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2. Author's personal copy
The cancer targeting potential of D-a-tocopheryl polyethylene glycol
1000 succinate tethered multi walled carbon nanotubes
Neelesh Kumar Mehra a
, Ashwni Kumar Verma b
, P.R. Mishra b
, N.K. Jain a,*
a
Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour Central University, Sagar 470 003, India
b
Pharmaceutics Division, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031, Uttar Pradesh, India
a r t i c l e i n f o
Article history:
Received 19 December 2013
Accepted 12 February 2014
Available online 4 March 2014
Keywords:
Carbon nanotubes
Doxorubicin hydrochloride
Vitamin E
KaplaneMeier survival
Tumor growth inhibition
Anticancer activity
a b s t r a c t
Our main aim in the present investigation was to explore the in vitro and in vivo cancer targeting po-
tential of the doxorubicin (DOX) laden D-a-tocopheryl polyethylene glycol 1000 succinate (vitamin E
TPGS) tethered surface engineered MWCNTs nanoformulation (DOX/TPGS-MWCNTs) and compare it
with pristine MWCNTs and free doxorubicin solution. The developed MWCNTs nanoformulations were
extensively characterized by Fourier-transform infrared, Raman spectroscopy, x-ray diffraction, electron
microscopy, and in vitro and in vivo studies using MCF-7 cancer cell line. The entrapment efficiency was
determined to be 97.2 Æ 2.50% (DOX/TPGS-MWCNTs) and 92.5 Æ 2.62% (DOX/MWCNTs) ascribed to p-p
stacking interactions. The developed formulations depicted the sustained release pattern at the lyso-
somal pH (pH 5.3). The DOX/TPGS-MWCNTs showed enhanced cytotoxicity, cellular uptake and were
most preferentially taken up by the cancerous cells via endocytosis mechanism. The DOX/TPGS-MWCNTs
nanoconjugate depicted the significantly longer survival span (44 days, p < 0.001) than DOX/MWCNTs
(23 days), free DOX (18 days) and control group (12 days). The obtained results also support the extended
residence time and sustained release profile of the drug loaded surface engineered nanotubes formu-
lations in body as compared to DOX solution. Overall we can conclude that the developed MWCNTs
nanoconjugate have higher cancer targeting potential on tumor bearing Balb/c mice.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Currently, surface engineered multifunctional carbon nanotubes
(CNTs) mediated targeted and controlled drug delivery has aroused
escalating attention as valuable, promising nano-architecture due
to its unique physicochemical properties in the treatment of cancer
and other dreadly diseases [1e4]. CNTs was originally discovered
and fully described by Prof. Sumio Iijima in 1991 and it is consid-
ered as promising targeted drug delivery vehicles because it can
easily cross cell membranes. CNTs is three dimensional, cylindrical,
sp2
hybridized carbon nanomaterial. CNTs can be subdivided into
single-, double-, triple-, and multi-walled CNTs [2,5e7].
The hydrophobic nature and inherent toxicity of first generation
pristine CNTs make them unsuitable for targeted/controlled drug
delivery. However, these major hurdles have been easily amelio-
rated by surface alteration through either covalent or non-covalent
approaches depending on the intermolecular interaction. The non-
covalent alteration is based on the extended p-system (p-orbital) of
the sidewall of the nanotubes bind with the guest moieties through
pep stacking interactions [2,6,8,9]. The surface engineered CNTs
serves as efficient multifunctional biological transporters devoid of
obvious toxicities. Iverson et al. reported that the alginate-
encapsulated single-walled carbon nanotubes (SWCNTs) did not
show any adverse response for more than 400 days [10]. Thus,
surface engineered CNTs has been designed and tested for targeted
delivery by conjugating targeting moieties and has proven non-
cytotoxic to human cells [11,12].
Doxorubicin (DOX), an anthracycline antibiotic, is a DNA-
interacting drug for treatment of various cancers especially
breast, ovarian, prostate, brain, cervix and lung cancers. Clinical
application of doxorubicin is limited because of its short half-life
and severe toxicity to normal tissues, especially gastrointestinal
toxicity and heart failure. The cardio-toxicity confines the cumu-
lative dose of DOX to 500e600 mg/m2
, which still can be increased
for tumor but not for heart disease [13e15]. Our group is continu-
ously working and exploring the drug delivery aspects employing
the surface engineered CNTs for targeting purpose including
* Corresponding author. Tel./fax: þ91 7582 265055.
