Substituted carborane-appended water-soluble single-wall carbon nanotubes: New approach to boron neutron capture therapy drug delivery

Article abstract of DOI:10.1021/ja0517116

Substituted C₂B₁₀ carborane cages have been successfully attached to the side walls of single-wall carbon nanotubes (SWCNTs) via nitrene cycloaddition. The decapitations of these C₂B ₁₀ carborane cages, with the

Full text of DOI:10.1021/ja0517116

Published on Web 06/16/2005
Substituted Carborane-Appended Water-Soluble Single-Wall
Carbon Nanotubes: New Approach to Boron Neutron Capture
Therapy Drug Delivery
Zhu Yinghuai,*,† Ang Thiam Peng,† Keith Carpenter,† John A. Maguire,*,‡
Narayan S. Hosmane,*,§ and Masao Takagaki*,|
Contribution from the Institute of Chemical and Engineering Sciences, No. 1 Pesek Road,
Jurong Island, Singapore 627833, Department of Chemistry, Southern Methodist UniVersity,
Dallas, Texas 75275, Department of Chemistry & Biochemistry, Northern Illinois UniVersity,
DeKalb, Illinois 60115, and Department of Neurosurgery, Aino College Hospital 4-5-4,
Higashioda, Ibaraki City, Osaka 567-0012, Japan
Received March 17, 2005; E-mail: hosmane@niu.edu
Abstract: Substituted C2B10 carborane cages have been successfully attached to the side walls of single-
wall carbon nanotubes (SWCNTs) via nitrene cycloaddition. The decapitations of these C2B10 carborane
cages, with the appended SWCNTs intact, were accomplished by the reaction with sodium hydroxide in
refluxing ethanol. During base reflux, the three-membered ring formed by the nitrene and SWCNT was
opened to produce water-soluble SWCNTs in which the side walls are functionalized by both substituted
nido-C2B9 carborane units and ethoxide moieties. All new compounds are characterized by EA, SEM, TEM,
UV, NMR, and IR spectra and chemical analyses. Selected tissue distribution studies on one of these
nanotubes, {([Na+][1-Me-2-((CH2)4NH-)-1,2-C2B9H10][OEt])n(SWCNT)} (Va), show that the boron atoms are
concentrated more in tumors cells than in blood and other organs, making it an attractive nanovehicle for
the delivery of boron to tumor cells for an effective boron neutron capture therapy in the treatment of cancer.
have been of special interest.5,9-25 It has recently been reported
that peptide-functionalized single-walled carbon nanotubes
(SWCNTs) were able to cross cell membranes and concentrate
Introduction
Carbon nanotubes (CNT) have attracted a great deal of
attention since their discovery in 1991.1 Not only are they
interesting in their own right,2 but methods have been developed
that led to chemically modified CNTs having useful properties,
such as solubility in polar and nonpolar solvents and moderate
biocompatibility that make them potentially important nono-
materials.2-8 Observations of enhanced water solubility of CNTs
through side-wall derivation with biologically important moieties
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† Institute of Chemical and Engineering Sciences.
‡ Southern Methodist University.
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Am. Chem. Soc. 2004, 126, 4669-4675.
§ Northern Illinois University.
| Aino College Hospital.
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Hirsch, A. J. Am. Chem. Soc. 2002, 124, 760-761.
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D. W.; Aijun, Y.; Xu, J. M.; Shana, O. K. J. Am. Chem. Soc. 2004, 126,
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Chem. Soc. 2004, 126, 15982-15983. (d) Heller, D. A.; Mayrhofer, R.
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10.1021/ja0517116 CCC: $30.25 © 2005 American Chemical Society
J. AM. CHEM. SOC. 2005, 127, 9875-9880
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A R T I C L E S
Yinghuai et al.
was maintained at -78 °C for 30 min and then allowed slowly to warm
to room temperature. The reaction mixture was stirred at that temper-
ature for 4 h and cooled to 0 °C, and then 2.6 mL (20.60 mmol) of
1-chloro-4-iodobutane was added through a syringe. After addition,
the reaction mixture was stirred for 30 min at 0 °C, slowly warmed to
room temperature, stirred for an additional 2 h, and then refluxed for
4 h. At this point, the mixture was cooled to 0 °C and then quenched
with deionized water. The organic phase was separated, and the aqueous
layer was extracted with diethyl ether (3 × 20 mL). The extract was
combined with the organic phase, and then the solvents were removed,
leaving a pale yellow sticky residue. This residue was purified with
thin-layer chromatography (SiO2), developed with a n-pentane/ethyl
acetate (v/v ) 6/1) mixture, to give 5.10 g of (Ia) as a colorless sticky
oil, which slowly solidified into wax during storage. Elemental Anal:
Found for Ia: C, 29.50; H, 7.40, calcd for a 1-Me-2-(CH2)4Cl-1,2-
C2B10H10/1-Me-2-(CH2)4I-1,2-C2B10H10 mixture in a 1/0.89 molar ratio,
in the cytoplasm of 3T6, 3T3 fibroblasts and phagocytic cells
without showing obvious toxic effects.9a Similar results were
obtained in HL60 cells, where it was found that functionalized
SWCNTs can help transport large attached groups into cells
without themselves exhibiting cell toxicity.9b These observations
raised the question whether suitably derived SWCNTs would
be useful boron delivery agents for use in boron neutron capture
therapy (BNCT). Such substances could prove to be a useful
addition to the group of tumor-targeting biomolecules, such as
porphyrin substrates, epidermal growth factors, liposomes, and
so forth, which have been investigated as possible BNCT drug
delivery agents, with varying degrees of success.26 It was this
speculation that led us to synthesize and characterize water-
soluble SWCNTs with appended monoanionic, substituted C2B9
carborane units and to study their boron tissue distributions.
