Modification of carboxylated multiwall nanotubes with benzotriazole derivatives and study of their anticancer activities

Article abstract of DOI:10.1007/s00044-013-0668-3

In vitro and in vivo results indicate that functionalized carbon nanotubes are promising for the development of unique anticancer drugs. Since therapeutic and pharmacologic agents could functionalize the structure of carbon nanotubes, we report for the first time three-component condensation reactions consisting of multiwalled carbon nanotube-COCl (MWNTCOCl), diazonium sulfonated salt and phenol derivatives to produce novel anticancer agents. The functionalized carboxylated multiwall nanotubes were then characterized by FT-IR, Raman, SEM, TEM, and solubility test. The functionalized MWNTs exhibited good aqueous solubility. Furthermore, the cytotoxic activity of the MWNT-COOH (A) as well as its functionalized carboxylated multiwall nanotubes (MWNT-CO-2-(1- hydroxynaphthalen-2-yl)-2H-benzo[d][1,2,3]triazole-5-sulfonic acid (B), MWNT-CO-2-(2-hydroxy-1-nitrosonaphthalen-3-yl)-2H-benzo[d][1,2,3] triazole-5-sulfonic acid (C) and MWNT-CO-2-(2-hydroxy-5-nitrophenyl)-2H-benzo[d] [1,2,3]triazole-5-sulfonic acid (D)) was tested against human gastric cancer MKN45 and human Colon cancer SW742. In vitro cytotoxicity studies represented a significant enhancement in the cytotoxic capability of f-MWNTs, suggesting these drugs improve drug antitumor activity. Therefore, the highly versatile functionalized carboxylated multiwall nanotubes could potentially be considered as anticancer candidates.

Full text of DOI:10.1007/s00044-013-0668-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0668-3
ORIGINAL RESEARCH
Modification of carboxylated multiwall nanotubes
with benzotriazole derivatives and study of their anticancer
activities
•
Mahdieh Entezari Mojdeh Safari
•
•
Malak Hekmati Shohreh Hekmat
•
Atena Azin
Received: 5 April 2013 / Accepted: 30 May 2013
Ó Springer Science+Business Media New York 2013
Abstract In vitro and in vivo results indicate that func-
tionalized carbon nanotubes are promising for the devel-
opment of unique anticancer drugs. Since therapeutic and
pharmacologic agents could functionalize the structure of
carbon nanotubes, we report for the first time three-com-
ponent condensation reactions consisting of multiwalled
carbon nanotube–COCl (MWNTCOCl), diazonium sulfo-
nated salt and phenol derivatives to produce novel anti-
cancer agents. The functionalized carboxylated multiwall
nanotubes were then characterized by FT-IR, Raman,
SEM, TEM, and solubility test. The functionalized
MWNTs exhibited good aqueous solubility. Furthermore,
the cytotoxic activity of the MWNT–COOH (A) as well as
its functionalized carboxylated multiwall nanotubes
(MWNT-CO-2-(1-hydroxynaphthalen-2-yl)-2H-benzo[d]
[1,2,3]triazole-5-sulfonic acid (B), MWNT-CO-2-(2-
hydroxy-1-nitrosonaphthalen-3-yl)-2H-benzo[d][1,2,3]tri-
azole-5-sulfonic acid (C) and MWNT-CO-2-(2-hydroxy-
5-nitrophenyl)-2H-benzo[d][1,2,3]triazole-5-sulfonic acid
(D)) was tested against human gastric cancer MKN45 and
human Colon cancer SW742. In vitro cytotoxicity studies
represented a significant enhancement in the cytotoxic
capability of f-MWNTs, suggesting these drugs improve
drug antitumor activity. Therefore, the highly versatile
functionalized carboxylated multiwall nanotubes could
potentially be considered as anticancer candidates.
Keywords Carboxylated multiwall nanotubes ꢀ
Functionalization ꢀ Characterization ꢀ Cell line ꢀ
Antitumor activity
Introduction
Cancer is a complex disease occurring as a result of a
progressive accumulation of genetic aberrations and epi-
genetic changes that enable escape from normal cellular
and environmental controls (Srinivas and Das, 2004).
