Parasitology International 63 (2014) 772–776

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Parasitology International
journal homepage: www.elsevier.com/locate/parint

Carbon nanotube based betulin formulation shows better efficacy against

Leishmania parasite
Prakash Saudagar, Vikash Kumar Dubey ⁎
Department of Biotechnology, Indian Institute of Technology Guwahati, Assam 781039, India

a r t i c l e i n f o a b s t r a c t

Article history: We report a novel antileishmanial formulation of betulin (BET) attached to functionalized carbon nanotubes
Received 11 February 2014 (f-CNTs). We conjugated betulin, a pentacyclic triterpenoid secondary metabolite, to carboxylic acid chains on
Received in revised form 15 June 2014 f-CNTs to obtain BET attached functionalized carbon nanotubes (f-CNT-Bet). The drug release profile demonstrat-
Accepted 8 July 2014
ed a fairly slow release of BET. The in-vitro cytotoxicities of BET, f-CNT and f-CNT-BET on J774A.1 macrophage cell
Available online 31 July 2014
line were 211.05 ± 7.14 μg/ml; 24.67 ± 3.11 μg/ml and 72.63 ± 6.14 μg/ml, respectively. The IC50 of BET and f-
CNT-BET against intracellular Leishmania donovani amastigotes were 8.33 ± 0.41 μg/ml and 0.69 ± 0.08 μg/ml,
respectively. The results demonstrate better antileishmanial efficiency of f-CNT-BET formulation than BET alone
and with no significant cytotoxicity observed on host cells.
© 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction utilization of nanotubes for drug administration. Various methods have

Leishmaniasis is caused by the protozoan parasites of genus Leishman-
ia. Among them, visceral leishmaniasis is the fatal infection of leishmani-
asis. Currently available treatments of leishmaniasis are known to
have high toxicity and cost. To overcome the drawbacks faced with avail-
able antileishmanial chemotherapeutics, new alternate drugs with
cheaper formulations have to be introduced. The recent advances in
antileishmanial drug development have mainly focused on identification
of new drug molecules and their formulations for specific targeting to in-
crease efficacy. Drug delivery systems based on nanocarriers have sub-
stantial potential for treatment of leishmaniasis, as evident from
liposomal formulation of amphotericin B [1]. But its high cost and toxicity
limit its use. Hence, the search for new drugs and their cost effective for-
mulations with increased efficacy is still desirable. Carbon nanotubes have
engrossed remarkable attention as the most promising nano-material in
the 21st century for numerous applications [2,3], neuro-engineering [4],
gene therapy [5,6], tissue engineering [7], electronics [8,9] and biosensor
technology [10]. CNTs have been exploited over current drug delivery ve-
hicles due to their high aspect ratio and their capability to bypass cell
membranes easily as well as high surface area, which offer multiple at-
tachment sites for drug targeting. Though the cell toxicity of CNTs is pro-
vocative, the surface functionalized CNTs are biocompatible. Therefore,
they are likely to be safe at the cellular level. It is reported by many groups
that surface functionalized CNTs are capable of reducing the toxic effect
[11] and also increase biocompatibility [12] thus, providing the potential
been used in order to attach drug molecules to the side walls of f-CNTs
[13,14]. Here, we report the formulation of antileishmanial agent, betulin
(BET) using functionalized carbon nanotubes (f-CNTs).
Betulin (a pentacyclictriterpenoid) has antileishmanial property
that is mediated through inhibition of trypanothione synthetase, one
of the key redox enzymes in parasite as reported by our group earlier
[15,16] and as well as its derivatives are reported to inhibit type lB
DNA topoisomerase of Leishmania donovani [17]. In this work we have
demonstrated the synthesis of f-CNT-BET and its effect on intracellular
amastigote parasite in vitro was investigated. Also the cytotoxicity stud-
ies on macrophage cells (J774A.1 cell line) were performed.
2. Materials and methods
2.1. Parasites, cell lines and chemicals
The L. donovani (BHU-1081) promastigote culture was donated by
Prof. Shyam Sundar, Banaras Hindu University, India and cultivated in
DMEM media as explained earlier [15,16]. Macrophage cell line
J774A.1 was taken from “National Centre for Cell Science” (NCCS),
Pune, India and cultivated in DMEM media as explained earlier [15,
16]. The multi-walled carbon nanotubes (MWCNTs) were purchased
from Sigma (98% purity). All other chemicals were of the highest
grade procured from Sigma-Aldrich or Merck.