E-mail addresses: neelesh81mph@gmail.com (N.K. Mehra), jnarendr@yahoo.co.in
(N.K. Jain).
Contents lists available at ScienceDirect
Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials
http://dx.doi.org/10.1016/j.biomaterials.2014.02.022
0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.
Biomaterials 35 (2014) 4573e4588

3. Author's personal copy
Doxorubicin [9,15,16], Gemcitabine [17], Sulfasalazine [5], and
Amphotericin B [12]. Recently, our group reported the targeted
delivery of DOX using folic acid conjugated PEGylated MWCNTs
with improved therapeutic outcomes [14].
D-a-tocopheryl polyethylene glycol 1000 succinate (vitamin E
TPGS; TPGS) is FDA approved water-soluble derivative of natural
Vitamin E (PEGylated vitamin E). TPGS is prepared by esterification
of D-a-tocopheryl acid succinate and PEG 1000 (amphiphilic
vitamin E) and moderately stable under normal conditions
[13,18,19]. TPGS is a promising surfactant for green processing of
the carbon based nanomaterials including CNTs and offers as an
alternative and valuable option enhancing the aqueous solubility,
avoiding multidrug resistance (MDR) and might elicit receptor-
mediated endocytosis (RME). Currently, TPGS is most widely used
to enhance the cellular uptake, cytotoxicity of the anticancer agents
like Doxorubicin, Paclitaxel and Vinblastine etc. The TPGS-based
conjugates are ideal solution for the bioactive(s), which have
obstacle in ADME characteristics [13].
In the present investigation, we surface engineered and deco-
rated MWCNTs with the targeting moiety TPGS laden with doxo-
rubicin (DOX/TPGS-MWCNTs) assessing the cancer targeting
potential and compared to free DOX solution in the tumor bearing
Balb/c mice. We also determined the pharmacokinetics, bio-
distribution, Kaplan Meier survival analysis, tumor growth inhibi-
tion study and toxicological aspects in terms of safety and efficacy.
It is believed that the TPGS anchored surface engineered MWCNTs
are adsorbed with apo-lipoproteins (ApoE), which interact with
LDL receptors and are internalized via RME mechanism [20,21].
2. Materials and methods
The pristine multi walled carbon nanotubes (MWCNTs) produced by Catalytic
Chemical Vapor Deposition (CCVD) with 99.3% purity, were purchased from Sigma
Aldrich Pvt. Ltd. (St. Louis, Missouri, USA) was used for the present studies. Cancer
cell lines were purchased from the National Centre for Cell Sciences (NCCS) Pune,
India. Poly-tetrafluoroethylene (PTFE) filters (0.22 mm pore size) were purchased
from Hangzhou Anow Microfiltration Co. Ltd., Hangzhou, China. Sulfuric acid, nitric
acid, thionyl chloride, ethylene diamine, succinic anhydride, dimethyl amino pyri-
dine, dichloromethane, dimethyl sulfoxide (DMSO), and 1-Ethyl-3-(3-
dimethylaminopro-pyl) carbodiimide (EDC) were purchased from HiMedia Pvt.
Ltd. Mumbai, India. All the reagents and solvents were used as received.
2.1. Purification and surface engineering of pristine MWCNTs
As-procured pristine MWCNTs were initially purified using vacuum oven and
microwave technique as previously reported by Mehra and Jain [14]. Purified
MWCNTs were then further used for functionalization in the subsequent steps like
carboxylation, acylation and amidation process, extensively characterized and re-
ported [5,9,12,14,16,17,22].
2.2. Conjugation of TPGS to surface engineered MWCNTs
2.2.1. Synthesis of succinoylated TPGS
The carboxylic derivative of TPGS (TPGS-COOH) was activated by succinic an-
hydride (SA) through ring-opening reaction in the presence of 4-
dimethylaminopyridine (DMAP) with slight modification of previously reported
method [13]. Briefly, TPGS (0.77 g, 0.5 mM), SA (0.10 g,1 mM) and DMAP (0.12 g,1 mM)
were mixed and heated at 100 Æ 5 C under nitrogen gas protection at room tem-
perature (RT) for 24 h; the mixture was cooled to room temperature (RT), taken up in
5.0 mL cold dichloromethane (DCM), filtered to remove excess SA and precipitated
in 100 mL diethyl ether at À10 C overnight. The obtained white precipitant was
filtered and dried in vacuum to obtain succinoylated TPGS (Scheme 1).