Gastric and colorectal cancers are among the most common
malignant cancers with poor prognoses and high mortality
rates worldwide (Xiao et al., 2011; Goldberg et al., 2004).
Despite recent advances in targeted therapy and improved
understanding of the biology and development of the
malignancy, progress in the treatment of these cancers has
been limited. Chemotherapy is an important therapeutic
approach in the treatment of a variety of cancers, but its
clinical applications are limited because of severe side
effects (Xiao et al., 2011; Safari et al., 2013). Recent
studies have shown that heterocyclic compounds contain-
ing nitrogen atoms have very promising biological activi-
ties such as antibacterial, antiviral and antifungal activity
(Swarnkar et al., 2007; Pandey and Negi, 2003; Chaudhary
et al., 2010). Benzotriazoles have become the most rapidly
expanding group of antifungal and antiviral compounds
(Wu et al., 2006; Handratta et al., 2005). More recently,
biotechnological applications of CNT are being anticipated
in a variety of fields ranging from microfluidics to bioin-
formatics (Kostarelos et al., 2005). In vitro antitumor
M. Entezari (&) ꢀ M. Safari ꢀ M. Hekmati
Department of Chemistry, Islamic Azad University,
Pharmaceutical Sciences Branch, Tehran, Iran
e-mail: mahdiehentezari63@yahoo.com
S. Hekmat
Department of Chemistry, Islamic Azad University,
North Tehran Branch, Tehran, Iran
A. Azin
Young Researchers Club, Islamic Azad University,
Mahshahr Branch, Mahshahr, Iran
123

Med Chem Res
properties of MWNTs have been investigated against a
wide panel of tumor cell lines of human origin and found to
be as effective as, or even better than standard anticancer
drugs (Iijima, 1991; Bachtold et al., 2000; McEuen et al.,
2002; Azizian et al., 2010). Very recent studies have
explored the possibilities for the use of CNT in tissue
regeneration after damage of the spinal cord, brain or bone
tissues and also in drug delivery system (Zhang et al.,
2006; Kam et al., 2005; Kostarelos et al., 2005). The
chemical modification of carbon nanotubes has received
much recent attention in recent decades (Chen et al., 1998;
Hamon et al., 1999). Chemical functionalization is a
common technique to enhance dispersion stability and
biocompatibility of CNTs (Georgakilas et al., 2002).
Moreover, these functionalized CNTs are capable of
forming chemical covalent or non-covalent bonds with
different pharmacologic agents, which makes them ideal
candidates for drug delivery systems. The covalent side
wall modifications of nanotubes have been well described
in several review papers (Gannon, 2007; Liu et al., 2007).
Keeping all these factors in mind, we aimed to attach the
new synthesized benzotriazole derivatives to the surface of
carboxylated multiwall nanotubes, to take advantage of the
MWNTs outstanding mechanical properties and to screen
the final products for their antitumor activity.
Fig. 1 Solubility test of MWNT–COOH (A) and functionalized
MWNTs (B–D)
with the functionalized MWNTs (Fig. 2). The two bands
indicative of C=O and C–O stretching vibrations give
valuable information about the behavior of the attached
functional groups to the MWCNTs. In spectrum A, the
characteristic absorption peaks of OH, C=O, and C–O
appear at 3,733, 1,704 and 1,204 cm-1, respectively. The
peak at 1,704 cm-1 assignable to a m (C=O) in MWNT–
COOH shifts to higher frequencies of 1,797, 1,719, and
1,718 cm-1 in functionalized MWCNTs (B–D), a result of
ester linkage formation. In the spectra of functionalized
MWCNTs (B–D), the bands at 1,111, 1,199, and
1,084 cm-1 apparently correspond to the stretching modes
of C–O, respectively. The new band at 1,512 cm-1 in the
spectrum of functionalized MWCNT (C) is attributed to
the nitroso group. The absorption bands at 1,489 and
1,262 cm-1 which are seen in the spectrum of functional-
ized MWNT (D) can be assigned to the nitro group. The
appearance of two bands in the range of 2,800–2,900 cm-1
are attributed to the C–C sp3 modes in the functionalized
products. In addition, the S–O stretching vibrations at
around 1,100–1,250 and 600–690 cm-1 are observed in the
IR spectra of all functionalized MWNTs (Hu et al., 2009;
Holzinger and coworkers, 2002; Zaragoza-Contreras et al.,
2009). Therefore, FT-IR spectra confirmed that MWNT–
COOH had been successfully modified by esterification.