2.2. Preparation of f-CNT-BET

Abbreviations: BET, betulin; f-CNTs, functionalized carbon nanotubes; f-CNT-BET,
betulin attached functionalized carbon nanotubes; MQ, macrophage cells.
⁎ Corresponding author. Tel.: +91 361 2582203. Synthesis methodology for f-CNT-BET is shown in Fig. 1. The carbox-
E-mail address: vdubey@iitg.ernet.in (V.K. Dubey). ylic functionalization of CNTs was prepared by the oxidation of raw

http://dx.doi.org/10.1016/j.parint.2014.07.008
1383-5769/© 2014 Elsevier Ireland Ltd. All rights reserved.
 P. Saudagar, V.K. Dubey / Parasitology International 63 (2014) 772–776 773

Fig. 1. Schematic representation of the synthesis of f-CNT attached BET: The carboxylic f-CNTs were prepared by acid oxidation of CNT. The f-CNT-BET were synthesized by carbodiimide-
activated esterification reaction [18,19].

CNTs with concentrated H2SO4/HNO3 (volumetric ratio 3:1) [18,19].
The carboxylic functionalized CNTs were conjugated with BET in a
carbodiimide-activated esterification reaction [19]. In a distinctive pro-
cedure, purified carboxylic f-CNTs (50 mg) were dissolved in 10 ml of
dimethyl sulfoxide (DMSO) and sonicated for 0.5 h to obtain a homoge-
neous suspension of carboxylic f-CNTs. Later, 5 ml of BET (100 mg/ml)
in DMSO was added drop by drop in a stirring mixture of f-CNTs. In a
separate beaker, 500 mg of N,N′-dicyclohexylcarbodiimide (DCC) and
75 mg of triethyl amine (Et3N) were added to the suspension of f-CNT
and stirred in flask for 24 h. After the completion of the reaction, the
DCC urea was separated by filtration. DMSO solution was washed with
ethyl acetate and brine solution. Combined organic layers were collect-
ed and evaporated using rotary vapor to obtain f-CNT-BET and dried in
vacuum. The functionalization of CNTs and attachment of BET to the f-
CNTs were confirmed by Fourier transform infrared (FTIR) spectroscopy
and transmission electron microscopy (TEM).
2.3. BET loading efficiency on f-CNTs
Loading efficiency of BET onto the f-CNTs was measured using spec-
troscopic method. In brief, BET was initially scanned to check for its
maximum absorbance and the same absorbance was used in quantify-
ing BET further. To calculate the amount of BET on to f-CNT, a suspen-
sion of f-CNT-BET dissolved in DMSO was centrifuged at 10,000 rpm
for 10 min to precipitate f-CNT-BET. The absorbance of BET in superna-
tant was measured using a UV–visible spectrophotometer and was
774 P. Saudagar, V.K. Dubey / Parasitology International 63 (2014) 772–776
subtracted from absorbance of BET in DMSO which was used as stan-
dard to calculate the relatively bound BET onto the f-CNTs. Three indi-
vidual preparations of f-CNT-BET were carried out for loading
efficiency to check reproducibility.
2.4. In vitro drug release experiments
Two dependent dialysis experiments each of 2 ml of f-CNT-BET
against 15 ml of phosphate buffer in two different flasks, one with pH
7.4 and the other with pH 5.8 at 37 °C shaken for 72 h were done to
evaluate BET release from f-CNT-BET. At different time points, 2 ml of di-
alysate was collected out from the flask and replaced with equal volume
of fresh phosphate buffer. The release of BET was quantified by spectro-
scopic measurement read at A210 nm as BET gave maximum absorbance
at 210 nm.
2.5. In vitro cytotoxicity on macrophage cells
The cytotoxic effect of BET, f-CNTs and f-CNT-BET was studied using
mouse macrophage cell line J774A.1 by MTT [3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide] assay [20] as described previous-
ly [15]. In brief, mouse macrophage cell line J774A.1 were cultured in
96-well plates, 2000 cells/well were seeded and allowed to adhere over-
night at 37 °C in a humid atmosphere of 5% CO2. The next morning the
un-adhered cells were removed by PBS washing and replaced with fresh
media containing defined concentration of either BET or f-CNTs or f-
CNT-BET and incubated for 48 h. After completion of the incubation pe-
riod, MTT assay was performed [15,20]. IC50 values for each of the com-
pound were calculated by plotting % cell viability vs. concentration.
2.6. In vitro antileishmanial activity assay
The macrophage cell line J774A.1 of cell density of 5 × 105 cells was
spread on a cover slip and incubated over night. The non-adherent cells
were removed by washing with PBS and a fresh medium was added.
Macrophages were infected with L. donovani promastigotes (parasite:
macrophage ratio of 10:1) and incubated at 37 °C in 5% CO2 for 6 h to
ensure parasite phagocytized by macrophage cells. Further, un-
phagocytized parasites were removed washing with PBS and a fresh
medium was added and incubated for 6 h. The graded concentrations
of BET or f-CNTs or f-CNT-BET were added and further incubated for
48 h. After completion of incubation with compounds, cells were then
fixed in methanol and Giemsa stained. The antileishmanial efficacy of
compounds was evaluated by microscopic counting of 100 macrophage