These results, reported herein, indicate that such modified
SWCNTs could prove to be new boron delivery agents for
effective BNCT in the treatment of cancer.
1
C, 29.52; H, 7.43. H NMR (CDCl3, relative to SiMe4, ppm): δ 3.41
(t, 2H, -CH2Cl), 3.05 (t, 2H, -CH2I), 2.90-1.20 (m, br, 19H, CH3-
C
cage, -(CH2)3-Ccage, B10H10). 13C NMR (CDCl3, relative to SiMe4,
ppm): δ 77.67, 77.59, and 74.74 for Ccage, 44.08 (-CH2Cl), 34.41,
33.96, 32.36, 31.66, 30.33, and 26.80 for Ccage-(CH2)3-, 23.14 and
23.09 for Ccage-CH3, 5.34 (-CH2I).
Experimental Section
Syntheses. All reactions were carried out under an argon atmosphere
using standard Schlenk techniques. Diethyl ether and benzene were
heated over sodium/benzophenone until a blue color was sustained,
and distilled under nitrogen just before use. 1,2-Dichlorobenzene was
dried over phosphorus pentoxide and distilled. N-Butyllithium (1.6 M
in hexanes), 1-chloro-4-iodobutane, 1,4-diiodobutane, sodium azide,
sodium iodide, and all other reagents (Aldrich), including organic
solvents, were used as received. 1-Methyl- and 1-phenyl-closo-1,2-
C2B10H11 were obtained from Katchem Ltd. and used as received.
Before use, the commercially available SWCNTs (Aldrich), character-
ized by a diameter of about 1 nm and a length in the range of 200-
1000 nm, were refluxed with 6 M HCl for 1 day and then centrifuged,
followed by repeated washing with deionized water until a pH of about
7 was reached, and then the residue dried in vacuo to ensure the
removable of any metal catalysts present in SWCNTs. FT-IR spectra
were measured using a BIO-RAD spectrophotometer with KBr pellets
or organic solvent films. Scanning electron microscopy (SEM) images
were obtained on a JSM-6700F field-emission microscope. The samples
were previously sputter-coated with a homogeneous palladium layer
for charge dissipation during the SEM imaging. Transmission electron
microscopy (TEM) measurements were carried out on a JEOL Tecnai-
G2, FEI analyzer at 200 kV; the samples for TEM measurements were
prepared by placing one drop of a diluted solution of the functionalized
SWCNTs onto a copper grid coated with carbon followed by the solvent
evaporation. Elemental analyses were measured on a EURO EA. UV-
visible spectra were recorded on a UV-2550, Shimadzu UV-visible
spectrophotometer. The samples for characterization by UV-visible
spectrophotometry were dispersed in C6H6 with the help of an ultrasonic
In a process similar to that described above for the synthesis of Ia,
4.41 g (20.00 mmol) of 1-Ph-closo-C2B10H11, 12.60 mL (20.16 mmol)
n-butyllithium, and 2.60 mL (20.61 mmol) of 1-chloro-4-iodobutane
produced 6.18 g of Ib, which was purified as described above.
Elemental Anal: Found for Ib: C, 41.58; H, 6.65, calcd based on a
1-Ph-2-(CH2)4Cl-1,2-C2B10H10/1-Ph-2-(CH2)4I-1,2-C2B10H10 molar ratio
of 1/0.82, C, 41.62; H, 6.70. 1H NMR (CDCl3, relative to SiMe4,
ppm): δ 7.60-7.27 (m, 5H, Ccage-C6H5), 3.24 (t, 2H, -CH2Cl), 2.88
(t, 2H, -CH2I), 3.30-1.20 (m, br, 16H, -(CH2)3-Ccage, B10H10). 13C
NMR (CDCl3, relative to SiMe4, ppm): δ 130.87, 130.86, 130.52, and
129.12 for C6H5, 83.82, 82.06, and 82.01 for Ccage, 44.05 (-CH2Cl),
34.22, 33.80, 32.37, 31.62, 30.26, and 26.71 for Ccage-(CH2)3-, 5.42
(-CH2I).
