27 Colloids and surface BIointerfaces

Colloids and Surfaces B: Biointerfaces 132 (2015) 17–26
Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces
journal homepage: www.elsevier.com/locate/colsurfb
Cancer targeting propensity of folate conjugated surface engineered
multi-walled carbon nanotubes
Neelesh Kumar Mehraa,b
, N.K. Jaina,b,∗
a
Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar 470 003, India
b
Pharmaceutical Nanotechnology Research Laboratory, ISF College of Pharmacy, Moga 142 001, India
a r t i c l e i n f o
Article history:
Received 4 November 2014
Received in revised form 25 April 2015
Accepted 27 April 2015
Available online 7 May 2015
Keywords:
Carbon nanotubes
Docetaxel
Pharmacokinetic
Kaplan–Meier survival
Tumor growth inhibition
Anticancer activity
a b s t r a c t
Our main aim in the present investigation was to investigate the cancer targeting potential of docetaxel
(DTX) loaded, folic acid (FA) terminated, poly (ethylene glycol) (PEG) conjugated, surface engineered
multi walled carbon nanotubes (DTX/FA-PEG-MWCNTs) in tumor bearing Balb/c mice. The percent load-
ing efficiency of DTX/FA-PEG-MWCNTs and DTX loaded MWCNTS (DTX/MWCNTs) was calculated to
be 93.40 ± 3.82% and 76.30 ± 2.62%, respectively. Flow cytometry analysis suggested that the DTX/FA-
PEG-MWCNTs arrested MCF-7 cells’ cycle in the G2 phase and was more cytotoxic as compared to
DTX/MWCNTs as well as free drug solution. The obtained pharmacokinetic parameters clearly describe
the biocompatibility of engineered nanotubes to degree of functionalization and ability for prolonged
residence inside the body. DTX/FA-PEG-MWCNTs was found to be significantly more efficient in tumor
suppression as compared with plain MWCNTs (non-targeted) as well as drug solution owing to the
enhanced drug release from endosomes after internalization. The DTX/FA-PEG-MWCNTs showed highly
significant prolonged survival span (40 days) as compared to DTX/MWCNTs (24 days), free DTX (19 days)
and control group (12 days). Overall, we can conclude that the DTX/FA-PEG-MWCNTs shows higher
cancer targeting propensity vis a vis minimal side effects in tumor bearing Balb/c mice.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
The development of ‘safe and effective’ nanomedicines for
the treatment of diseases including diabetes, acquired immune
deficiency syndrome, tuberculosis and cancer still remains the fore-
most challenging task to researchers, scientists and academicians,
worldwide. Cancer accounted for 7.6 million deaths (approximately
13% of all deaths) in 2008 according to recent Fact Sheet of World
Health Organization (WHO) wherein approximately 70% deaths
occurred in low- and middle-income countries. In 2030, cancer
deaths are projected to rise over 13.1 million, worldwide [1]. The
failure of chemotherapy is due to the non-selectivity as well as
inability to target the anticancer agent(s) to the cancerous cells.
The various available nano-sized carrier systems including den-
drimers [2], nanoparticles [2] and carbon nanotubes [3–8] are being
∗ Corresponding author at: Pharmaceutics Research Laboratory, Department
of Pharmaceutical Sciences, Dr. H. S. Gour University, Sagar 470 003, India.
Tel.: +91 7582 265055; fax: +91 7582 265055.
E-mail addresses: neelesh81mph@gmail.com (N.K. Mehra),
jnarendr@yahoo.co.in (N.K. Jain).
continuously explored for improved specificity and targeting as
well as realizing the attributes of the ‘magic bullet’ concept.
In the past two decades, surface engineered carbon nanotubes
(CNTs) have been explored designed and considered as valuable,
promising, ‘safe and effective’ alternative nano-architecture for
pharmaceutical and biomedical applications due to their unique
physicochemical properties. CNTs comprise of thin graphite sheets
of condensed benzene rings rolled upon into the nanoneedle, seam-
less tubular hollow cylinder. CNTs can be distinguished on the basis
of their lengths, diameters, and most importantly, presence of walls
and are categorized into single-, double-, triple-, and multi-walled
carbon nanotubes [3,9–11]. The pristine CNTs (first generation;
untreated CNTs) are not suitable for drug delivery on account of
their hydrophobicity and toxicity due to the presence of impurities,
which can fortunately be overcome by surface functionalization.
Higher degree of functionalization (hence lower toxicity) makes
nanotubes better, safer and effective drug delivery system [12].
The surface alterations of CNTs can be performed either by cova-
lent or non-covalent interactions depending on the intermolecular
interaction. The non-covalent modifications, based on the extended
␲-system (p-orbital) of the nanotubes sidewall, interact with the
guest chemical moieties through ␲–␲ stacking interactions. Cur-
rently, surface engineered CNTs are being explored for targeted
http://dx.doi.org/10.1016/j.colsurfb.2015.04.056
0927-7765/© 2015 Elsevier B.V. All rights reserved.

18 N.K. Mehra, N.K. Jain / Colloids and Surfaces B: Biointerfaces 132 (2015) 17–26
delivery and have been claimed to be non-toxic to human cells
[9,12,13].
Docetaxel (N-debenzoyl-N-tert-butoxycarbonyl-10-deacetyl-
paclitaxel) used in the present investigation is a semi-synthetic
taxane, derived from the precursor 10-deacetyl baccatin III and
extracted from the European yew tree Taxus baccata for targeting
to ␤-subunit of tubulin. It entered into clinical trials in 1990 and
demonstrates the efficacy in the treatment of several malignancies
including prostate, small and non-small cell lung cancer and breast
cancer etc. [14].
In the present investigation we intended to explore the can-
cer targeting potential of the docetaxel (DTX) bearing surface
engineered MWCNTs. The developed DTX bearing surface engi-
neered MWCNTs nanoconjugates were characterized for loading
efficiency, in vitro release, hemocompatibility and toxicity in tumor
bearing Balb/c mice.
2. Experimental
2.1. Materials
Multi Walled Carbon Nanotubes (MWCNTs) produced by chem-
ical vapor deposition (CVD) with 99.3% purity, were purchased from
Sigma Aldrich Pvt. Ltd. (St. Louis, Missouri, USA). Docetaxel was
received as a benevolent gift from M/s Fresenius Kabi Oncology Ltd;
(FKOL) (formerly Dabur Research foundation), Sahibabad, India. All
reagents and solvents were used as received.