cells. The parasite density in treated cells was expressed as a percentage
of control and the IC50 value for each of the compound was calculated by
plotting % amastigotes/100 macrophage vs. concentration.
2.7. Statistical analysis
Data analysis was performed as reported in our earlier publication
[16]. The data were expressed as mean ± standard deviation. Differ-
ences between two groups were analyzed for statistical significance by
unpaired Student's t test using SigmaPlot software. Differences were
considered significant at 0.05 and 0.001 levels of confidence.
3. Results
3.1. Conformation of f-CNT-BET construct as characterization by FTIR and
TEM
The carboxylic acid functionalization of CNTs (f-CNTs) and attach-
ment of BET on to the f-CNTs were successfully achieved as evident
from FTIR spectroscopic analysis (Supplementary material Fig. 1).
The strong band at wavelength 3426 cm–1 corresponds to the presence
of \OH moieties on the surface of CNTs (Supplementary material Fig. 1).
Further, after functionalization of CNTs, the new peak at wavelength
1768 cm–1 due to C_O stretching of carboxyl functional groups
(\COOH) has emerged (Supplementary material Fig. 1). The peaks at
wavelength 2920 cm–1 and 2849 cm–1 were assigned to O\H stretch
of carboxyl functional groups. The distinctive peaks observed in FTIR
spectra of f-CNTs have confirmed the carboxylic functionalization of
CNTs. The attachment of BET on to f-CNT was achieved by carbodimide
based esterification reaction. The synthesis of f-CNT-BET was confirmed
by the characteristic ester C_O asymmetric stretching as studied by
FTIR spectra. The peak at 1741 is due to the C_O asymmetric stretch
of the ester linkage present in synthesized f-CNT-BET (Supplementary
material Fig. 1).
The physical characterization of f-CNTs and f-CNT-BET was done
by studying micrograph structures using TEM (Supplementary ma-
terial Fig. 2). The micrograph of f-CNTs as seen Fig. 3 A and C shows
the length and slightly rough surface caused by functionalization
with acid treatment. The functionalization of CNTs improves its sol-
ubility nature and also provides the surface anchors to attach drug
molecules. The micrograph of f-CNT-BET as seen in Fig. 3B and D
was also characterized by TEM using the same image scale to under-
stand the morphological changes. The formation of a very rough sur-
face on the exterior and interior walls of the f-CNT-BET can be very
clearly seen. The diameter of f-CNT-BET is remarkably increased
due to attachment of BET onto the f-CNT surface, forming a layer.
The presence of dark spots indicated by arrow marks shows the
drug attachment sites. The successful synthesis of f-CNT-BET is evi-
dent from FTIR and TEM results.
3.2. BET loading efficiency onto f-CNTs
BET gave the maximum absorbance of 210 nm (Fig. 2A). Hence, the
loading efficiency of BET was spectroscopically measured at the same
wavelength. The absorbance of 3.846 and 0.833 at 210 nm was recorded
by 1 mg/ml BET and unbound BET from the supernatant of f-CNT-BET,
respectively. Thus, the percentage of BET loaded on to the f-CNTs was
calculated to be 78.3% (Fig. 2B). The percentage of BET loading onto
the f-CNTs was reproducible.
3.3. In vitro release of BET
In vitro drug release profiles of BET from f-CNT-BET in phosphate
buffer with pH 7.4 and 5.8 at 37 °C are shown in Fig. 2C. The drug release
was tested under acidic and neutral pH conditions to mimic the cytosol
and lysosome conditions of the macrophage cells. The release of BET
reached up to 38.4 ± 1.8% in acidic pH 5.8 in a 72 h incubation period,
whereas only 12.5 ± 1.1% of BET release was observed in neutral
pH 7.4 condition with same incubation time. The more release of BET
in acidic condition is observed compared to neutral pH condition be-
cause the ester bond degrades rapidly under acidic condition and slowly
in the neutral pH condition.
3.4. In vitro cytotoxicity
In the host, Leishmania parasite thrives and proliferates inside the
macrophage cells and hence, it is the main site for drug delivery. Nano for-
mulation mediated drug delivery is specific for macrophages due to their
phagocytosis nature. Therefore it is important to assess the cytotoxicity of
f-CNT-BET formulation to the macrophage cells themselves. The cytotox-
icity was studied using standard MTT assay which is also used by other
groups to assess the toxic response on CNTs to cells [21]. To understand
the cytotoxic effect of BET, f-CNT and f-CNT-BET on host cells, the
in vitro cytotoxicity studied on the macrophage cell line J774A.1 was eval-
uated at different concentrations and the fifty percent inhibition concen-
tration was calculated for all the three compounds (Fig. 3). The IC50 value
of BET, f-CNT and f-CNT-BET was calculated to be 211.05 ± 7.14 μg/ml,
24.67 ± 3.11 μg/ml and 72.63 ± 6.14 μg/ml respectively. The data clearly
 P. Saudagar, V.K. Dubey / Parasitology International 63 (2014) 772–776 775