The characteristic IR frequencies (cm-1) of the studied
compounds (A–D) are listed in Table 1.
Results and discussion
Solubility of functionalized MWNTs
Functionalized MWNTs were prepared using esterification
of MWNT to obtain a homogenous distribution of MWCNTs
in solvent. Controlling the degree of dispersion of nanotubes
is difficult because of the strong intra-molecular forces that
exist between carbon nanotubes which are responsible for
the formation of 10–50 nm nanotube bundles. The bundles
are difficult to exfoliate since the nanotubes may be hundreds
to thousands of nano meters long (Szymczyk et al., 2011).
Noticeably, before any biology-related application can even
be envisaged, the aqueous solubility of carbon nanotubes has
to be resolved. Herein, the existence of extremely large
hydrophilic groups containing sulfonic acid in modified
CNTs gives good dispersion in water for almost a month, as
shown in Fig 1.
Raman spectroscopy
Spectroscopic studies
Raman spectroscopy is a powerful technique used to char-
acterize structural changes of carbon nanotubes, specifically
changes owing to significant sidewall functionalization.
As shown in Fig. 3, the D and G bands of the MWNT
at around 1,300 and 1,500 cm-1, attributed to defects,
Infrared spectroscopy
FT-IR bands related to different groups have been assigned
by comparison of the FT-IR spectra of the MWNT–COOH
123

Med Chem Res
Fig. 2 FT-IR spectra of MWNT–COOH (a) and functionalized MWNTs (b–d)
Table 1 Diagnostic IR bands
Compound
m (C=O)
m (C–O)
m (OH)
m (NO)
m (NO2)
m (S–O)
of the MWNT–COOH and
functionalized MWNTs
MWNT–COOH A
Compound B
1,704
1,797
1,719
1,718
1,204
1,111
1,199
1,084
3,733
_
_
–
_
_
_
–
–
1,056,624
1,137,613
1,262,693
Compound C
Compound D
1,512
–
–
14,891,262
disorder-induced peaks, and tangential-mode peaks, can be
clearly observed forboth MWNT–COOH and functionalized
MWNTs. More importantly, D- to G-band intensity ratios
(ID/IG) for functionalized MWNTs (B–D) are around 1.66,
1.50, 1.62, respectively, which are greater than that for
MWNT–COOH (0.50). The increase in intensity of the
defect mode at 1,300 cm-1 was related to sp3 hybridization
of carbon (Jorio et al., 2004; Dresselhaus et al., 2005). This
band is barely observable in MWNT–COOH but is clearly
detectable after functionalization, indicating an increase in
defects along the nanotube.
products have a rough surface. The changes in the mor-
phology of products are remarkable.
Transmission electron microscopy (TEM) is the most
powerful tool that reveals the diameters of the single-wall
and multiwall CNTs, the number of walls and the distance
between the walls. Moreover, the coating on the surface of
nanotubes can be observed by the aid of TEM. The TEM
image of A shows that MWNT–COOH exist as bundles or
ropes of MWNT. These bundles dissociate in the modified
nanotubes, indicating functionalization prevents MWNT
from aggregation and enhances the diameter (Najafi et al.,
2006) (Fig. 5).