2.2.2. Conjugation of TPGSeCOOH to amine terminated MWCNTs
The TPGSeCOOH was reacted with amine terminated MWCNTs (MWCNTse
NH2) using EDC chemistry with slight modifications [14,23]. The amine terminated
MWCNTs and TPGSeCOOH were reacted in DMSO at 1:2 ratio (excess amount of
TPGSeCOOH) with continuous magnetic stirring for 2 days at room temperature.
The unconjugated TPGS-COOH was removed by dialysis method and MWCNTs
conjugate was collected, freeze dried and characterized (Scheme 2).
2.3. Drug loading
Briefly, DOX was dissolved in acetone (10 mg/mL), and approximately 1.2 mL
aqueous triethyl amine (TEA) solution was added in a molar ratio of 2:1 (DOX:TEA).
The solution was magnetically stirred overnight using Teflon bead and mixed with
the dispersion of MWCNTs in PBS (pH 7.4) with the same ionic strength adjusted by
addition of sodium chloride (NaCl). The DOX: MWCNTs mixture in optimized 1:2 (w/
w) ratio was magnetically stirred overnight (50 rpm; Remi, India) at 37 Æ 0.5 C for
24 h using Teflon bead to facilitate entrapment of DOX. Thereafter, DOX laden
MWCNTs were separated by the centrifugation to remove free/unbound DOX until
solution became color free, and measured at lmax 480.2 nm spectrophotometrically
(Shimadzu 1601, UV-Visible Spectrophotometer, Shimadzu, Japan) using a calibra-
tion curve prepared under the same condition [14,24]. The DOX loading efficiency
was calculated spectrophotometrically using the following formula:
% Loading Efficiency ¼
Weight of loaded DOX À Weight of free DOX
Weight of loaded DOX
 100
The product was collected, dried and lyophilized (Heto dry winner, Denmark,
Germany) and stored at 5 Æ 3 C for further use of studies.
2.4. Characterization of pristine and engineered MWCNTs
The pristine MWCNTs (as procured) and TPGS functionalized MWCNTs were
characterized using Fourier Transform Infrared (FTIR), Raman, x-ray diffraction
(XRD), particle size and particle size distribution measurement.
2.4.1. FTIR spectroscopy
The FTIR spectroscopy were performed using compressed KBr pellet method in
Perkin Elmer FTIR spectrophotometer (Perkin Elmer 783, Pyrogon 1000 Spectro-
photometer, Shelton, Connecticut) and scanned in the range from 4000 to 500 cmÀ1
.
2.4.2. Average particle size and particle size distribution (PSD) measurement
The average particle size and particle size distribution (PSD) of pristine and
surface engineered MWCNTs were determined by photon correlation spectroscopy
in a Malvern Zetasizer nano ZS90 (Malvern Instruments, Ltd, Malvern, UK) at room
temperature (RT).
2.4.3. Electron microscopy
The size and surface morphology were characterized by Transmission Electron
Microscopy (TEM; Morgagni 268-D, Fei Electron Optics, Holland) after drying on
carbon-coated copper grid and staining negatively by 1% phosphotungstic acid (PTA)
by metal shadowing technique.
2.4.4. X-ray diffraction (XRD) analysis
The X-ray diffraction (XRD) analysis of the pristine and surface engineered
MWCNTs was carried out using X-ray diffractometer (PW 1710 Rigaku, San Jose, CA)
Scheme 1. Schematic representation of the activation of the TPGS (TPGSeCOOH).
N.K. Mehra et al. / Biomaterials 35 (2014) 4573e45884574

4. Author's personal copy
by adjusting X-ray power of 40 kV and 40 mA. Three hours of exposure time was
taken to analyze samples and X-ray diffraction data was obtained by general area
detector diffraction system at 25 C.
2.4.5. Raman spectroscopy
The order-disorder hexagonal carbon and the Raman spectra (Strokes lines)
were recorded by a Raman micro-spectroscopy RINSHAW, inVia Raman Spec-
trophotometer (Renishaw, Gloucestershire, UK). The micro-spectrophotometer
was operated exciting with the 532 nm laser radiation under objective lens of
20Â magnification (Olympus BX 41, USA) with a slit of 1 Â 6 mm and an incident
power was around 1 mW. The exposure time was 30 s and three scans were
accumulated for each spectrum. All the spectra were recorded at 0.1 cmÀ1
step
intervals at RT. Additionally, to protect from damage by the laser beam, the
sample was embedded into a KBr pellet and low power of 1.2 mW was employed
on the surface of sample to minimize appreciable peak shift or peak broadening
caused by the laser heating.