2.2. Surface engineering of the pristine MWCNTs
Firstly, the procured pristine MWCNTs were purified by treat-
ing in a microwave oven (GEM Insta Cook, Gurgaon, India) at
400 ± 2 ◦C for 2 h. The microwave treated MWCNTs (500 mg) were
refluxed with a mixture of concentrated Nitric and Sulphuric acid
(HNO3:H2SO4::1:3 ratio) in a flat bottom flask (equipped with
the reflux condenser and thermometer) with continuous mag-
netic stirring (100 RPM; Remi, Mumbai, India) at 120 ± 5 ◦C for
6 h; washed, ultra centrifuged (20,000 rpm for 15 min; Z36HK,
HERMLE LaborTchnik GmbH, Germany), vacuum dried (Jyoti Sci-
entific Industries, Gwalior, India), lyophilized (Heto dry Winner,
Denmark, Germany), and collected [9,13,15].
2.3. Folic acid (FA) conjugation with surface engineered MWCNTs
The MWCNTs were conjugated with folic acid (FA) using PEG
spacer and characterized following the method reported by us ear-
lier [9].
2.4. Loading efficiency
The MWCNTs: DTX in optimized ratio (1:2) were added to
anhydrous ethanol (0.5 mL) in an ultrasonic bath for about 15 min
with drop-wise addition of PBS (pH 7.4) solution and ultrasoni-
cated using an ultrasonic probe (400 W) for approximately 10 min
(Lark, Chennai, India). The resultant suspension was ultracen-
trifuged at 10,000 rpm for 10 min until the MWCNTs were fully
separated, and the obtained supernatants were discarded. The
remaining solids were thoroughly rinsed with anhydrous ethanol
and deionized water to remove excess docetaxel. The amount of
unbound DTX in the solution was determined by measuring the
absorbance at max 230 nm in a spectrophotometer (Shimadzu
1601, UV–Visible Spectrophotometer, Shimadzu, Japan) and the
DTX loading efficiency was calculated (n = 3). The product was col-
lected, dried, lyophilized (Heto dry winner, Denmark, Germany)
and stored at 5 ± 3 ◦C for further studies. Finally, the DTX loaded
MWCNTs formulations i.e. DTX loaded MWCNTs (DTX/MWCNTs)
and DTX loaded FA-PEG-MWCNTs (DTX/FA-PEG-MWCNTs) were
prepared.
2.5. Characterization of pristine and functionalized MWCNTs
The pristine and surface engineered MWCNTs were extensively
characterized using different analytical characterization tools. The
surface topography of the pristine and surface engineered MWC-
NTs was determined through transmission electron microscopy
(TEM; Morgagni 268-D, Fei Electron Optics, Holland) after drying
on carbon-coated copper grid and negative staining with 1% phos-
photungstic acid (PTA) [13].
The surface fracture of the nanotubes nanoformulations was
studied using atomic force microscopy (AFM) in a tapping mode
with Digital Nanoscope IV Bioscope (Veeco Innova Instruments,
Santa Barbara, CA, USA) after drying in air.
The average particle size and size distribution were determined
by photon correlation spectroscopy in a Malvern Zetasizer nano
ZS90 (Malvern Instruments, Ltd, Malvern, UK) at room temperature
(RT) after addition of surfactant.
The Raman spectra of the pristine and f-MWCNTs for
order-disorder hexagonal carbon were recorded using Raman
micro-spectroscopy RINSHAW, inVia Raman Spectrophotometer
(RENISHAW, Gloucestershire, UK). The microspectrophotometer
was operated with 532 nm laser radiation under objective lens of
20× magnification (Olympus BX 41, USA) with a slit of 1 × 6 mm
whereas the incident power was approximately 1 mW with 30 s
exposure time [13].
The X-ray diffractograms (XRD) were recorded (X-ray diffrac-
tometer, PW 1710 Rigaku, San Jose, CA) by adjusting X-ray power
of 40 kV and 40 mA of MWCNTs formulations [13].
2.5.1. In vitro release studies
The release of docetaxel from the developed MWCNTs formu-
lations (DTX/FA-PEG-MWCNTs and DTX/MWCNT) was monitored
separately in sodium acetate buffer saline pH 5.3 (lysosomal pH),
and phosphate buffer saline pH 7.4 (physiological pH) through
a modified dialysis diffusion technique while maintaining the
physiological temperature 37 ± 0.5 ◦C throughout the study (n = 3)
[13,14,16–19]. The known amount of DTX loaded MWCNTs for-
mulations (10 mL) was added in the dialysis sac (MWCO, 12 kDa),
hermetically tied and placed into the receptor compartment
(ethanol: phosphate buffer pH 5.3:7.4::3:7 containing Tween 80)
with slow and continuous magnetic stirring at 37 ± 0.5 ◦C under
strict sink condition. Tween 80 was used in the release medium to
solubilize the DTX and to facilitate the passage across the dialysis
membrane. Aliquots were withdrawn at definite time points from
the mixture and immediately replenished with an equal volume
of fresh medium for estimation of the concentration of DTX using
UV/Visible spectrophotometer at max 230.0 nm (UV/Vis, Shimadzu
1601, Kyoto, Japan).
2.5.2. Accelerated stability study
The DTX/FA-PEG-MWCNTs and DTX/MWCNTs were stored in
tightly closed glass vials separately in dark as well as in amber col-
ored and colorless glass vials at 5 ± 3, 25 ± 2 and 40 ± 2 ◦C for a
period of six months in stability chambers (Remi CHM-6S, India)
(n = 3) [13,20]. The MWCNTs formulations were analyzed initially
and periodically up to six months for any change in particle size,
drug content and organoleptic features like aggregation, precipita-
tion, color and odor, if any.
2.6. Comparison of hemolytic toxicity
The hemolytic toxicity of the administered MWCNTs formula-
tions was assessed in vitro [13,19]. Briefly, fresh whole human blood

N.K. Mehra, N.K. Jain / Colloids and Surfaces B: Biointerfaces 132 (2015) 17–26 19
was collected in Hi-Anticlot blood collecting vials (HiMedia, Mum-
bai, India) and centrifuged at 3000 rpm (Remi, Mumbai, India) for
15 min in an ultracentrifuge (Z36HK, HERMLE LaborTchnik GmbH,
Germany). The red blood corpuscles (RBCs) were collected from
the bottom and separated out, washed with physiological normal
saline (0.9%; w/v) until clear and the colorless supernatant was
obtained above the cell mass. The RBCs suspension (1 mL) was
mixed separately with 0.9% (w/v) normal saline (4.5 mL), free DTX,
DTX/MWCNTs and DTX/FA-PEG-MWCNTs dispersions (0.5 mL) and
incubated for 60 min, to allow interaction. After incubation, sam-
ples were centrifuged for 15 min at 1500 rpm and the supernatant
was taken to quantify the hemoglobin content at max 540 nm
spectrophotometrically considering 0.9% w/v NaCl solution (nor-
mal saline) and deionized water, respectively as zero and 100%
hemolytic (n = 3).