SEM and TEM characterization of the modified
MWNTs
Pharmacology: antiproliferative activity in vitro
More direct evidence for the functionalization of MWNTs is
manifested by scanning electron microscopy (SEM) images
(Zheng et al., 2003). Figure 4 shows SEM micrographs of
fracture surfaces of A (MWNT–COOH) and B, C, and
D (modified nanotubes). The images clearly indicate that
compound A has a smooth surface but functionalized
The in vitro cytotoxicities of MWNT–COOH as well as
corresponding functionalized MWNTs against tumor cell
lines of human gastric cancer MKN45 and human colon
cancer (SW742) were determined using the MTT micro-
culture colorimetric assay. This study was designed to
evaluate the antitumor activity of MWNT–COOH and
123

Med Chem Res
Fig. 3 Raman spectra of MWNT–COOH (a) and functionalized MWNTs (b–d)
Fig. 4 Scanning electron
microscopy (SEM) images for
MWNT–COOH (a) and
functionalized MWNTs (b–d)
modified MWNTs to observe the effect of different sub-
stituents attached to the surface of MWNTs on tumor cell
lines. An improved activity was observed for the func-
tionalized MWNTs of the present investigation. The results
of the in vitro cytotoxicity tests are expressed as IC50, the
concentration of compound (lg/mL) that inhibits prolifer-
ation rate of the tumor cells by 50 % as compared to
control untreated cells (Table 2). Furthermore, the screen-
ing results are compared with the standard drugs cisplatin
and doxorubicin that are in current clinical use as antitu-
mour agents.
From the derived IC50 values, it is concluded that the
cytotoxic effect of the MWNT–COOH and modified
MWNTs on tested cell lines was strictly concentration
dependent (Fig. 6).
Estimates based on IC50 values shows that MWNT–
COOH is less cytotoxic compared to modified MWNTs
(B–D) and more cytotoxic than cisplatin and doxorubicin
123

Med Chem Res
Fig. 5 Transmission electron
microscopy (TEM) of MWNT–
COOH (a) and functionalized
MWNTs (b–d)
From those results one can envisage that modification of
carboxylated multiwall nanotube led to a significant
decrease of the final cytotoxic activity. Further study on the
antitumor effects of these compounds is ongoing based on
these very positive preliminary results.
Table 2 IC50 (lg/mL) for the 48 h of action of the studied com-
pounds, cisplatin and doxorubicin on MKN-45 and SW742 deter-
mined by MTT test
Compound
MKN-45
SW742
MWNT–COOH A
Compound B
Compound C
Compound D
Cisplatin
0.026
0.005
0.06
0.002
5.11
_
0.002
0.001
0.001
0.001
_
Experimental section
Material and physical measurement
Doxorubicin
0.45
All chemicals and reagents were purchased from Merck
and Fluka and used without further purification. MWNT–
COOH (95 % purity, 20–30 nm; Netrino Co. Ltd) were
purchased and used as received. The FT-IR spectra were
recorded on a Nexus 870 FT-IR spectrometer using KBr
pellets (Thermo Nicolet, Madison, WI). FT-Raman spectra
were recorded on 960 ES spectrometer (Thermo Nicolet).
SEM was used to study the morphology of the MWNTs
and carried out on the XL30 electron microscope (Philips,
Amsterdam, Netherlands). TEM images were recorded
using JEOL 6400. Elemental analyses of carbon, hydrogen,
and nitrogen were performed using a Series II 2400 (Perkin
Elmer, Waltham, MA).
against the investigated cell lines. Compounds (A–D)
present lower IC50 values than those of cisplatin and
doxorubicin, which indicates their higher activity against
the tumoral cell lines evaluated. The IC50 values of the
compounds B, C, and D against SW742 were in a similar
range, and were 450 times better than that of the antitumor
drug doxorubicin.
Compounds B and D exhibited higher activity (0.005
and 0.002 lg/mL) than compound C (0.06 lg/mL) against
MKN-45 cell line.