2.5. In vitro release studies
The in vitro release of DOX from DOX/MWCNTs and DOX/TPGS-MWCNTs
nanoformulation was studied in sodium acetate buffer saline (pH 5.3) and phos-
phate buffer saline (pH 7.4) as recipient media in a modified dissolution method
maintaining the physiological temperature (37 Æ 0.5 C) throughout the study [14].
The dialysis membrane (MWCO 5e6 kDa, HiMedia, India) filled with the developed
optimized nanotubes formulations separately, hermetically tied at both ends and
immediately placed into the receptor media maintaining strict sink conditions with
constant stirring using magnetic stirrer at RT adjusted to 37 Æ 0.5 C (100 RPM;
Remi, Mumbai, India). The aliquots were withdrawn at different time points and
volume of recipient compartment was maintained by replenishing with fresh sink
solution. The DOX concentration was determined in triplicate at different time
points after appropriate dilutions by UV/Visible spectrophotometer at lmax 480.2 nm
(UV/Vis, Shimadzu 1601, Kyoto, Japan).
2.6. Stability study
The DOX laden optimized developed MWCNTs nanoformulations (DOX/
MWCNTs and DOX/TPGS-MWCNTs) were stored in dark and in amber colored and
colorless glass vials at 5 Æ 3 C, room temperature (25 Æ 2 C) and at 40 Æ 2 C for a
period of six months in stability chambers (Remi CHM-6S, India) as per “Interna-
tional Conference on Harmonization of Technical Requirements for Registration of
Pharmaceuticals for Human Use” (ICH) guidelines for finished pharmaceutical drug
products [25]. The MWCNTs nanoformulations were analyzed initially and period-
ically upto six months for any change in particle size, drug content and organoleptic
features like aggregation and precipitation, color and odor changes if any.
Scheme 2. Schematic representation of the TPGS functionalized MWCNTs.
N.K. Mehra et al. / Biomaterials 35 (2014) 4573e4588 4575

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2.7. Comparison of hemolytic toxicity study
The erythrocytes-nanotubes formulations interaction was performed in vitro
following previously reported procedure, with minor modification [14,17]. Briefly,
fresh whole human blood was collected in HiAnticlot blood collecting vials
(HiMedia, Mumbai, 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, colorless superna-
tant was obtained above the cell mass. The RBCs suspension (1 mL) was mixed with
the 0.9% w/v normal saline (4.5 mL), free DOX, DOX/MWCNTs and DOX/TPGS-
MWCNTs dispersions (0.5 mL) incubated for 60 min; and allowed to interact. After
incubation, samples were centrifuged for 15 min at 1500 rpm and supernatant was
taken to quantify the hemoglobin content at lmax 540 nm spectrophotometrically
considering 0.9% w/v NaCl solution (normal saline) and deionized water as nil and
100% hemolysis, respectively. The percent hemolysis was calculated using the
formula.
Hemolysis % ¼
ðAbs À Abs0Þ
ðAbs100 À Abs0Þ
 100
where, Abs, Abs0 and Abs100 represent the absorbance of samples, a solution of 0%
hemolysis and a solution of 100% hemolysis, respectively.
2.8. Cell line studies
The MCF-7 (Michigan Cancer Foundation-7; an estrogen receptor positive hu-
man breast cancer cell line derived from pleural effusion) cell line was procured
from National Center for Cell Sciences (NCCS), Pune, India for the present study. The
MCF-7 cells were cultured in a humidified atmosphere containing atmosphere at 5%
CO2 at 37 C in Dulbecco’s Modified Eagle Medium (DMEM; HiMedia, Mumbai, In-
dia) containing 10% fetal bovine serum (FBS; HiMedia, Mumbai, India) supple-
mented with 2 mM L-glutamine, 1% penicillinestreptomycin mixture (Sigma, St
Louis, Missouri) incubated for 24 h for more than 80% confluence. The medium was
changed two to three times in a week [14,26,27].
2.8.1. Methylthiazole tetrazolium (MTT) cytotoxicity assay
The methylthiazole tetrazolium (MTT) cytotoxicity assay was performed by
cleavage of tetrazolium salt [{3-(4,5 dimethyl thiazole-2 yl)-2,5-diphenyl tetrazo-
lium bromide} (MTT)] to a blue formazan derivative by living cells [14,23,26,28].