Synthesis of IIa,b from NaI and Acetone. A 2.00 g sample of Ia,
or 3.00 g sample of Ib, 6.50 g (43.15 mmol) of sodium iodide, and
120 mL of HPLC grade acetone were refluxed for 3 days and then
cooled to room temperature. All volatiles were removed by pumping,
and the residue was extracted with diethyl ether. The extract was then
dried in vacuo to obtain a pale yellow sticky residue that was then
purified by flash column chromatography (SiO2, 2.5 × 30 cm, eluted
with mixed solvents n-pentane/ethyl acetate in v/v ) 5/1 ratio) to obtain
1-Me-2-(CH2)4I-1,2-C2B10H10 (IIa) (2.30 g, 92% yield, based on
chloride content in (Ia)) or 1-Ph-2-(CH2)4I-1,2-C2B10H10 (IIb) (3.38 g,
95% yield based on chloride content in (Ib)) as colorless oils.
IIa: Elemental Anal: Calcd for C7H21B10I: C, 24.71; H, 6.22.
Found: C, 24.60; H, 6.20. 1H NMR (CDCl3, relative to SiMe4, ppm):
δ 3.12 (t, 2H, -CH2I); 2.90-1.20 (m, br, 19H, CH3-Ccage, -(CH2)3-
C
cage, B10H10). 13C NMR (CDCl3, relative to SiMe4, ppm): δ 76.63,
1
bath. H, 13C, and 11B NMR spectra were recorded on a Bruker 400
73.79 (Ccage); 32.98 (-CH2CH2CH2CH2I); 31.38 (-CH2CH2CH2CH2I);
29.36 (-CH2CH2CH2CH2I); 22.18 (Ccage-CH3); 4.50 (-CH2CH2-
CH2CH2I). 11B NMR (CDCl3, relative to BF3‚OEt2, ppm): δ -3.70
analyzer at 400.13, 100.62, and 128.38 MHz, respectively. Inductively
coupled plasma-optical emission spectroscopy (ICP-OES) measurements
were made on a VISTA-MPX machine.
(1) Synthesis of IIa,b from 1-Chloro-4-iodobutane and ortho-
Carborane. Synthesis of Intermediates Ia,b. A dry 250-mL three-
necked round-bottom flask equipped with a magnetic stirring bar was
charged with 1-methyl-1,2-C2B10H11 (3.17 g, 20.00 mmol) and dissolved
in 100 mL of a diethyl ether/benzene (v/v ) 2/1) mixture. The solution
was cooled to -78 °C, and then 12.6 mL (20.16 mmol) of the
n-butyllithium solution was added dropwise with a syringe. The mixture
(1B, 1JBH ) 161 Hz); -5.04 (1B, 1JBH ) 171 Hz); -8.57 (2B, 1JBH
)
90 Hz); -9.14 (2B, 1JBH ) 108 Hz); -10.01 (4B, 1JBH ) 151 Hz). IR
(film on KBr, cm-1) 3005 (s, s), 2983 (s, s), 2869 (m, s), 2587 (vs, s,
νBH), 1955 (w, s), 1852 (w, s), 1452 (s, s), 1385 (m, s), 1296 (m, s),
1261 (m, s), 1221 (s,s), 1172 (s, s), 1022 (s, s), 947 (m, s), 789 (m,
br), 729 (vs, s), 665 (w, s), 600 (w, s), 502 (m, s).
IIb: Elemental Anal: Calcd for C12H23B10I: C, 35.83; H, 5.76.
Found: C, 35.76; H, 5.70. 1H NMR (CDCl3, relative to SiMe4, ppm):
δ 7.60-7.29 (m, 5H, C6H5), 3.30-1.20 (m, br, 18H, -(CH2)4-Ccage
,
(26) (a) Soloway, A. H.; Tjarks, W.; Barnum, B. A.; Rong, F.-G.; Barth, R. F.;
Codogni, I. M.; Wilson, J. G. Chem. ReV. 1998, 98, 1515-1562. (b)
Hawthorne, M. F.; Lee, M. W. J. Neuro-Oncol. 2003, 62, 33-45. (c)
Hawthorne, M. F.; Shelly, K. J. Neuro-Oncol. 1997, 33, 53-58. (d) Shelly,
K.; Feakes, D. A.; Hawthorne, M. F.; Schmidt, P. G.; Krisch, T. A.; Bauer,
W. F. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 9039-9043.
B10H10). 13C NMR (CDCl3, relative to SiMe4, ppm): δ 130.06, 130.04,
129.72, 127.97 (C6H5); 82.66, 80.81 (Ccage); 32.71 (-CH2CH2CH2CH2I);
31.30 (-CH2CH2CH2CH2I); 29.13 (-CH2CH2CH2CH2I); 4.15 (-CH2-
CH2CH2CH2I). 11B NMR (CDCl3, relative to BF3‚OEt2, ppm): δ -2.98
9
9876 J. AM. CHEM. SOC. VOL. 127, NO. 27, 2005

Substituted Carborane-Appended Water-Soluble SWCNTs
A R T I C L E S
(2B, 1JBH ) 150 Hz); -9.10 (2B, 1JBH ) 112 Hz); -9.72 (6B, 1JBH
)
(-CH2CH2CH2CH2Nd); 27.90 (-CH2CH2CH2CH2Nd); 22.76 (Ccage-
107 Hz). IR (film on KBr, cm-1) 3437 (m, br), 2956 (w, s), 2587 (vs,
s, νBH), 2253 (m, s), 1620 (m, br), 1493 (m, s), 1447 (s, s), 1221 (m,
s), 1171 (m, s), 1071 (m, s), 999 (w, s), 918 (s, s), 751 (s, s), 716 (s,
s), 694 (s, s), 651 (s, s), 498 (w, s).