123

Med Chem Res
H2SO4
NH2
NO2
HO3S
NH2
NO2
N2
NO2
MKN-45
120
100
80
60
40
20
-
+
HO3S
HO3S
NH2
NO2
NaNO2/ HCl
A
B
C
D
Scheme 1 Sulfonation of 2-nitrobenzenamine and preparation of
diazonium salt
O
O
O
O
Cl
Cl
MWNT
MWNT
0
0.001
0.01
0.1
OH
Cl
Concentration µg/ml
Scheme 2 Preparation of MWCNT–COCl
SW741
120
100
80
60
40
20
-
Esterification of carboxylated multiwall nanotubes
The mixture of diazonium salt (1 mmol) and phenol
derivatives (1 mmol) dissolved in DMF was added to the
MWNTCOCl suspension followed by stirring for 15 h at
90 °C. 20 % w/w aqueous solution of NaOH was then
added dropwise to the reaction mixture within 1 h in the
presence of catalyst (Zn powder). The mixture was stirred
for 6 h at 95 °C. After cooling to room temperature, the
mixture was filtered and washed thoroughly with tetrahy-
drofuran. Subsequently, the black solid was vacuum-dried
at room temperature for 5 h (Scheme. 3).
A
B
C
D
0
0.001
0.01
0.1
Concentraition µg/ml
Fig. 6 Cytotoxicity graphs from typical MTT assay showing the
effect of MWNT–COOH (a) and functionalized MWNTs (b–d) on
the viability of MKN-45 and SW742
In vitro studies
Preparation of drug solutions
Preparation of 3-amino-4-nitrobenzenesulfonic acid
and its diazonium salt
Stock solutions of the studied compounds were prepared in
dimethyl sulfoxide (DMSO, Sigma Aldrich) at a concen-
tration of 1,000 lg/mL, sterilized by filtration through
Millipore filter, 0.22 lm, before use, and diluted by cell
culture medium to various working concentration. Nutrient
medium was RPMI-1640 (Gibco BRL, Scotland) supple-
mented with 10 % fetal bovine serum (FBS-Gibco BRL/
Scotland). MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide was dissolved (5 mg/mL) in phos-
phate buffer saline pH 7.2, and filtered through Millipore
filter, 0.22 lm, before use.
The reaction of 2-nitrobenzenamine with concentrated
sulfuric acid for 3 h led to the formation of 3-amino-4-
nitrobenzenesulfonic acid (Riggs et al., 2000; Fu et al.,
2001; Sun et al., 2002; Czerw et al., 2001). Diazonium salt
was then prepared by adding HCl/NaNO2 to 3-amino-4-
nitrobenzenesulfonic acid at 0 °C, as shown in Scheme 1.
Preparation of MWCNT–COCl
MWNT–COOH (20 mg) were sonicated in 30 mL of N,N-
dimethylformamide (DMF) in three separate pots for
45 min to give a homogeneous suspension. Oxalyl chloride
(1 mL) was added dropwise to any MWNT suspension at
0 °C under a constant flow of nitrogen. The mixture was
stirred at 0 °C for 2 h and followed at room temperature for
the same duration. Finally, the temperature was increased
to 70 °C, and the mixture was stirred overnight to remove
excess oxalyl chloride (Scheme. 2).
Cell line and cell culture
The MKN-45 (human gastric cancer) cells (NCBI
C115615, National Cell Bank of Iran) and SW742 (human
colon cancer) cells (NCBI C146, National Cell Bank of
Iran), were obtained from Pasteur Institute of Iran. Cells
were cultured in RPMI-1640 medium supplemented with
10 % heat inactivated fetal calf serum (FCS- Gibco BRL,
123

Med Chem Res
O
O
OH
R3
MWNT
R1
HO3S
NO2
MWNT
R1
O
+
O
R1
R2
One Pot
HO3S
N2
O
N
N
zn powder
HO3S
+
N
N
+
Cl
MWNT
NO2
N
R2
R2
R3
R3
(1)
(5)
(2)
(4)
(3)
5
B
C
D
R1
NO
H
H
phenyl
H
R2
R3
phenyl
NO2
Scheme 3 Preparation of anti cancer drug candidate by esterification of carboxylated multiwall nanotubes
Scotland) 2 mM L-glutamine (Gibco BRL, Scotland) and
antibiotics, including streptomycin (100 lg/mL) and pen-
icillin (100 IU/mL) (Sigma, USA) and incubated at 37 °C
in a humidified 5 % CO2 atmosphere.