Briefly, MCF-7 cells were seeded in 96-well plates with density 1 Â104
cells per well
and allowed to adhere for 24 h at 37 C prior to assay. Then the cells in quadruplet
wells were treated with free DOX, DOX/MWCNTs, and DOX/TPGS-MWCNTs at
concentrations-0.01, 0.1, 10, 100 mM for 24 h. Thereafter, medium was decanted and
50 mL of methylthiazole tetrazolium (MTT) (1 mg/mL) in DMEM ((10 mL; 5 mg/ml in
Hank’s Balanced Salt Solution; without phenol red) was added to each well and
incubated at 37 C for 4 h. MTT is reduced by mitochondrial dehydrogenase activity
in metabolically active cells to form insoluble formazan crystals. The formazan
crystals were solubilized in 50 mL isopropanol by shaking at room temperature for
10 min. Absorbance was measured at 570 nm. The absorbance given by untreated
cells was taken as 100% cell survival and the relative (%) cell viability was calculated
using following formula:
Cell viabilityð%Þ ¼
½AŠtest
½AŠcontrol
 100
where, [A]test is the absorbance of the test sample and [A]control is the absorbance of
control samples.
2.8.2. Cell cycle analysis and sub-G1 DNA measurement
The cultured MCF-7 cells were seeded in 1 Â 104
cells per well in 6-well plates
and incubated for 24 h. MWCNTs formulations (2 nM/mL concentration) were added
into each well and incubated for 24 h. After incubation the cells were harvested by
centrifugation at 1000 Â g for 10 min, washed with ice-cold PBS and fixed using 70%
cold ethanol overnight. The fixed cells were suspended in pre-cold PBS and further
treated with RNase (DNase free, 100 mg/mL) and propidium iodide (PI; 50 mg/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 on ice till used. The number of cells in
different phases of the cell cycle was determined using the cell cycle analysis soft-
ware with FACSCalibur Flow Cytometer (Becton, Dickinson Systems, FACS cantoÔ,
USA) [29].
2.8.3. Cell uptake/fluorescence microscope studies
The qualitative and quantitative cellular uptake of the DOX from the DOX loaded
nanotubes formulation was performed using FACSCalibur Flow Cytometer (Becton,
Dickinson Systems, FACS cantoÔ, USA). The developed formulations and free drug
solution were incubated as in case of DNA cell cycle content, for 4 h, and then the
medium was removed, the cells were washed with cold-PBS three times and
analyzed quantitatively (FACSCalibur Flow Cytometer (Becton, Dickinson Systems,
FACS cantoÔ, USA) and qualitatively (Inverted microscope; Leica, Germany)
[13,21,23].
2.9. In vivo studies
The Balb/c mice of either sex (20e25 g) were used for present in vivo studies in
accordance with the guidelines by Committee for the Purpose of Control and Su-
pervision of Experiments on Animals (CPCSEA) Registration No. 379/01/ab/CPCSEA/
02 of Dr. H.S. Gour Vishwavidyalaya, Sagar, (M.P.). India. All the experimental animal
protocols were approved by the Institutional Animal Ethics Committee and animals
were acclimatized at room temperature by maintaining the relative humidity (RH)
55e60% under natural light/dark condition prior to studies. The tumor model was
generated by injected serum-free MCF-7 cells (1 Â 107
cells) using hypodermic
needle subcutaneously in the right hind leg of the mice and routinely monitored for
tumor development by palpating the injected area with index finger and thumb for
the presence of the tumor (approximately 100 mm3
) [14,30].
2.9.1. Analysis of pharmacokinetic parameter after intravenous (i.v.) administration
The different pharmacokinetic parameters were determined after i.v. adminis-
tration of free DOX and DOX laden MWCNTs nanoformulations with the same i.v.
dose (5.0 mg/kg body weight dose). The blood samples were collected from the
retro-orbital plexus of eyes with mild anesthesia conditions into the Hi-Anticlot
blood collecting vials (HiMedia, Mumbai, India) at predetermined time points
upto 48 h. The collected of blood samples were centrifuged to separate RBCs and
supernatant (serum) was collected, 100 mL trichloro acetic acid (TCA) in methanol
(10% w/v) was added, vortexed and ultracentrifuged (Z36HK, HERMLE LaborTchnik
GmbH, Germany). The clear supernatant was collected and DOX concentration was
determined by High Performance Liquid Chromatography (HPLC) method and
different pharmacokinetic parameters were calculated [14,31e33].