2.7. Cell culture experiment
The MCF-7 (Michigan Cancer Foundation; folate overexpress-
ion) derived from pleural effusion was procured from National
Center for Cell Sciences (NCCS), Pune, India. Before starting cell
culture experiments, the cells were pre-cultured until approxi-
mately 80% confluence was reached. The cell lines were cultured
in Dulbecco’s Modified Eagle Medium (DMEM; HiMedia, Mum-
bai, India) containing 10% fetal bovine serum (FBS; HiMedia,
Mumbai, India) supplemented with 2 mM l-glutamine and 1%
penicillin–streptomycin mixture (Sigma, St Louis, Missouri) in
humidified atmosphere containing 5% CO2 at 37 ◦C [13].
2.7.1. Methylthiazole tetrazolium (MTT) cytotoxicity assay
The MCF-7 cells were incubated in 96-well transparent tissue
culture plates at density of 1 × 104 cells/well. After 12 h the old
medium was removed and cells were incubated separately with the
DTX, DTX/MWCNTs and DTX/FA-PEG-MWCNTs at equivalent DTX
concentrations (0.001, 0.01, 1, 10, 100 ␮M) and allowed to adhere
for 24 and 48 h at 37 ◦C prior to assay. The medium was decanted
and 50 ␮L of methylthiazole tetrazolium (MTT) (1 mg/mL) in DMEM
(10 ␮L; 5 mg/mL in Hank’s balanced Salt Solution; without phenol
red) was added and incubated. The experiment was performed in
triplicate. The absorbance of the wells was measured at 570 nm and
the percent cell viability was calculated using following formula:
Cell viability (%) =
[A]test
[A]control
× 100
where [A]test is the absorbance of the test sample and [A]control is
the absorbance of control samples.
2.7.2. Cell cycle analysis
Cell cycle distribution of the developed nanotubes formula-
tions in different phases was studied using the cultured MCF-7
cells (n = 3). Briefly, the cultured MCF-7 cells were treated with
the developed MWCNTs formulations (2 nM/mL) for 24 h while
the cells treated with culture medium served as control. The cells
were harvested by centrifugation, washed with ice-cold PBS and
fixed using 70% cold ethanol overnight. Subsequently cells were
trypsinized, washed and fixed in 70% ethanol and further treated
with Ribonuclease A (DNase free, 100 ␮g/mL) and propidium iodide
(PI; 50 ␮g/mL) for 30 min at 37 ◦C in dark. The treated cells were
centrifuged and obtained cell pellets were re-suspended with PBS
and kept until use. The percent of cells arrested in different phases
(G1, G2, and S phase) of the cell cycle event was counted using
cycle analysis software with FACSCalibur Flow Cytometer (Becton,
Dickinson Systems, FACS cantoTM, USA) [13,21–23].
2.7.3. Cellular uptake of the formulations through flow cytometry
The cellular uptake study of free DTX and DTX loaded MWCNTs
formulations labeled in 5:1 ratio of DTX with fluorescein isothio-
cyanate (FITC) solution (100 ␮g/mL in DMSO) using MCF-7 cell line
was performed in a flow cytometer for quantitative analysis (n = 3).
The developed formulations and free DTX were incubated for 4 h
as in case of DNA cell cycle content. After 3 h exposure, cells were
washed with cold-PBS, trypsinized, centrifuged and analyzed quan-
titatively using FACSCalibur Flow Cytometer (Becton, Dickinson
Systems, FACS cantoTM, USA).
2.8. In vivo studies
The in vivo studies were carried out with prior approval of
Institutional Animal Ethics Committee as per guidelines of the Com-
mittee for the Purpose of Control and Supervision of Experiments
on Animals (CPCSEA) of Dr. H.S. Gour Vishwavidyalaya, Sagar (M.P.),
India (Registration No. 379/01/ab/CPCSEA/02). The Balb/c mice of
uniform weight (20–25 g) were housed in ventilated plastic cages
and allowed access to water ad libitum. The Balb/c mice were fed on
a special low-folate diet and acclimatized at 25 ± 2 ◦C and 50–60%
relative humidity under natural light/dark condition prior to in vivo
study [9].
The tumor was developed using right flank method by injecting
serum-free cultured MCF-7 cells (1 × 107 cells) in the right hind leg
of the mice. The tumor development was monitored by palpating
the injected area with index finger and thumb for the presence of
the tumor (approximately 100 mm3) [9,13,24].
2.8.1. Determination of pharmacokinetic parameters after
intravenous administration
The pharmacokinetic parameters after intravenous (i.v.) admin-
istration of free DTX and DTX loaded nanotubes formulations
(30 mg/kg body weight) were determined. The blood samples were
collected in Hi-Anticlot blood collecting vials (HiMedia, Mumbai,
India) at different time points (0.25, 0.5, 1, 2, 3, 6, 12, 18, 24 and
48 h) from the retro-orbital plexus of eyes (animals) under mild
anesthesia. The supernatant (serum) was collected after centrifu-
gation of blood, vortexed and ultracentrifuged (Z36HK, HERMLE
LaborTchnik GmbH, Germany), and finally the concentration of
drug was determined by High Performance Liquid Chromatography
(HPLC) method and different pharmacokinetic parameters were
calculated.
2.8.2. Tissue/organ biodistribution study
The organ biodistribution study of the free DTX, DTX/MWCNTs
and DTX/FA-PEG-MWCNTs formulations was performed on tumor
bearing Balb/c mice (n = 3). The formulations (equivalent dose of
DTX = 30.0 mg/kg body weight) were sterilized using 0.2 ␮m milli-
pore filter and administered intravenously through caudal tail vein
route. The mice were carefully sacrificed by decapitation at 1, 6, 12
and 24 h time points for the collection of organs like liver, spleen,
kidney, heart, and tumor. The collected organs were washed in
Ringer’s solution, dried with the help of tissue paper, weighed and
stored frozen till used. The required quantity of ethanol was added
and homogenized (York Scientific Instrument, New Delhi, India),
vortexed and ultracentrifuged at 3000 rpm for 15 min (Z36HK,
HERMLE LaborTchnik GmbH, Germany). The clear supernatant was
collected, injected into an HPLC system (Shimadzu, C18, Japan) and
assayed for DTX content wherein mobile phase consisted of ace-
tonitrile:methanol:0.02 M ammonium acetate buffer (pH 5.0) in
20:50:30; v/v/v ratio at 1 mL/min flow rate at 102/101 bar pressure.