CH3) (Here, medium 13C chemical shifts are used to describe broad
peaks). 11B NMR (C6D6, relative to BF3‚OEt2, ppm): δ -5.01 (br);
-9.99 (br); -10.45 (br). IR (KBr pellet, cm-1) 3413 (w, br), 2940 (s,
s), 2865 (m, s), 2582 (vs, s, νBH), 1704 (w, s), 1648 (m, br), 1558 (m,
br), 1440 (s, s), 1363 (s, br), 1175 (m, s), 1020 (s, s), 946 (m, s), 728
(s, s), 678 (s, s), 501 (w, br).
(2) Synthesis of IIa,b from 1,4-Diiodobutane and ortho-
Carborane. In a process similar to the preparation of Ia, 3.88 g of IIa
was synthesized in 86% yield from 2.10 g (13.27 mmol) of 1-Me-
closo-C2B10H11, 8.70 mL (13.92 mmol) of n-BuLi (1.6 M in hexanes),
and 1.86 mL (13.96 mmol) of 1,4-diiodobutane after purification by
TLC as described above. In the same way, IIb was produced in 90%
yield (2.47 g) from 1.50 g (6.81 mmol) of 1-Ph-closo-C2B10H11, 4.38
mL (7.01 mmol) of n-BuLi (1.6 M in hexanes), and 0.93 mL (6.98
mmol) of 1,4-diiodobutane. The NMR spectra of IIa and IIb obtained
by the two methods were identical.
Synthesis of IIIa,b. A 2.00-g (5.88 mmol) sample of IIa, or a 2.26-g
(5.62 mmol) sample of IIb, was mixed with 3.90 g (59.39 mmol) of
sodium azide and 120 mL of HPLC grade acetone and refluxed in the
dark for 3 days in a 250-mL three-necked round-bottom flask equipped
with a magnetic stirring bar. After cooling to room temperature, all
solvents were removed by pumping, and the residue was extracted with
diethyl ether. The diethyl ether was then removed from the extract to
give the crude product that was later purified by TLC (SiO2, developed
with n-pentane/ethyl acetate in 5:1 ratio) to produce 1.31 g of 1-Me-
2-(CH2)4N3-1,2-C2B10H10 (IIIa) (87% yield) or 1.59 g of 1-Ph-2-
(CH2)4N3-1,2-C2B10H10 (IIIb) (89% yield) as colorless waxy solids.
IIIa: Elemental Anal: Calcd for C7H21B10N3: C, 32.92; H, 8.29;
N, 16.46. Found: C, 32.88; H, 8.26; N, 16.40. 1H NMR (CDCl3, relative
to SiMe4, ppm): δ 3.25 (t, 2H, -CH2N3), 2.90-1.20 (m, br, 19H,
-(CH2)3-Ccage, B10H10). 13C NMR (CDCl3, relative to SiMe4, ppm):
δ 79.01, 76.12 (Ccage); 52.22 (-CH2CH2CH2CH2N3); 36.18 (-CH2-
CH2CH2CH2N3); 29.83 (-CH2CH2CH2CH2N3); 28.21 (-CH2CH2CH2-
CH2N3); 24.49 (Ccage-CH3). 11B NMR (CDCl3, relative to BF3‚OEt2,
In a procedure identical to the one described above for the preparation
of IVa, 98.80-mg functionalized SWCNTs {(1-Ph-2-[(CH2)4Nd]-1,2-
C2B10H10)n(SWCNT)} (IVb) were obtained from the reaction involving
1.83 g (5.76 mmol) of IIIb, 80.0 mg of pretreated SWCNTs, and 66
mL of 1,2-dichlorobenzene. The amount of carborane cage loading was
calculated to be 0.81 mmol of carborane per gram of SWCNTs or 1.07
× 103 carborane cages per SWCNT. 1H NMR (C6D6, relative to SiMe4,
ppm): δ 7.30-6.40 (m, br, 5H, C6H5); 4.10-0.80 (m, br, 18H,
-(CH2)4-Ccage, B10H10). 13C NMR (C6D6, relative to SiMe4, ppm): δ
129.40, 129.26, 127.68, 127.28 (C6H5); 66.65 (-CH2CH2CH2CH2Nd);
37.81 (-CH2CH2CH2CH2Nd); 28.80 (-CH2CH2CH2CH2Nd); 22.76
(-CH2CH2CH2CH2Nd) (Here, medium 13C chemical shifts are used
to describe broad peaks). 11B NMR (C6D6, relative to BF3‚OEt2, ppm):
δ -3.94 (br); -11.33 (br). IR (KBr pellet, cm-1): 3439 (w, br), 3062
(w, s), 2929 (s, br), 2862 (m, s), 2575 (vs, s, νBH), 1648 (w, s), 1580
(m, br), 1494 (m, s), 1446 (s, s), 1367 (m, br), 1183 (w, br), 1109 (w,
s), 1065 (m, s), 1032 (m, s), 1002 (m, s), 931 (w, s), 882 (w, s), 802
(w, s), 756 (m, s), 729 (s, s), 691 (s, s), 571 (w, br), 495 (w, s).
