/kg, which was well
tolerated by the animal. Images were acquired on a 1.5 T GE LX
EchoSpeed scanner (GE Medical Systems, Milwaukee, WI) using a
custom spiral surface coil. The animal was scanned in the prone position
Separation of the Gd@C60 fraction. All soluble fullerenes were
then removed from the anaerobically collected sublimate by repeatedly
washing with o-dichlorobenzene inside the argon-filled glovebox using
a continuous-cycling (Soxhlet-style) extractor operating at 40 Torr and
100 °C, until the washings were colorless. Next, the solids were
extracted in dichloromethane suspension with a solution of excess tris-
(p-bromophenyl)aminium hexachloroantimonate, which solubilized
small amounts of oxidizable Gd@C2n species (e.g., Gd@C82 and other
Gd@C2n).20 The solids were separated from the dark-brown filtrate and
(44) Wharton, T.; Kini, V. U.; Mortis, R. A.; Wilson, L. J. Tetrahedron Lett.
2001, 42, 5159.
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in the MRI experiment. Prior to contrast agent administration, coronal
flow-compensated T1-weighted fast spin-echo images were acquired
from 18 2-mm contiguous sections (echo time of 15 ms, repetition time
of 400 ms, echo train length of 2, field-of-view of 12 cm × 12 cm,
256 × 192 matrix, four averages). Immediately before, during, and
after contrast agent administration, a dynamic fast spin-echo sequence
was used to acquire T1-weighted images from 5 2-mm sections (with
2-mm gaps) with a temporal resolution of 11 s (echo time of 14 ms,
repetition time of 400 ms, echo train length of 4, field-of-view of 12
cm × 9 cm, 256 × 128 matrix, one average). The total duration of the
dynamic scanning sequence was 5 min. Following the dynamic
acquisition, fast spin-echo T1-weighted images were acquired from
the 18 2-mm contiguous sections (using the parameters listed above)
at 10-, 20-, 30-, and 45-min post-contrast agent infusion.
Acknowledgment. We thank the NIH for support (SBIR
Grant 5-R44-CA66383 and Grant 1-R01-EB000703). L.J.W.
thanks the Robert A. Welch Foundation (Grant C-0627) for
additional support.
Supporting Information Available: Mass spectra of Gd@C2n
containing arc-produced soot and sublimate (PDF). This material
is available free of charge via the Internet at http://pubs.acs.org.
JA0340984
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