2.9.2. Tissue/organ biodistribution study
The in vivo biodistribution of the DOX laden MWCNTs formulations and free DOX
were studied on tumor bearing Balb/c mice. The sterilized free DOX, DOX/MWCNTs
and DOX/TPGS-MWCNTs conjugates after dispersion in normal saline (0.9%; w/v)
were administered intravenously through caudal tail vein route (equivalent dose of
DOX ¼ 5.0 mg/kg body weight) into animals. Each mice was administered the same
i.v. dose and carefully sacrificed by decapitation method at time intervals of 1, 6, 12
and 24 h for the collection of visceral organs like liver, spleen, kidney, heart, and
tumor immediately. The collected organs were washed with Ringer’s solution to
separate any adhered debris and dried with the help of tissue paper, weighed and
stored under frozen till used. Tissues were homogenized (York Scientific Instrument,
New Delhi, India) and vortexed after addition of chloroform (CHCl3) and methanol
(CH3OH) mixture and ultracentrifuged at 3000 rpm for 15 min (Z36HK, HERMLE
LaborTchnik GmbH, Germany). After centrifugation, obtained supernatant was
decanted into another vial and evaporated to dryness under nitrogen gas in a bath at
60 Æ 2 C temperature. The dried residue was collected in vials and injected in to an
HPLC and analyzed for DOX content by HPLC (Shimadzu, C18, Japan) method,
wherein mobile phase consisted of buffer pH 4.0/acetonitrile/methanol (60:24:16; v/
v/v) with 1.2 mL/min flow rate at 102/101 bars pressure with adjusting 20 min
runtime and peak at 480.2 nm was considered with its retention time (RT) and area.
2.9.3. Assessment of anti-tumor cancer targeting efficacy
The in-vivo anti-tumor cancer targeting efficacy of the DOX laden MWCNTs
formulation was assessed in the tumor bearing Balb/c mice. The initial tumor size
was taken approximately 100 mm3
in size. The tumor bearing mice were randomly
divided into four treatment groups (control, free DOX, DOX/MWCNTs and DOX/
TPGS-MWCNTs) for treatment with 5.0 mg/kg body weight dose equivalent to
DOX. At predetermined time intervals the tumor volume (cubic millimeters) was
measured by measuring its dimension (major and minor axis) using electronic
digital Vernier Caliper. The formula used to compute tumor volume was similar to
volume of an ellipsoid, where V ¼ 4/3 p(1/2 length  1/2 width  1/2 depth) with an
assumption that width is equal to depth and p equals 3, and so the final formula
used was V ¼ 1/2  length  width2
. The median survival time was also recorded.
The study was terminated 45 days post treatment. All animals were accommodated
in a pathogen-free laboratory environment during the tenure of the studies.
2.9.4. Toxicological assessment
The various toxicological aspects like hematological parameters and hepato-
toxicity were determined.
2.9.4.1. Hematological studies. The hematological parameters were estimated
following the earlier reported method [5,14]. The mice were divided in respective
groups and administered the same i.v. dose of the developed nanotubes formula-
tions and free drug solution and maintained on same regular diet upto 7 days. After 7
days blood samples were collected from the mice and the red blood corpuscles
(RBCs), white blood corpuscles (WBCs) and differential count of monocytes, lym-
phocytes and neutrophils, % Hb, MCH and HCT were determined.
2.9.4.2. Hepatotoxicity. The serum enzyme activities such as creatinine levels,
lactate dehydrogenase (LDH), blood urea nitrogen (BUN), SGPT (Serum glutamic
pyruvic transaminase) were assayed using commercially available kit (Crest Bio-
system, India) [26,27,34].
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2.10. Statistical analysis
The results are expressed as mean Æ standard deviation (ÆSD) (n ¼ 3) and
statistical analysis was performed with Graph Pad Instat Software (Version 3.00,
Graph Pad Software, San Diego, California, USA) by one-way ANOVA followed by
TukeyeKramer test for multiple comparisons. The pharmacokinetic data analysis of
plasma concentration time profile was conducted using the Kinetica software
(Thermo scientific), USA followed by non-compartment analysis. A probability
p 0.05 was considered while significant and p 0.001 was considered as
extremely significant.
3. Results and discussion
The surface engineered carbon nanotubes (CNTs) play a pivotal
role and present new opportunities for research and development
including drug targeting arena. We procured pristine MWCNTs,
purified using microwave oven (separate the impurities) and cut
(shorten nanotubes) prior to use. The longer nanotubes are unable
to enter most of the cancerous cells and may be toxic. Oxidation is
one of the most common and prerequisite technique for intro-
ducing the hydrophilic functional groups (carboxylic, phenolic and
lactone etc) at the ends and side wall of the nanotubes through
strong oxidizing acid treatment increasing aqueous dispersibility of
the nanotubes [14,35,36].