Synthesis of Va,b. A 2.00-g sample of sodium hydroxide was
dissolved in 60 mL of 95% ethanol, and the resulting solution was
added to 60.00 mg of IVa or 60.00 mg of IVb with constant stirring
using an ultrasonic bath for 30 min. The resulting mixture was heated
to reflux for 3 days and cooled to 0 °C, and the solution was then
neutralized with aqueous HCl to a pH equal to about 5.0 to remove
any unreacted NaOEt. Removal of all the volatiles under reduced
pressure and washing with small amounts of cold water to remove
sodium chloride produced a residue that was dried in vacuo for 3 days
to yield 62.0 mg of {([Na+][1-Me-2-((CH2)4NH-)-1,2-C2B9H10][OEt])n-
(SWCNT)} (Va) or 62.3 mg of {([Na+][1-Ph-2-((CH2)4NH-)-1,2-
C2B9H10][OEt])n(SWCNT)} (Vb).
1
1
ppm): δ -3.82 (1B, JBH ) 167 Hz); -5.08 (1B, JBH ) 157 Hz);
1
1
-8.25 (2B, JBH ) 94 Hz); -9.05 (2B, JBH ) 112 Hz); -10.06 (4B,
1JBH ) 149 Hz). IR (film on KBr, cm-1) 3404 (vw, br), 2941 (s, s),
2872 (m, s), 2589 (vs, s, νBH), 2253 (w, s), 2098 (vs, s, νN≡N), 1455 (s,
s), 1383 (w, s), 1283 (s, s), 1256 (s, s), 1178 (w, s), 1026 (m, s), 923
(s, s), 749 (s, s), 650 (s, s), 556 (w, s).
Va: 1H NMR (DMSO-d6, relative to SiMe4, ppm): δ 3.22 (br, 2H,
-OCH2-); 2.62 (br, 2H, -CH2-NH); 2.30 to -0.40 (m, br, 21H,
-(CH2)3-Ccage, CH3-Ccage, B9H9, -OCH2CH3); -2.50 to -3.30 (br,
1H, BHbridge). 13C NMR (DMSO-d6, relative to SiMe4, ppm): δ 60.81
(br), 56.00 (br) (Ccage); 55.98 (-OCH2-); 35.02, 27.10, 21.83
(-CH2CH2CH2CH2NH-, CH3-C(cage), 2C is covered by DMSO-d6
peaks); 18.80 (-OCH2CH3). (Here, medium 13C chemical shifts are
used to describe broad peaks). 11B NMR (DMSO-d6, relative to BF3‚
OEt2, ppm): δ -11.40, -19.69, -35.63, -38.22 (br). IR (KBr pellet,
cm-1) 3577 (s, br), 3213 (s, s), 2930 (s, s), 2866 (s, s), 2514 (vs, s,
IIIb: Elemental Anal: Calcd for C12H23B10N3: C, 45.40; H, 7.30;
N, 13.24. Found: C, 45.29; H, 7.26; N, 13.20. 1H NMR (CDCl3, relative
to SiMe4, ppm): δ 7.60-7.30 (m, 5H, C6H5); 3.30-1.20 (m, br, 18H,
-(CH2)4-Ccage, B10H10). 13C NMR (CDCl3, relative to SiMe4, ppm):
δ 131.06, 130.68, 130.57, 128.90 (C6H5); 83.57, 81.76 (Ccage); 50.59
(-CH2N3), 34.43 (-CH2CH2CH2CH2N3); 28.11 (-CH2CH2CH2-
CH2N3); 26.52 (-CH2CH2CH2CH2N3). 11B NMR (CDCl3, relative to
BF3‚OEt2, ppm): δ -3.48 (2B, 1JBH ) 148 Hz); -9.50 (2B, 1JBH ) 80
Hz); -10.19 (6B, 1JBH ) 78 Hz). IR (film on KBr, cm-1) 2938 (m, s),
2871 (m, s), 2587 (vs, s, νBH), 2254 (w, s), 2098 (vs, s, νN≡N), 1493
(m, s), 1452 (s, s), 1350 (m, s), 1282 (s, s), 1189 (w, s), 1066 (m, s),
919 (s, s), 754 (s, s), 652 (s, s).
νBH), 1610 (s, s), 1453 (s, br), 1196 (m, s), 1023 (m, br), 799 (w, br),
751 (w, br).
1
Vb: H NMR (DMSO-d6, relative to SiMe4, ppm): δ 7.50-6.70
(m, br, 5H, C6H5); 3.46 (br, 2H, -OCH2-); 2.60-0.30 (m, br, 20H,
-(CH2)4-Ccage, B9H9, -OCH2CH3); -1.80 to -2.80 (br, 1H, BHbridge).