(Ohno and Abe, 1991), which measures the reduction of
the yellow tetrazolium salt MTT (3-[4,5-dimethylthiazol-
2-yl]-2, 5-diphenyltetrazolium bromide; SIGMA) into a
purple formazan crystal, mainly by the activity of the
mitochondrial enzymes, cytochrome oxidase and succi-
nate dehydrogenase. In brief, cells were incubated for
48 h and then, 20 lL of MTT solution (5 mg/mL) in
phosphate buffer saline (1/10 of total volume in a well)
was added to wells. Samples were incubated for further
4 h at 37 °C in humidified atmosphere with 5 % CO2.
Supernatants were removed and 100 lL of DMSO was
added to the plate as a solvent to each well. The plate was
shaken for 15 min by a shaker incubator to dissolve the
formazan crystals.
Trypan blue exclusion
The loss of membrane integrity, as a morphological charac-
teristic for cell death, was assayed by trypan blue dye exclu-
sion (Moldeus et al., 1978). The alive cells were estimated
through a hemocytometer and phase-contrast microscopy.
Cell sensitivity analysis
The optical density (OD) value was defined as the
absorbance of each individual well, minus the blank value
(blank is the mean optical density of the control cells).
Finally, the absorbance at 570 nm (test wavelength) and
with a reference filter of 630 nm was measured using an
ELISA microplate reader (Stat Fax-2100, USA). All
experiments were performed three times, and the percent-
age of cytotoxicity was calculated according to following
formula:
MKN-45 and SW742 cells were seeded (15,000 cells per
well) into 96-wells flat-bottom microtiter plates and incu-
bated for 4 h prior to the addition of filtered 3 different
concentrations of the studied compounds. Final concen-
trations achieved in treated wells were 0.001, 0.01, and
0.1 lg/mL. Each concentration was tested in quadruplicate
on each cell line. The final concentrations (\0.1 %) of
DMSO, were non-toxic to the cells. Each assay included a
Mean absorbance of toxicant treated cells
% Cytotoxicity ¼ 1 ꢁ
ꢂ 100
Mean absorbance of negative control
blank containing complete medium without cells. The
incubation time was 48 h, during the period the control
cells showed exponential growth.
% Viability ¼ 100 ꢁ % Cytotoxicity
Data analysis
Determination of target cell survival
After subtracting the solvent toxicity, the concentration
giving 50 % inhibition (IC50) was determined for the test
samples by nonlinear regression analysis of curves. Graph
Cell survival was determined by MTT test according to
the method of (Mosmann, 1983) and modified by
123

Med Chem Res
Gannon GI (2007) Carbon nanotube-enhanced thermal destruction of
cancer cells in a noninvasive radiofrequency field. Cancer
110:2654–2655
Georgakilas V, Kordatos K, Prato M, Guldi DM, Holzinger M, Hirsch
A (2002) Organic functionalization of carbon nanotubes. J Am
Chem Soc 124:760–761
Goldberg RM, Sargent DJ, Morton RF, Fuchs CS, Ramanathan RK,
Williamson SK, Findlay BP, Pitot HC, Alberts SR (2004) A
randomized controlled trial of fluorouracil plus leucovorin,
irinotecan, and oxaliplatin combinations in patients with previ-
ously untreated metastatic colorectal cancer. J Clin Oncol
22:23–30
Hamon MA, Chen J, Hu H, Chen Y, Itkis ME, Rao AM, Eklund PC,
Haddon RC (1999) Dissolution of single-walled carbon nano-
tubes. Adv Mater 11:834–840
Handratta VD, Vasaitis TS, Njar VCO, Gediyal K, Kataria R, Chopra
P, Newman D, Farquhar R, Guo Z, Qiu Y, Brodie AMH (2005)
Novel C-17-Heteroaryl Steroidal CYP17 inhibitors/antiandro-
gens: synthesis, in vitro biological activity, pharmacokinetics,
and antitumor activity in the LAPC4 human prostate cancer
xenograft mode. J Med Chem 48:2972–2984
Hu CY, Xu YJ, Duo SW, Zhang RF, Li MS (2009) One-step
electrodeposited carbon nanotube/zirconia/myoglobin film.