Fig. 2. Measuring BET loading efficiency onto the f-CNT and its relea: (A) spectral scan of BET compound. The maximum absorbance read by BET is 210 nm; (B) loading efficiency BET on to
f-CNT. Around 78.3% of the drug loaded efficiency was achieved; (C) the drug release profile of BET studied at acidic and neutral pH conditions. Around 38.4 ± 1.8% of BET was released in
acidic pH condition whereas only around 12.5 ± 1.1% of BET release in neutral pH condition.

indicates that BET, f-CNT and f-CNT-BET did not have any significant
toxicity and thereby indicating their safe application in antileishmanial
drug formulation.
3.5. The f-CNT-BET has better efficacy than BET
The antileishmanial activity in the form of parasite burden in para-
site infected macrophage cells was studied microscopically as explained
in the Methods section. There is a significant difference observed in par-
asite burden in cells treated with BET and f-CNT-BET. It was observed
that nearly 80% of the amastigotes were inhibited at 3.91 μg/ml of f-
CNT-BET whereas only 30% inhibition was observed with BET at same
concentration (Fig. 4). The IC50 value for BET and f-CNT-BET calculated
was 8.33 ± 0.41 μg/ml and 0.69 ± 0.08 μg/ml respectively. There is
more than tenfold difference in the IC50 value for BET and f-CNT-BET,
which clearly indicates the increased efficacy of drug due to their
targeted site of delivery. The antileishmanial activity of f-CNTs was
also tested and there is no notable antileishmanial activity detected
even at the high concentration of 15.63 μg/ml of f-CNTs (Fig. 4). There-
by, revealing that the improved efficacy is due to the action of the drug
attached to f-CNT but not by f-CNT alone.
4. Discussion
In the last decade several drug formulations like liposomal, emul-
sions, micelles and nanoparticles have been employed for targeted de-
livery of drug to the particular tissue [22–25]. Unfortunately, most of
these formulations fail to reach the target as they are readily engulfed
by the macrophage cell that recognizes them as foreign particles. Fortu-
nately this nature of macrophages facilitates the antileishmanial drug
delivered to the parasite infected site which is macrophage cells itself.
Although the most successful drug formulation for leishmaniasis liposo-
mal amphotericin B is available in the market, its use is limited due to its
high cost, in spite of preferential pricing of US$18/50 mg negotiated by
the WHO. A new drug with cost effective formulation can change the
present drawbacks with chemotherapeutics against leishmaniasis.
BET, a pentacyclictriterpenoid widely found in the bark of white birch
trees, is a widely studied anticancer compound can emerge as new
antileishmanial drug due to its selective toxicity toward parasite cells.
We have explored the properties of f-CNTs and BET to construct f-
CNT-BET which was achieved by carbodimide based esterification reac-
tion. The drug release was more at acidic conditions, thereby favoring
high release of drug at targeted site i.e. lysosomal complex inside the
macrophage cells where the parasite thrives and proliferates.