13C NMR (DMSO-d6, relative to SiMe4, ppm): δ 131.36, 127.13. 125.69
(C6H5); 67.10, 62.92 (Ccage); 55.99 (-OCH2-); 35.08, 32.94, 27.20,
and 26.82 (-CH2CH2CH2CH2NH-); 18.50 (-OCH2CH3). (Here,
medium 13C chemical shifts are used to describe broad peaks). 11B NMR
(DMSO-d6, relative to BF3‚OEt2, ppm): δ -10.90, -19.58, -35.79,
-38.91 (br). IR (KBr pellet, cm-1) 3582 (s, br), 3218 (s, br), 2933 (s,
s), 2860 (m, s), 2515 (vs, s, νBH), 1600 (s, br), 1491 (s, s), 1443 (s, s),
1378 (w, s), 1180 (w, s), 1034 (m, s), 873 (w, s), 761 (m, s), 701 (s,
s), 487 (w, s).
Tissue Distribution. The biodistributions of Va in both saline and
dimethyl sulfoxide (DMSO) solvents were measured using six-week-
old female BALB/c mice (provided by Shanghai Pharmaceutical
Institute) in a method similar to that of the literature.26 The mice were
housed and treated humanely under standard conditions. EMT6 tumor
Synthesis of IVa,b. An 80.00-mg sample of pretreated SWCNTs
was placed in a dry 100-mL three-necked round-bottom flask, equipped
with a magnetic stirring bar and reflux condenser, suspended in 60
mL of 1,2-dichlorobenzene and irradiated in an ultra sonic bath for 30
min. At this point, 1.50 g (5.87 mmol) of IIIa was added, and the
mixture was refluxed for 1 week. The solvent was removed, and the
resulting residue was washed with n-hexane (5 × 30 mL) and then
dried in high vacuum for 3 days, resulting in 93.30 mg of {(1-Me-2-
[(CH2)4Nd]-1,2-C2B10H10)n(SWCNT)} (IVa). This yield gave a loading
of 0.73 mmol of carborane per gram of SWCNTs or 9.67 × 102
carborane cages per SWCNT (based on the mass of a typical 1-µm-
1
long and about 1-nm-diameter nanotube as 2.2 × 10-18g). H NMR
(C6D6, relative to SiMe4, ppm): δ 3.50-0.80 (m, br, 21H, -(CH2)4-
C
cage, CH3-Ccage, B10H10). 13C NMR (C6D6, relative to SiMe4, ppm):
δ 66.66 (-CH2CH2CH2CH2Nd); 37.81 (-CH2CH2CH2CH2Nd); 29.42
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A R T I C L E S
Yinghuai et al.
substituted carborane units is shown in Scheme 1. The formation
of the lithium salt of [1-R-closo-1,2-C2B10H10]- (R ) Me, Ph)
followed literature procedures.29 The monolithium compound
was not isolated but was reacted, in situ, with 1-chloro-4-
iodobutane to give a mixture of 1-R-2-(CH2)4Cl-1,2-C2B10H10
and 1-R-2-(CH2)4I-1,2-C2B10H10 in Cl/I molar ratios of 1:0.89
and 1:0.82 for R ) Me and Ph, respectively. The approximately
equimolar ratios of the two alkyl halides reflect the extremely
high reactivity of the monolithium carborane. Because of this
halide distribution, an extra step, that of converting the chloride
to the iodide by refluxing with NaI, had to be introduced.
Although this step was lengthy (3 days), the reaction did not
adversely affect the overall yields of IIa,b (92-95%). Com-
pounds IIa,b were also synthesized using 1,4-diiodobutane
instead of 1-chloro-4-iodobutane. The methods are comparable,
with 1,4-diiodobutane giving slightly lower yields of IIa and
IIb (86 and 90%, respectively). However, because of the length
of the former synthesis, the latter method would be preferable
for standard syntheses of IIa and IIb. The subsequent conversion
of the alkyl iodide to the corresponding azides (IIIa,b)
proceeded in high yields (87-89%). The precursors IIa,b and
IIIa,b were characterized by chemical analysis, infrared spectra,
and NMR spectra. All data are consistent with the formulations
shown in Scheme 1. The 13C NMR spectra show the presence
of the carborane cage carbons at δ 73-83 ppm, which is in the
range of the reported Ccage resonances of other C2B10 systems,29
in addition to those of the Ccage-substituted moieties. The 11B
NMR spectra are also in accord with literature values.29,30 In
addition to showing infrared peaks at 2587 cm-1, assigned to
the B-H bond stretching, compounds IIIa and IIIb show strong
absorptions at 2098 cm-1 due to the NdN stretching mode of
vibrations (Figure 3). The attachment of the substituted car-
borane units to the SWCNTs was accomplished by the cyclo-
addition reaction of the nitrenes, IIIa,b, to the side walls of the
SWCNTs, through thermally induced N2 extrusion (see Scheme
1). This is a standard method for attaching groups to the side
walls of SWCNTs.2b,31 The loading of the carborane cage per
gram of SWCNTs is 0.73 and 0.81 mmol for IVa and IVb,
respectively. The absence of the NdN absorption bands in the
IR spectra of IV confirm successful attachment of the carborane
moiety to the SWCNTs. The 13C NMR spectra of IVa and IVb
show a shift of about 14.4 and 16.1 ppm in the resonances of
the carbons R to the nitrogen atoms at δ ) 66.66 and 66.65
ppm, respectively, which is in the range for carbons bonded to
sp3-hybridized nitrogen atoms. In addition, there is a significant
broadening of the peaks in the 11B NMR spectra, which has
been observed for other groups on attachment to CNTs.31 The
possibility of a [3 + 2] cycloaddition of the azides to SWCNTs
is excluded in our experiments by the long-term refluxing at
high temperature. This was confirmed by the absence of -Nd
N-N- absorption in their IR spectra (Figure 3) and the presence
of N2 as a product. The carborane-functionalized SWCNTs were
analyzed by SEM and TEM (see images in the Supporting
Information). The functionalized SWCNTs are apparently
bundled, which may partially be caused by aggregation of the
Figure 1. Boron tissue distributions of Va in saline.