2.8.3. Assessment of anti-tumor targeting efficacy
The in vivo cancer targeting efficacy of the DTX/FA-PEG-
MWCNTs and DTX/MWCNTs formulations was determined in

tumor bearing Balb/c mice. The Balb/c mice were accommodated in
a pathogen-free laboratory environment during the tenure of the
studies. Tumor measurement was performed using electronic digi-
tal Vernier Caliper and the tumor volume at the longest and a widest
two dimension point was measured. The tumor size was calculated
using the formula = 1/2 × length × width2 and median survival time
was also recorded. The in vivo tumor study was terminated 45 days
post-treatment [13].
The hematological study was performed following a reported
method for DTX/FA-PEG-MWCNTs, DTX/MWCNTs and free DTX
and analyzed at a local pathology laboratory [9,13,25].
2.9. Statistical analysis
The results were expressed as mean ± standard deviation (n = 3)
and statistical analysis was performed with Graph Pad Instat Soft-
ware (Version 3.00, Graph Pad Software, San Diego, California, USA)
by one-way ANOVA followed by Tukey–Kramer test for multiple
comparison. The pharmacokinetic data analysis of plasma concen-
tration time profile was conducted using the Kinetica software
(Thermo scientific, USA) followed by non-compartment analysis.
A probability of p ≤ 0.05 was considered significant while p ≤ 0.001
was considered to be extremely significant.
3. Results and discussion
Folic acid (FA), also known as folate, vitamin Bc (folacin), vitamin
B9, M, pteroyl-l-glutamic acid, is a water-soluble vitamin, neces-
sary for the synthesis of purines and pyrimidines. FA conjugated
nanocarriers are known to exhibit ligand–receptor interactions,
internalized through caveolae-mediated endocytosis mechanism,
and release the drug molecules into the cytoplasm. Numerous
study reports are already available on folate-mediated targeting
of anticancer bioactives [9,16,21,26,27]. Castilo et al. reported the
non-covalent conjugate of SWCNTs and FA aimed to interact with
cells over-expressing folate receptors using rapid ‘one pot’ syn-
thesis method. The low toxicity of SWCNTs-FA by cancer cells
suggested their potential use in drug delivery and diagnosis of can-
cer or treatment of tropical diseases such as leishmaniasis [28].
In the current scenario, surface tailored carbon nanotubes (CNTs)
are attracting great attention in the treatment of cancer including
theragnostic applications. We have initially functionalized the pro-
cured MWCNTs with PEG spacer and appended the folic acid (FA) as
a targeting ligand for specific targeting [9]. The high drug loading
ability of the surface engineered MWCNTs suggests the potential
application of CNTs as a targeted drug delivery system. The percent
loading efficiency of DTX in DTX/MWCNTs and DTX/FA-PEG-
MWCNTs formulations determined through modified dissolution
method was found to be 76.30 ± 2.62% and 93.40 ± 3.82%, respec-
tively. This high percent loading efficiency was achieved due to
strong hydrophobic, electrostatic and ␲–␲ stacking interactions
among CNTs and DTX. The high loading efficiency of engineered
nanotubes makes it a better carrier with better stability at normal
pH and sustained release in acidic microenvironments (lower pH).
Our in vitro results are in good agreement with previous reports
[19,28,29].
The TEM and AFM were used to investigate the surface
morphology in terms of size, shape and topography of the devel-
oped engineered MWCNTs formulations. TEM photomicrographs
suggest that the nanotube formulations were tubular and in nano-
metric size range (Fig. 1A and B). AFM analysis also revealed the
nanoneedle tubular structure of the DTX/FA-PEG-MWCNTs formu-
lation (Fig. 1C).
The average particle size (nm) and size distribution (PSD)
with polydispersity index (PDI) were determined in a Malvern
Zetasizer nano ZS90 (Malvern Instruments, Ltd, Malvern, UK) at
room temperature (RT). The particle size of the DTX/MWCNTs
and DTX/FA-PEG-MWCNTs was found to be 220.41 ± 9.50 (PI-
0.27 ± 0.02) and 240.28 ± 8.60 nm (PI-0.42 ± 0.06), respectively.
Ren and co-workers reported the particle size and polydisper-
sity index (PI) of the doxorubicin loaded angiopep-2 modified
PEGylated oxidized MWCNTs (DOX-O-MWCNTs-PEG-ANG) to be
202.23 ± 3.43 nm and 0.342 ± 0.01, respectively [24].
The XRD analysis of functionalized MWCNTs and FA-PEG-
MWCNTs clearly precludes any change in the original seamless
tubular structure as in case of pristine MWCNTs (Fig. 2A and B).
Raman spectroscopy provides information about the hybridiza-
tion state and the defect chemistry of the CNTs. CNTs have four main
bands in Raman spectrum (i) radial breathing mode (RBM), (ii) G-
band, (iii) D-band, and (iv) D mode [13,29]. The Raman spectrum
of the purified MWCNTs showed the Raman shift at 1579.85 cm−1
and at 1346.15 cm−1, which correspond to the G band (graphite-
like mode) and D band (disorder-induced band), respectively. The
Raman spectrum of the DTX/FA-PEG-MWCNTs showed the G band
around 1565 cm−1 and D band around 1310 cm−1. The shifting of
the G and D band to the lower Raman intensity in the DTX/FA-PEG-
MWCNTs is mainly due to increase in the extension of conjugation
of FA with functionalized MWCNTs, which would increase
the single bond characteristic in the functionalized systems
(Fig. 2C and D).
Fig. 1. Transmission electron microscopic image of (A) DTX/FA-PEG-MWCNTs, (B) DTX/MWCNTs and (C) atomic force microscopic image of DTX/FA-PEG-MWCNTs.

Fig. 2. X-ray diffraction pattern of (A) pristine, and (B) DTX/FA-PEG-MWCNTs and Raman spectra of (C) pristine, and (D) DTX/FA-PEG-MWCNTs conjugate.