J Chin Chem Soc 56:234–238
Iijima S (1991) Helical microtubules of graphitic carbon. Nature
354:56–58
Jorio A, Saito R, Dresselhaus G, Dresselhaus MS (2004) Determi-
nation of nanotubes properties by Raman spectroscopy. Phys
Eng Sci 362:2311–2336
Pad Prism version 4.00 was used to calculate IC50. Mean
difference among groups was calculated by paired t test,
one-way and repeated measures ANOVA (p \ 0.05).
Conclusions and outlook
This study is the first of its kind to present esterification of
carboxylated multiwall nanotubes with benzotriazole
derivatives to modify MWNT–COOH for biomedical stud-
ies. The functionalized MWNTs have been characterized by
FT-IR and Raman spectroscopies, SEM and TEM. The
in vitro cytotoxic activity of the studied compounds has been
evaluated against human cancer cell lines: MKN-45 and
SW742. The screening results have shown that modified
MWNTs have better anticancer activity than MWNT–
COOH. The most outstanding results were obtained from the
activity of compounds B, C, and D against SW742 cell line,
which exhibited much higher cytotoxicity than doxorubicin.
Our findings are of considerable value for selecting the most
appropriate material and functionalization protocol for
in vitro applications. Therefore, these functionalized multi-
wall carbon nanotubes are envisaged to become very
promising candidates for further in vivo tests which will be
carried out in the near future.
Kam NW, O’Connell M, Wisdom JA, Dai H (2005) Carbon
nanotubes as multifunctional biological transporters and nearin-
frared agents for selective cancer cell destruction. J Am Chem
Soc 127:12492–12493
Kostarelos K, Lacerda L, Partidos CD, Prato M, Bianco A (2005)
Carbon nanotube-mediated delivery of peptides and genes to
cells: translating nanobiotechnology to therapeutics. J Drug Del
Sci Tech 15:41–47
Acknowledgments The authors like to express their profound
gratitude to the Islamic Azad University, Pharmaceutical Sciences
Branch, Iran for providing necessary research facilities and financial
assistance. The authors also gratefully acknowledge the valuable help
of Dr. Hilary Jenkins, research scientist at McMaster University,
Canada.
Liu Z, Sun X, Nakayama-Ratchford N, Dai H (2007) The supramo-
lecular chemistry of organic-inorganic hybrid materials. ACS
Nano 1:50–56
McEuen PL, Fuhrer MS, Park H (2002) Single-walled carbon
nanotube electronics. IEEE Trans Nanotech 1:78–185
Moldeus T, Hogberg J, Orrhenius S, Fleischer S, Packer L (1978)
Isolation and use of liver cells. Meth Enzymol 52:60–71
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays.
J Immunol Meth 65:55–63
References
Azizian J, Tahermansouri H, Biazar E, Heidari S, Chobfrosh Khoei D
(2010) Functionalization of carboxylated multiwall nanotubes
with imidazole derivatives and their toxicity investigations. Int J
Nanomedicine 5:907–914
Najafi E, Kim J, Han S (2006) UV-ozone treatment of multi-walled
carbon nanotube for enhanced organic solvent dispersion. J Coll
Surf 284(A):373–378
Bachtold A, Fuhrer MS, Plyasunov
S (2000) Scanned probe
microscopy of electronic transport in carbon nanotubes. Phys
Rev Lett 84:6082–6608
Ohno M, Abe T (1991) Rapid colorimetric assay for the quantification
of leukemia inhibitory factor (LIF) and interleukin-6 (IL-6).