Figure 2. Boron tissue distributions of Va in DMSO.
cells, a mammary carcinoma,27 were then transplanted into the right
flank of the young female BALB/c mice of ∼20-g body weight one
week before testing. A 200 µL of a saline solution of Va at a
concentration of 23 mg/mL, or 200 µL of a DMSO solution of Va at
a concentration of 50 mg/mL, was slowly injected into the tail vein of
the mice. For comparison, four tissues, tumor, blood, lung, liver, and
spleen samples were collected and analyzed with ICP-OES. The mice
were anesthetized (diethyl ether) and bled into heparinized syringes
via cardiac puncture before surgery to collect blood in a method
described in the literature.28 The collected blood was then placed into
tared cryogenic tubes and kept frozen at -70 °C. The mice were later
sacrificed via cervical dislocation while anesthetized. The tumor and
organs samples (liver, lung, and spleen) were collected, placed in tared
cryogenic tubes, and kept frozen at -70 °C before being subjected to
analysis with ICP-OES. The results are shown in Figures 1 and 2, for
saline and DMSO, respectively. Each data point represents the average
of five mice. For clarity, error bars are not shown in the graphical data;
standard deviations were typically ∼5-15% of the average values.
Results and Discussion
Syntheses and Spectra. The sequence of reactions leading
to the side-wall functionalization of the SWCNTs with the
(29) Spielvogel, B. F.; Rana, G.; Vyakaranam, K.; Grelck, K.; Dicke, K. E.;
Dolash, B. D.; Li, S.-J.; Zheng, C.; Maguire, J. A.; Takagaki, M.; Hosmane,
N. S. Collect. Czech. Chem. Commun. 2002, 67, 1095-1108.
(30) Plesek, J.; Jelinek, T.; Hermanek, S.; Stibr, B. Collect. Czech. Chem.
Commun. 1986, 51, 81-89.
(31) Holzinger, M.; Vostrowsky, O.; Hirsch, A.; Hennrich, F.; Kappes, M.;
Weiss, R.; Jellen, F. Angew. Chem., Int. Ed. 2001, 40, 4002-4005.
(27) Rockwell, S. C.; Kallman, R. F.; Fajardo, L. P. J. Natl. Cancer Inst. 1972,
49, 735-746.
(28) Hoff, J. Lab. Anim. 2000, 29, 47-53.
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9878 J. AM. CHEM. SOC. VOL. 127, NO. 27, 2005

Substituted Carborane-Appended Water-Soluble SWCNTs
A R T I C L E S
Scheme 1. Syntheses of Substituted Carborane-Appended SWCNTs
functionalized SWCNTs and sample preparation process. Al-
though it was not possible to judge by TEM if the functional
groups were covalently attached to the tubes, the solubility of
the material in organic solvents, the NMR, IR, and ICP-OES
data are decisive arguments in support of the carborane
functionalization of the tubes.
resonances at δ -2.50 to -3.30 ppm and -1.80 to -2.80 ppm
for Va and Vb, respectively. All of these data are consistent
with the successful syntheses of a hitherto unknown set of stable,
water-soluble, carborane-appended SWCNTs, and thus this
constitutes a new synthetic approach that can be extended to
other systems of practical significance.