The cumulative in vitro release of DTX from the surface tailored
MWCNTs formulations was studied at the normal physiological
and lysosomal pH for determining the overall pharmaceutical ther-
apeutic efﬁcacy in blood stream and at target site (Fig. 3). The
pH of the cytosol is neutral to mildly alkaline (7.4–7.8) while
lysosomal pH is acidic (4–5.5). During the internalization of the
DTX/MWCNTs into the target MCF-7 cells, initially the drug has
to be released from the nanotubes formulations in order to exert
its overall therapeutic effect. The in vitro release behavior of
DTX from the surface engineered MWCNTs formulations exhib-
ited biphasic pattern that was characterized by an initial faster
followed by sustained release. At pH 5.3 and 7.4 the cumulative
percent DTX release was found to be 93.20 ± 3.76, 33.20 ± 1.88
and 85.90 ± 3.82, 17.40 ± 0.10 for DTX/MWCNTs, and DTX/FA-PEG-
MWCNTs, respectively in 24 h whereas in 200 h cumulative DTX
release from DTX/FA-PEG-MWCNTs was found to be 70.22 ± 3.02
and 54.60 ± 1.45 at pH 5.3 and 7.4, respectively. The sustained
release of DTX was observed due to the limited solubility and
strong hydrophobic interaction among DTX and surface engineered
MWCNTs. Arora and co-workers reported the development of
MWCNTs-docetaxel conjugates by covalent interaction, involving
nucleophilic substitution reaction mechanism and reported that
the drug release from the docetaxel-MWCNTs conjugate was faster
in acidic pH, as compared to that in buffer of normal cell pH [29].
Our results are in good agreement with previous reports [29,31,32].
The developed nanotube formulations were found to be most
stable in dark at 5 ± 3 ◦C. However, on storage in light at 25 ± 2 ◦C,
slight turbidity was observed, which might be due to aggregation
of nanotubes (Tables 1 and 2). At 40 ± 2 ◦C, all the formulations
showed higher turbidity that may be ascribed to the formation of
larger aggregates and bundling of nanotubes [20]. The drug leakage
from the developed nanotubes formulations is another important
parameter, which was measured to assess the stability. The drug
leakage from the developed nanotube formulations was found to be
negligible at 5 ± 3 ◦C, hence considered being most stable at 5 ± 3 ◦C
temperature in dark condition. Our results are in good agreement
with previous reports [9,30]. The percent hemolysis of pristine
MWCNTs (18.0 ± 0.50%), oxidized MWCNTs (15.50 ± 0.56%), DTX
(19.20 ± 0.45%), DTX/MWCNTs (14.87 ± 0.44%) and DTX/FA-PEG-
MWCNTs (9.20 ± 0.14%) was determined on collected whole human
blood. Pristine MWCNTs showed highest (18.0 ± 0.50%), while
DTX/FA-PEG-MWCNTs showed minimum (9.20 ± 0.14%) hemolytic
toxicity. The pristine nanotubes exhibit high hemolytic toxicity due
to their inherent toxicity while surface engineering of MWNCTs
Fig. 3. Cumulative amount of DTX released from the DTX/MWCNTs and DTX/FA-PEG-MWCNTs nanoconjugates at 37 ± 0.5 ◦
C in phosphate buffer solution (pH = 5.3 and 7.4).
(Values represented as means ± SD; n = 3).

Table 1
Accelerated stability studies for the DTX/MWCNTs formulations.
Stability parameter DTX/MWCNTs after 6 months
Dark Light
T1 T2 T3 T1 T2 T3
Turbidity − − ++ + ++ +++
Precipitation − − ++ + ++ ++
Change in color − − + + + ++
Crystallization − − + + + ++
Change in consistency − − + + + ++
Percent drug leakage (after months)
1 1.40 ± 0.02 1.80 ± 0.03 2.62 ± 0.04 2.90 ± 0.05 3.51 ± 0.04 8.83 ± 0.44
2 2.82 ± 0.03 2.21 ± 0.03 4.62 ± 0.04 3.80 ± 0.04 5.02 ± 0.03 10.85 ± 0.04
4 3.81 ± 0.06 3.02 ± 0.04 5.03 ± 0.04 5.62 ± 0.02 7.83 ± 0.02 13.47 ± 0.02
6 5.02 ± 0.02 4.61 ± 0.08 5.42 ± 0.06 7.82 ± 0.07 9.64 ± 0.07 15.63 ± 0.08
Table 2
Accelerated stability studies for the DTX/FA-PEG-MWCNTs formulations.
Stability parameter DTX/FA-PEG-MWCNTs after 6 months
Dark Light
T1 T2 T3 T1 T2 T3
Turbidity − − ++ + ++ +++
Precipitation − − ++ + ++ ++
Change in color − − + + + ++
Crystallization − − + + + ++
Change in consistency − − + + + ++
Percent drug leakage (after months)
1 0.40 ± 0.02 1.41 ± 0.05 1.92 ± 0.04 1.42 ± 0.03 1.80 ± 0.02 5.71 ± 0.06
2 1.10 ± 0.03 1.80 ± 0.04 2.91 ± 0.05 1.73 ± 0.07 2.21 ± 0.08 6.02 ± 0.02
4 1.90 ± 0.03 2.10 ± 0.07 3.30 ± 0.07 2.21 ± 0.04 3.00 ± 0.03 6.51 ± 0.06
6 2.10 ± 0.05 2.50 ± 0.06 3.81 ± 0.04 2.60 ± 0.09 3.42 ± 0.08 7.33 ± 0.03
T1, T2 and T3 represent 5 ± 3, 25 ± 2, and 40 ± 2 ◦
C temperatures, respectively. Values represented as mean ± S.D. (n = 3) “−, +, ++ and +++” indicate no change, small change,
considerable change and major change, respectively.
and conjugation of FA-PEG drastically reduced erythrocytes toxic-
ity and improved biocompatibility. The degree of functionalization
considerably reduced hemolysis by nearly 50% in case of DTX/FA-
PEG-MWCNTs, possibly due to the enhanced aqueous solubility and
separation of impurities.
MTT assay is a simple, non-radioactive, colorimetry based assay
for determining the relative percent cell viability. The cytotoxic-
ity of DTX loaded nanotubes formulations at different micromolar
concentration against MCF-7 cells after 24 and 48 h was deter-
mined using MTT cytotoxicity assay. MTT assay revealed that upon
increasing the concentration from 0.001 to 100 ␮M of DTX loaded
nanotubes formulations the relative percent cell viability of the
cancerous cells was decreased following initial 24 h treatment.