In this purification, carboxyl (eCOOH) functional groups were
generated onto the CNTs surface making them more safe with
improved aqueous solubility for precise and targeted drug delivery.
We previously reported the purification, oxidation of pristine
MWCNTs and determined the total functional groups and carbox-
ylic acid (eCOOH) by Boehm Titration method [14].
The TPGSeCOOH was conjugated to the amine terminated
MWCNTs through EDC chemistry. The FTIR spectra of TPGS, pristine
MWCNTs and functionalized MWCNTs are shown in Fig. 1.
The FTIR spectrum of procured unmodified (pristine) MWCNTs
depicts absorption peak at 1626 cmÀ1
, confirming the presence of
carbon residue on the nanotubes surface. A clear single peak at
2400.24 cmÀ1
, which could be ascribed to the stretching of the
carbon nanotubes backbone is another important characteristic
(Fig. 1 A).
The acid functionalized MWCNTs (MWCNTseCOOH) shows the
peaks at around 3425.6, 1637.4 and 1370.1 cmÀ1
. The peak at
1637.4 cmÀ1
is attributable to asymmetrical stretching of C]O
stretching vibration mode that was ascribed the expansion of
carboxylation on the MWCNTs, and a peak at 3425.6 cmÀ1
was
ascribed to the OeH stretching vibration (Fig. 1B). The blue shift
observed in the carboxyl stretching may be a consequence of
introduction of hydrogen bond amongst the surface carboxylic
functional groups.
The FTIR spectrum of as-received TPGS shows characteristic
peak at 3088.17, 2876.31, 2788.22, 1682.21, 1513.21, 1428.12,
1243.64, and 1182.93 cmÀ1
. The aromatic stretching was found at
3088.17 cmÀ1
, while 2876.31 cmÀ1
and 2788.22 cmÀ1
shows the
aliphatic stretching for asymmetric and symmetric characteristic
peak (CeH stretching of the CH3). The C]O stretching shows peak
Fig. 1. FTIR spectra of (A) pristine MWCNTs, and (B) oxidized MWCNTs. FTIR spectra of (C) TPGS, and (D) TPGS-MWCNTs.
N.K. Mehra et al. / Biomaterials 35 (2014) 4573e4588 4577

7. Author's personal copy
at 1682.21 cmÀ1
, approximately. The C]C characteristic ring
stretching was observed at 1513.21 and 1428.12 cmÀ1
. The CeO
stretching was observed at 1243.64 and 1182.93 cmÀ1
. The obtained
characteristic peaks suggest that the TPGS is pure and authentic
(Fig. 1C).
The FTIR spectrum of the TPGS conjugated MWCNTs shows
characteristic peaks at 1689.20 cmÀ1
of eC]O stretching of
amide bond formation, 2943.41 cmÀ1
due to CeH stretching of
CH2 functional group, and 3412.76 cmÀ1
of NeH stretching. The
obtained characteristic peaks of the as-received TPGS indicate
that the TPGS was successfully conjugated with the carboxylic
group (-COOH) of the MWCNTs through amide bond formation
(Fig. 1D).
The average particle size (nm) and particle size distribution with
polydispersity index (PDI) were determined by photon correlation
spectroscopy in a Malvern Zetasizer nano ZS90 (Malvern In-
struments, Ltd, Malvern, UK) at room temperature (RT). The par-
ticle size of the purified MWCNTs through microwave treatment
was found to be 1254 Æ 5.88 nm with polydispersity index (PDI)
0.429 Æ 0.23, however upon chemical treatment the size of the
functionalized MWCNTs reduced. The average particle size and
particle size distribution of the DOX/MWCNTs and DOX/TPGS-
MWCNTs were found to be 230.41 Æ 1.3 and 250.18 Æ 5.5 nm
with polydispersity index (PDI) of 0.27 Æ 0.010 and 0.32 Æ 0.008,
respectively (Table 1). The average particle size and PDI clearly
suggest that the developed nanotubes formulation have narrow
Fig. 1. (continued).
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particle size distribution with low polydispersity index (PDI). Ren
et al. reported the size PDI of DOX loaded oxidized angiopep-2
conjugated PEG-MWCNTs (DOX-O-MWCNTs-PEG-ANG) to be
202.23 Æ 3.43 nm and 0.342 Æ 0.016, respectively [30].