The carborane cages attached to SWCNTs can be decapitated
by refluxing for 3 days with alcoholic base to produce the water-
soluble SWCNTs, Va,b. Compounds Va,b were found to be
soluble in polar and moderately polar solvents such as DMSO,
tetrahydrofuran, water, acetone, and dimethylformamide. For
example, Va could be dissolved in water and DMSO to a
concentration of 24 and 53 mg/mL, respectively. The NMR
spectra of Va,b in DMSO-d6 are clear enough to confirm that
the three-membered ring formed by nitrene and SWCNTs has
been opened by an ethoxide ion to give an ethoxo- and amido
sidewall-functionalized SWCNTs. The 1H NMR spectra of Va,b
show the presence of the ethoxy groups and the broad B-H-B
2. Biological Results. Tissue distribution studies were
conducted as a function of time after tail injection of Va,
dissolved in both saline and DMSO solvents, in mice using a
literature method.26 In brief, EMT6 tumor cells, a mammary
carcinoma, were transplanted into the right flank of the young
female BALB/c mice of ∼20 g body weight one week before
testing. Boron concentrations in blood and four tissues (tumor,
lung, liver and spleen) were analyzed to gauge the relative
advantage of SWCNTs delivery. Typical time course tissue
distribution experiments examined tissue boron concentration
at four time points over 48 h. The saline solution results, shown
in Figure 1, demonstrate that maximum boron concentrations
in the tumor (22.8 µg(boron/g(tissue))) were achieved after 30
h, then dropped very slowly until, after 48 h, the value was
21.5 µg(boron)/g(tissue), which is slightly lower than the desired
value of 30 µg(boron)/g tumor for an effective BNCT. Interest-
ingly, the boron concentration in blood drops rapidly and reaches
a value of 6.9 µg(boron)/g(tissue) to give a tumor-to-blood ratio
of 3.12, which is favorable for BNCT. The low boron
concentration in the other tissues shows that there is a
preferential uptake of Va by the tumor cells with a long retention
time of over 48 h. This is the most important requirement of a
successful BNCT drug. The same general results were found
in DMSO solutions; there is enhanced boron uptake and
retention by tumor cells of the carboranes attached to SWCNTs
(see Figure 2). The main difference is that in DMSO the
carborane is assimilated faster (a maximum concentration of
27.9 µg(boron)/g(tissue) in 16 h versus 22.8 µg(boron)/g(tissue)
in 30 h in saline). The long-time (48 h) tumor-to-blood boron
ratio in DMSO is 6.13, which is significantly greater than that
Figure 3. IR spectra of IVa, IVb, Va, Vb, and SWCNTs.
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A R T I C L E S
Yinghuai et al.
found in water. Given that the DMSO solution is more
concentrated in Va than the saline solution (50 versus 23 mg/
mL) the increased rate of boron uptake and retention is not
suprising. In view of the fact that a number of studies show
that unbound borane and carborane anions show no preferential
absorption or retention in tumor cells,26d,32 the use of SWCNTs
as boron delivery vehicles shows promise. The actual mecha-
nism of the accumulation of carborane-modified SWCNTs in
tumors has not yet been determined. It could be the result of
the increased and immature vasculature of the rapidly growing
tumor cells. It has been shown that nonspecific hydrophobic
bonding exists between nanotubes and proteins.33 The nanotubes
could nonspecifically associate with hydrophobic regions of the
cell surfaces and then be internalized by endocytosis, and such
a mechanism has been found in HL60 cells and in a number of
other cell lines.9c The phenomenon of the enhanced permeability
and retention effect (EPR), due to the increased vascular
permeability and a decrease in the lymphatic drainage system
in tumor cells, has been recognized as a general effect leading
to the passive accumulation of macromolecular drugs in tumor
cells.34 It could well be that such an effect is operable in the
accumulation of the Va in tumor cells. Whatever the mechanism,
the data in Figures 1 and 2 clearly demonstrate the enhanced
accumulation and retention of the carborane-attached SWCNTs
in tumor tissue, compared to blood and to the other tissues tested.
Conclusions
It has been shown that it is possible to link the substituted
nido-carborane units to the side walls of SWCNTs. The resulting
water-soluble nanotubes have been found to be tumor-specific
and thus absorbed preferentially by EMT6 tumor cells. These
results indicate that a further investigation of these functionalized
SWCNTs as effective boron delivery agents for BNCT in cancer
treatment is warranted. More complete biodistribution studies
and cytotoxicity studies based on cell culture are needed. Such
studies are currently underway in our laboratory.
Acknowledgment. This work was supported by grants from
the Institute of Chemical and Engineering Sciences Ltd.
Singapore, the National Science Foundation (CHE-0241319),
the donors of the Petroleum Research Fund, administered by
the American Chemical Society, The Robert A. Welch Founda-
tion (N-1322 to J.A.M.), and Northern Illinois University
through a Presidential Research Professorship. N.S.H. grate-
fully acknowledges the Forschungspreis der Alexander von
Humboldt-Stiftung and the Gauss Professorship of the Go¨ttingen
Academy of Sciences.
(32) (a) Freakes, D. A.; Shelly, K.; Knobler, C. B.; Hawthorne, M. F. Proc.
Natl. Acad. Sci. U.S.A. 1994, 91, 3029-3033. (b) Freakes, D. A.; Shelly,
K.; Hawthorne, M. F. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 1367-1370.
(33) Chen, R. J.; Bangsaruntip, S.; Drouvalakis, K. A.; Kam, N. W. S.; Shim,
M.; Li, Y. M.; Utz, P. J.; Dai, H. J. Proc. Natl. Acad. Sci. U.S.A. 2003,
100, 4984-4989.
Supporting Information Available: NMR, IR, and UV-vis
spectra of I-V and SEM, TEM of IVa. This material is
available free of charge via the Internet at http://pubs.acs.org.
(34) Maeda, H.; Seymour, L. W.; Miyamoto, Y. Bioconjugate Chem. 1992, 3,
351-362.
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