After 48 h, DTX exerted higher cytotoxicity as compared to 24 h
treatment due to sustained release of drug from the nanotubes
formulations. The DTX/FA-PEG-MWCNTs exerted higher cytotox-
icity as compared to DTX/MWCNTs and DTX solution and the
increased cytotoxic response was found to be concentration and
exposure duration dependent. Folate receptors (FRs) are common
tumor marker highly over-expressed on the cancerous cells surface
that facilitates cellular internalization (Fig. 4). Thus, DTX/FA-PEG-
MWCNTs formulation could efﬁciently deliver DTX to the nucleus of
the cell possibly by nanoneedle-transporter or receptor-mediated
endocytosis (RME) mechanism [9,11,13,33].
We examined the effects of the developed nanotubes formula-
tions on cell cycle in MCF-7 cells through ﬂow cytometry. Generally,
cell cycle analysis could be characterized by the four distinct phases
in proliferating cell population: G1-, S-(DNA synthesis phase), G2-
and M-phase (mitosis), while G2- and M-phases have an iden-
tical DNA content and could not be discriminated on the basis
Fig. 4. Percent cell viability of MCF-7 cells after treatment with free DTX, DTX/MWCNTs and DTX/FA-PEG-MWCNTs at (A) 24, and (B) 48 h. Values represented as mean ± SD
(n = 3).

Fig. 5. DNA content and cell cycle analysis (above) and quantitative cell uptake of the (A) control, (B) DTX, (C) DTX/MWCNTs and (D) DTX/FA-PEG-MWCNTs formulations on
MCF-7 cell lines using flow cytometry. Values represented as mean ± SD (n = 3).
of the DNA content [13,21,34]. The DNA flow cytometric analy-
sis (Fig. 5A–D) indicated that the treatment of MCF-7 cells with
the nanotubes formulations in 10 nM concentration caused 24 h
arrest in G2 phase of the cell cycle. The control cells showed
86.50 ± 3.22%, 8.15 ± 0.16% and 5.35 ± 0.08% population arrest in
the G1, G2 and S-phase, respectively. Percentage of cell arrest in
the G2 phase was found to be 42.29 ± 2.12% (DTX), 56.22 ± 1.56%
(DTX/MWCNTs), and 60.67 ± 2.02% (DTX/FA-PEG-MWCNTs) lead-
ing to mitotic arrest in G2/M phase of the cell cycle that ultimately
leads to cell death. The DTX/FA-PEG-MWCNTs cells arrest was
approximately 30.70 ± 1.24%, 60.67 ± 2.76% and 8.64 ± 0.22% in G1,
G2 and S-phase, respectively. Thus the DNA cell cycle analysis sug-
gests that the cancerous cells were arrested significantly in the G2
phase when treated with the DTX loaded formulation.
The DTX destroys cell’s ability to use its cytoskeleton in a flexible
manner binding with ␤-subunit of tubulin. DTX acts by binding
to microtubules and inhibits microtubule depolymerization to free
tubulin. It has been reported that the nanotube formulation belongs
to cell-cycle specific anticancer drug, which mainly arrest the cells
in G2 phase of the cell cycle [14].
The quantitative cell uptake studies of developed nanotube
formulations were performed using flow cytometry to investi-
gate the cellular uptake in MCF-7 cells. Fluorescence Activated
Cell Sorting (FACS) is a special type of flow cytometry, which
can quantitatively measure the cell uptake. A flow cytometer
analyses particles by passing them in single file through a laser
beam and counts upto 1000 cells/s of fluorescence intensities.
The quantitative cellular uptake of the DTX from the developed
nanotubes formulations in MCF-7 cell is shown in Fig. 5A–D. In
FACS chromatograms, control group showed 69.16 ± 2.32% fluo-
rescence intensity in R1 region. The observed percent fluorescent
intensity of the DTX, DTX/MWCNTs and DTX/FA-PEG-MWCNTs was
found to be 62.10 ± 3.04, 66.10 ± 3.22 and 77.72 ± 2.88%, respec-
tively shifted toward R2 region. The observed higher fluorescence
intensity clearly suggests higher uptake of the DTX/FA-PEG-
MWCNTs formulation.
The FA-terminated poly(ethylene glycol) (PEG-FA) coated on
SWCNTs (DOX/PEG-FA/SWCNTs) in a facile non-covalent method
was designed and constructed for targeting delivery of DOX to
cancer cells. The DOX/PEG-FA/SWCNTs exhibit excellent stability
under neutral pH condition and selectively attach onto cancer cells
and enter the lysosomes or endosome by clathrin-mediated endo-
cytosis [27]. Receptor-mediated cellular trafficking can facilitate
cellular internalization of the drug loaded MWCNTs after conju-
gation with targeting moiety. The in vitro release of FITC from the
MWCNTs formulations showed negligible release (<1%) in 3 h, sug-
gesting that only nanotubes formulations are internalized into the
MCF-7 cell lines. FITC dye was covalently attached to nanotubes and
also on to interior wall of the nanotubes [5]. The obtained fluores-
cence indicates the rapid internalization of nanotube formulations.
The free FITC was washed away from the FITC loaded nanotube for-
mulations prior to cellular uptake study. The FA-targeted MWCNTs
may increase the therapeutic index in a greater affinity for colorec-
tal cancer cells than un-targeted MWCNTs [29]. Recently, Arora and
co-workers reported the translocation and toxicity of the docetaxel
(DTX) conjugated MWCNTs employing MCF-7 and MDA-MB-231
human breast cancer cells. The DTX-MWCNTs conjugates indicate
increased efficacy over the drug in terms of cytotoxicity and thereby
enriching cancer therapies [35].
The pharmacokinetic study was performed to assess the effect
of surface engineering on different pharmacokinetic parameters
like half value duration (HVD), area under the curve (AUC), area

Table 3
Pharmacokinetic parameters of free DTX and DTX loaded MWCNTs formulations.
Parameters HVD (h) AUC(0–t)
(␮g/mL h)
AUC(0–∞)
(␮g/mL h)
AUMC(0–t)
(␮g/mL h2
)
AUMC(0-∞)
(␮g/mL h2
)
t1/2 (h) MRT (h)
Free DTX 0.34 ± 0.03 10.56 ± 0.65 11.02 ± 0.10 30.70 ± 0.46 38.15 ± 1.28 2.70 ± 0.02 3.45 ± 0.01
DTX/MWCNTs 0.90 ± 0.05 22.17 ± 0.10 22.77 ± 0.18 127.24 ± 2.24 145.75 ± 4.55 4.64 ± 0.04 6.40 ± 0.02
DTX/FA-PEG-MWCNTs 1.10 ± 0.08 33.67 ± 0.16 34.95 ± 0.34 328.35 ± 4.50 405.56 ± 6.54 8.81 ± 0.03 11.60 ± 0.07
Mean ± S.D. (n = 3); probability p < 0.001; standard deviation <5%.