J Immunol Meth 145:199–203
Pandey VK, Negi HS (2003) Synthesis and biological activity of
some new benzophenothiazines. Indian J Chem (B) 42:206–210
Riggs JE, Walker DB, Carroll DL, Sun YP (2000) Optical limiting
properties of suspended and solubilized carbon nanotubes. J Phys
Chem 104:7071–7076
Safari M, Yuosefi M, Jenkins HA, Bikhof Torbati M, Amanzadeh A
(2013) Synthesis, spectroscopic characterization, X-ray structure
and in vitro antitumor activities of new triorganotin(IV) com-
plexes with sulphur donor ligand. Med Chem Res. doi:
10.1007/s00044-013-0549-9
Chaudhary M, Pareek D, Ojha KG, Pareek A (2010) Synthesis and
antibacterial activity of 1-(2-Diazo- 6-ethoxybenzothiazolyl)
substituted benzene derivatives. Int J Curr Chem 1:175–179
Chen J, Hamon MA, Hu H, Chen Y, Rao AM, Eklund PC, Haddon
RC (1998) Solution properties of single-walled carbon. Nano-
tubes Sci 282:95–98
Czerw R, Guo Z, Ajayan PM, Sun YP, Carroll DL (2001)
Organization of polymers onto carbon nanotubes: a route to
nanoscale assembly. Nano Lett 1:423–427
Dresselhaus MS, Dresselhaus G, Saito R, Jorio A (2005) Raman
spectroscopy of carbon nanotubes. Phys Rep-Rev Sect Phys Lett
409:47–99
Fu K, Huang W, Lin Y, Riddle LA, Carroll DL, Sun YP (2001)
Defunctionalization of functionalized carbon nanotubes. Nano
Lett 1:439–441
Srinivas KVNS, Das B (2004) Iodine catalyzed one-pot synthesis of
3,4-dihydropyrimidin-2(1H)-ones and thiones: a simple and
123

Med Chem Res
efficient procedure for the Biginelli reaction. Synthesis
13:2091–2093
Xiao XY, Hao M, Yang XY, Ba Q, Li M, Ni SJ, Wang LS, Du X
(2011) Licochalcone A inhibits growth of gastric cancer cells by
arresting cell cycle progression and inducing apoptosis. Cancer
Lett 302:62–75
Zaragoza-Contreras EA, Lozano-Rodrıguez ED, Roman-Aguirre M,
Antunez-Flores W, Hernandez-Escobar CA, Sergio G, Aguilar-
Elguezabal A (2009) Evidence of multi-walled carbon nanotube
fragmentation induced by sonication during nanotube encapsu-
lation via bulk-suspension polymerization. Micron 40:621–627
Zhang Z, Yang X, Zhang Y (2006) Delivery of telomerase reverse
transcriptase small interfering RNA in complex with positively
charged single-walled nanotubes suppresses tumor growth. Clin
Cancer Res 12:4933–4939
Sun YP, Fu K, Lin Y, Huang W (2002) Functionalized carbon
nanotubes: properties and applications. Acc Chem Res 35:1096
Swarnkar PK, Kriplani P, Gupta GN, Jha KGO (2007) Synthesis and
antibacterial activity of some new phenothiazine derivatives. E J
Chem 4:14–20
´
Szymczyk A, Roslaniec Z, Zenker M, Garcıa-Gutierrez MC,
Hernandez JJ, Rueda DR, Nogales A, Ezquerra TA (2011)
´
´
Preparation and characterization of nanocomposites based on
COOH functionalized multi-walled carbon nanotubes and on
poly (trimethylene terephthalate). eXPRESS. Polymer Lett
5:955–995
Wu CY, King KY, Kuo CJ, Fang JM, Wu YT, Ho MY, Liao CL, Shie
JJ, Liang PH, Wong CH (2006) Stable benzotriazole esters as
mechanism-based in activators of the severe acute respiratory
syndrome 3CL protease. Chem Biol 13:261–268
Zheng M, Jagota A, Strano MS, Santos AP, Barone P, Chou SG
(2003) Structure-based carbon nanotube sorting by sequence-
dependent DNA assembly. Science 302:1545–1548
123