Fig. 3. In vitro assessment of cytotoxicity studied on macrophage cells (J774A.1 cell line): (A) cytotoxic effect of BET on macrophage cells and the IC50 value observed to be 211.05 ±
7.14 μg/ml; (B) cytotoxic effect of f-CNTs on macrophage cells and the IC50 value observed to be 24.67 ± 3.11 μg/ml; and (C) cytotoxic effect of f-CNT-BET on macrophage cells and
the IC50 value observed to be 72.63 ± 6.14 μg/ml. Statistical analysis of data was performed as mentioned in the Methods section.
776 P. Saudagar, V.K. Dubey / Parasitology International 63 (2014) 772–776
Fig. 4. In vitro antileishmanial activity on intracellular amastigote cells: The antileishmanial
activity of BET, f-CNTs, andf-CNT-BET was studied using microscopic counting of amastigote
cell inside the infected macrophage cells. A dose dependent response is observed with BET
and f-CNT-BET, whereas the f-CNT did not show any antileishmanial effect on parasite, re-
vealing that the f-CNTs alone are not antileishmanial and the improved efficacy is due to
the impact of the drug attached to f-CNT-BET. The IC50 value for BET and f-CNT-BET is
found to be 8.33 ± 0.41 μg/ml and 0.69 ± 0.08 μg/ml, respectively. Statistical analysis of
data was performed as mentioned in the Methods section.
We have selected CNTs as the nanocarriers for the attachment of
BET, primarily due to their ability to cross the cell membrane and have
a large surface area where a large quantity of drug can be loaded and
thereby increasing the drug efficacy. This work shows a new approach
to leishmaniasis cure. By employing f-CNTs as nanocarriers for the
drug delivery system of new antileishmanial drug BET, its efficacy is sig-
nificantly increased as observed in in vitro settings. There is no obvious
toxicity measured when tested on macrophage cell, thus indicating
their biocompatibility at cellular level. The f-CNTs have several advan-
tages as nanocarriers of drug compared to other materials as the surface
of CNTs can be designed according to our need for anchoring the drug
stably onto them. The chemical synthesis of f-CNT-BET is rather cost ef-
fective than macromolecules, which can be produced in low cost com-
pared to existing liposomal formulation.
5. Conclusion
We conclude that, we have successfully synthesized f-CNT-BET by
carbodimide based esterification reaction and investigated loading effi-
ciency and release prolife of the drug, BET. We have studied the cytotox-
icity of BET, f-CNT and f-CNT-BET on macrophage cell which
demonstrated their biocompatibility nature at cellular level. The f-
CNT-BET showed high antileishmanial activity with increased efficacy
compared to BET as evidenced from in vitro antileishmanial studies on
intracellular amastigote. Taken together our results f-CNT-BET can
emerge as a new therapy in treating leishmaniasis. The potency of the
compound has to be further validated on in vivo model. Studies with
in vivo model may further help in better assessing the f-CNT-BET in de-
veloping improved chemotherapeutics for treating leishmaniasis.
6. Author's contribution
VKD has conceptualized the idea. PS has performed the experiments
and wrote the manuscript.
Acknowledgments
Research fellowship to PS by IIT Guwahati is acknowledged. Finan-
cial support by the Department of Biotechnology, Government of India
in the form of research grant (Project no: BT/PR152/MED/29/282/
2011) to VKD is also acknowledged.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.parint.2014.07.008.
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