Abbreviations: Cmax = peak plasma concentration; Tmax = time taken to reach Cmax; t1/2 = elimination half life; MRT = mean residence time; AUC(0–∞) area under plasma drug
concentration over time curve; HVD: half value duration.
under the first moment plasma concentration curve (AUMC), half-
life (t1/2), and mean residence time (MRT) (Table 3 and Fig. 6). The
mean residence time (MRT) and t1/2 of free DTX, DTX/MWCNTs and
DTX/FA-PEG-MWCNTs were found to be 3.46 ± 0.01, 6.40 ± 0.02,
11.60 ± 0.07 h and 2.70 ± 0.02, 4.64 ± 0.04, 8.81 ± 0.03 h, respec-
tively. The MRT of DTX/FA-PEG-MWCNTs was found to be
approximately 3.5 and 1.85 folds higher compared to free DTX and
DTX/MWCNTs, respectively. The obtained results are attributed to
the biocompatibility of engineered nanotubes upon surface func-
tionalization (degree of functionalization) and ability to reside
for longer time inside the body. The improved pharmacokinetic
data make nanotubes as most promising, smart, and ideal nano-
biocarrier for site-specific targeting. The sustained drug release
patterns in blood were achieved to a greater extent for DTX/FA-
PEG-PMWCNTs as against DTX/MWCNTs and free DTX. It clearly
suggests the improved pharmacokinetic parameters with better
bioavailability and prolonged retention in systemic circulation than
that resulting from administration of drugs-MWCNTs and free drug
solution to mice.
A comparative biodistribution study was performed to assess
the amount of drug that reaches in to different organs like liver,
spleen, kidney, lungs and tumor after intravenous (i.v.) adminis-
tration of free DTX, DTX/MWCNTs and DTX/FA-PEG-MWCNTs into
the tumor bearing Balb/c mice. In case of DTX/FA-PEG-MWCNTs
formulation, higher concentration of DTX uptake was observed
in tumor in 24 h. The DTX concentrations (percent injected dose
per organ) from the DTX/MWCNTs determined at 1, 6, 12, and
24 h were found to be 31.57 ± 0.18, 30.24 ± 0.17, 26.44 ± 0.15 and
20.44 ± 0.88 in liver and 3.88 ± 0.06, 5.56 ± 0.86, 7.47 ± 0.65, and
7.67 ± 0.86 in tumor, respectively. The DTX concentrations in liver
were found to be 38.67 ± 0.06, 36.65 ± 0.94, 32.34 ± 0.74, and
28.54 ± 0.32, respectively at 1, 6, 12 and 24 h from DTX/FA-PEG-
MWCNTs. The DTX concentrations in tumor were determined to
be 9.45 ± 0.24, 12.56 ± 0.63, 13.01 ± 0.24, and 17.65 ± 0.18 from
DTX/FA-PEG-MWCNTs, respectively at 1, 6, 12 and 24 h time points
(Fig. 7).
The higher levels of the surface engineered MWCNTs observed
at initial time point of administered dose in kidney and the rapid
decline in the overall formulation thereafter suggest that most of
the nanotubes were eliminated through the renal excretion route.
Researchers have reported no signs of toxicity due to accumulation
in body/organs suggesting the utility of such systems in thera-
peutic delivery [12]. In vitro drug release data suggested initial
rapid release followed by gradual slow release; similar pattern was
observed in in vivo study. The obtained data from the DTX/FA-PEG-
MWCNTs formulations are in good agreement with the previously
published reports [9,13,26,29].
The in vivo tumor targeting efficacy of the DTX/MWCNTs and
DTX/FA-PEG-MWCNTs was assayed on breast tumor model. The
starting tumor size was 100 mm3 for all dose receiving groups
including developed nanoconjugates as well as normal saline
and control group. The size of the tumor volume (mm3) at 30
days after treatment of DTX/FA-PEG-MWCNTs was calculated to
be 57.0 ± 3.56. The reduced size of the tumor clearly suggests
the better and efficient targeting of the developed nanotube
formulations. The DTX/FA-PEG-MWCNTs (targeted, stealth, long
circulatory nature) was found to be more active than DTX/MWCNTs
and free DTX solution with significant reduction in tumor growth.
The DTX loaded nanotubes formulations could be ranked in the
following order:
(DTX/FA-PEG-MWCNTs > DTX/MWCNTs > free DTX)
(Maximum inhibitory Minimum inhibitory)
The higher antitumor activity of the targeted stealth nanotube
formulations could be ascribed to higher accumulation in cancer-
ous cells via receptor-mediated endocytosis (R-ME) and passive
diffusion (tiny nanoneedle) mechanism. However, FA appended
stealth nanotubes formulations were found to be significantly
more efficient in tumor suppression compared with plain MWC-
NTs (non-targeted) and drug solution owing to the accelerated drug
release from endosomes after internalization. Significant reduction
in subsequent growth in tumor was probably due to ligand-driven
Fig. 6. Plasma profile of free DTX and various nanotubes formulations. Values represented as mean ± SD (n = 3).

Fig. 7. Biodistribution of DTX after intravenous administration of DTX solution, DTX/MWCNTs and DTX/FA-PEG-MWCNTs formulation in tumor bearing Balb/c mice). *p ≤ 0.05;
**p ≤ 0.01; ***p ≤ 0.001. ns: not significant vs. Free DTX. (Values represented as means ± SD; n = 3).
Fig. 8. Kaplan–Meier survival curves of MCF-7 bearing Balb/c mice analyzed by Log-
rank (Mental-Cox) test with normal saline group as control.
internalization of the surface engineered nanotubes at the tar-
get site/tissue(s), which was accompanied by slow and sustained
release of the drug. The tumor growth inhibition study clearly indi-
cates that inclusion of the pH-responsive characteristics increases
the overall targeting efficiency of the targeted and non-targeted
nanotubes formulations. The prepared surface engineered MWC-
NTs formulations did not elicit any change in body weight of mice.
The DTX loaded NGR peptide conjugated SWCNTs (DTX-
NGR–SWCNTs) enhanced the targeting efficiency compared with
DTX loaded SWCNTs (SWCNTs-DTX) and DTX alone [32].
Kaplan–Meier survival curves based on survival time were plot-
ted for different groups of animals using Log-rank test. The curves
suggested that the tumor bearing mice of DTX/FA-PEG-MWCNTs
exhibited significantly longer median survival time span (40 days,
p < 0.001) than DTX/MWCNTs (24 days), free DTX (19 days) and con-
trol group (12 days) (Fig. 8). These results further confirmed the
higher tumor treatment potential possessed by the surface engi-
neered MWCNTs, which resulted in longer survival span of tumor
bearing mice. The longest survival span was observed in case of
DTX/FA-PEG-MWCNTs.
Hematological parameters (RBCs, WBCs and differential counts)
were determined to assess the relative effect of MWCNTs formu-
lations (DTX/MWCNTs, and DTX/FA-PEG-MWCNTs) compared to
free DTX on different components of blood. The RBCs and WBCs
counts in free DTX, MWCNTs, DTX/MWCNTs and DTX/FA-PEG-
MWCNTs formulations treated blood sample were found to be
5.82 ± 0.22, 7.50 ± 0.56, 8.02 ± 0.36, 8.81 ± 0.88 and 10.05 ± 0.32,
8.60 ± 0.62, 9.06 ± 0.62, and 10.20 ± 0.60, respectively as shown in
Table 4. The differential counts i.e. monocytes, lymphocytes and
neutrophiles of DTX/FA-PEG-MWCNTs formulation were found to
be 1.02 ± 0.38, 7.71 ± 0.6 and 1.31 ± 0.12 × 103/␮L, respectively.
The DOX/FA/CHI/SWCNTs did not exhibit obvious liver toxicity by
blood routine and serum biochemical parameters on female nude
Balb/c mice [4]. The extensive data from the serum biochemical
Table 4
Serum biochemical parameters of Balb/c mice treated with free DTX, MWCNTs, DTX/MWCNTs and DTX/FA-PEG-MWCNTs formulations after 7 days.
Group RBCs (×106
/␮L) WBCs (×106
/␮L) Differential counts (×103
/␮L) Hb (g/dL) HCT
Monocytes Lymphocytes Neutrophils
Control 9.21 ± 0.40 10.80 ± 0.40 1.40 ± 0.60 7.92 ± 0.42 1.62 ± 0.42 12.40 ± 0.33 35.50 ± 0.65
Normal saline 8.40 ± 0.32 9.63 ± 0.42 0.91 ± 0.34 6.11 ± 0.44 1.00 ± 0.66 10.50 ± 0.22 34.40 ± 0.25
Free DTX 5.82 ± 0.22 10.05 ± 0.32 0.92 ± 0.55 7.93 ± 0.12 1.41 ± 0.60 10.22 ± 0.15 32.40 ± 0.16
MWCNTs 7.50 ± 0.56 8.60 ± 0.62 0.71 ± 0.76 5.91 ± 0.88 0.90 ± 0.85 9.80 ± 0.94 33.62 ± 0.12
DTX/MWCNTs 8.02 ± 0.36 9.06 ± 0.62 0.90 ± 0.12 6.82 ± 0.90 1.01 ± 0.40 10.81 ± 0.22 33.82 ± 0.45
DTX/FA-PEG-MWCNTs 8.81 ± 0.88 10.20 ± 0.60 1.02 ± 0.38 7.71 ± 0.60 1.31 ± 0.12 11.80 ± .90 34.02 ± 0.12
Values are expressed as mean ± SD. Number of animals per time points were three (n = 3); WBCs: white blood corpuscles, RBCs: red blood corpuscles, Hb; hemoglobin, HCT;
haematocrit.

parameters suggest that the RBCs, WBCs and differential count of
the DTX/FA-PEG-MWCNTs were almost similar to the control and
normal saline treated groups. Similarly, the differential counts i.e.
leucocytes, monocytes and lymphocytes were found almost similar
in case of DTX/FA-PEG-MWCNTs nanoconjugates to normal values.
The results clearly establish the superior biocompatibility of the
folate appended MWCNTs than the pristine MWCNTs and free DTX
solution.
4. Conclusion
The highly effective novel targeted drug delivery system based
on FA conjugated and PEGylated MWCNTs was developed and
evaluated in facile strategy for cancer treatment. The DTX/FA-PEG-
MWCNTs formulation showed higher cytotoxicity as compared to
DTX/MWCNTs and free drug solution and arrested cell death in G2
phase. In contrast, quantitative cell uptake demonstrated signifi-
cantly higher uptake of FA conjugated nanotubes formulations. The
ex vivo studies such as MTT cytotoxicity, DNA cell cycle, and quan-
titative cell uptake clearly revealed that the targeted drug delivery
along with specific targeting moiety increases the receptor interac-
tion for selective killing of MCF-7 cells. The pharmacokinetic studies
also revealed the long circulatory (stealth) nature of the devel-
oped MWCNTs formulations. In vivo toxicity studies suggest that
the surface engineered MWCNTs formulations easily escaped from
the excretory organ. The degree of functionalization minimizes the
toxicity of the carbon nanotubes. The developed surface engineered
MWCNTs nanoconjugates have shown promising potential in can-
cer therapy and to deliver significantly higher concentration of DTX
to the cancerous tissue than pristine MWCNTs and free drug. Thus,
it may be concluded that the DTX laden FA-PEG-MWCNTs holds
strong targeting potential in cancer treatment.
Declaration of interest
The authors report no conflict of interest.
Acknowledgement
One of the author Dr. Neelesh Kumar Mehra is thankful to the
University Grants Commission (UGC), New Delhi, India for provid-
ing the Senior Research Fellowship during the tenure of the studies.
The authors also acknowledge Dr. Ranveer Kumar, Department of
Physics, Dr. H. S. Gour University, Sagar, India for Raman spec-
troscopy; Central Instruments Facilities (CIF), National Institute of
Pharmaceutical Education and Research (NIPER), Mohali, Chandi-
garh, India for Particle Size analysis; Central Drug Research Institute
(CDRI), Lucknow, India for FTIR spectroscopy; All India Institute
of Medicine and Sciences (AIIMS), New Delhi, India for electron
microscopy; Indian Institute of Technology (IIT), Indore, India for
AFM analysis; Diya Laboratory, Mumbai, India for XRD analysis and
National Center for Cell Sciences (NCCS), Pune, India for providing
the cell line.
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