Transmission electron microscopy (TEM) was used to investi-
gate the possible morphological changes of MWCNTs depending on
the severity of each oxidizing treatment and find out any increase
in size owing TPGS conjugation on functionalized MWCNTs. The
TEM photomicrographs of pristine, oxidized and TPGS tethered
MWCNTs are shown in Fig. 2. Results showed that the size was
reduced after oxidation and increase in time of oxidation,
decreased the size of MWCNTs. The TEM photomicrographs of the
TPGS-MWCNTs clearly revealed that the developed optimized
nanotubes nanoformulation was in nanometric size range even
after conjugation. The developed TPGS-MWCNTs conjugate showed
good dispersibility and stability at RT.
Raman spectroscopy technique is a very valuable and commonly
used spectroscopic method for characterization of CNTs. It provides
the information on the hybridization state and defect concentration
of the CNTs. It also gives the information about slight structural
changes of MWCNTs, and changes in electronic structure of the
attached functional moieties [22,37,38].
Carbon nanotubes usually have following four bands in Raman
spectra: (1) Radial breathing mode (RBM) in 100e400 cmÀ1
region,
which is inversely proportional to the diameter of the nanotubes;
(2) the disorder-related so-called D mode, approximately at 1330e
1360 cmÀ1
provides information regarding amorphous impurities
and carbon nanotubes wall disorders; (3) high-energy mode
known as tangential G band (HEM, often called G mode in the re-
gion of 1500e1600 cmÀ1
) caused by stretching along the CeC
bonds in the graphitic plane; and (4) the D’mode at approximately
1615 cmÀ1
. The RBM mode is a characteristic for single walled
carbon nanotubes (SWCNTs) only that is caused by uni-axial vi-
brations of CNTs. In most cases, MWCNTs do not show this signal,
instead they show the D’band, which is assigned to the in-plane
vibrations of graphite [37e39]. The Raman spectra of the pristine
and surface engineered MWCNTs are shown in Fig. 3. The Raman
spectrum of the pristine MWCNTs shows the Raman shift at
1579.85 cmÀ1
and at 1346.15 cmÀ1
, which correspond to the G band
(graphite-like mode) and D band (disorder-induced band),
respectively. As the MWCNTs were oxidized (carboxylated), G band
was shifted to 1584.73 cmÀ1
and D band to 1352 cmÀ1
. The Raman
spectrum of the TPGS-MWCNTs shows the G band around
1565 cmÀ1
and D band around 1310 cmÀ1
. The shifting of the G and
Table 1
Physicochemical characterization of different MWCNTs complexes (Values repre-
sented as mean Æ SD; n ¼ 3).
Formulations Particle size
(nm)
Polydispersity
index
% Entrapment
efficiency
DOX/MWCNTs 230.41 Æ 1.3 0.27 Æ 0.010 92.5 Æ 2.62
DOX/TPGS-MWCNTs 250.18 Æ 5.5 0.32 Æ 0.008 97.2 Æ 1.20
Fig. 2. Transmission Electron Microscopic images: (A) Pristine, (B) Oxidized, and (C) TPGS conjugated MWCNTs.
Fig. 3. Raman spectra of (A) Pristine, (B) Oxidized, and (C) TPGS/MWCNTs.
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D band toward lower Raman shift in TPGS-MWCNTs is mainly due
to increase in the extent of conjugation of TPGS with surface
engineered MWCNTs, which would increase the single bond char-
acteristics in the functionalized system.
X-ray diffraction analysis (XRD) is another valuable tool for
characterizing the MWCNTs and surface functionalization, which
depicts the geometry or shape using X-rays. It is a non-destructive
analytical technique based on the elastic scattering of X-rays. It re-
veals information about the crystallographic structure, chemical
composition, physical properties and degree of crystallinity [22,40].
The Fig. 4 shows the XRD pattern of the pristine, oxidized and TPGS
conjugated surface engineered MWCNTs. The XRD analysis of func-
tionalized MWCNTs and TPGS-MWCNTs clearly shows that there was
no change in the seamless tubular structure and were found similar
with procured pristine and purified MWCNTs. Jain co-workers
previously reported the XRD pattern of the pristine and surface
functionalized MWCNTs and our results are in agreement [17,22].
The drug loading efficiency and in vitro release behavior are the
most important, prerequisite characteristic in the development and
characterization of targeted drug delivery system. The drug
entrapment efficiency was studied following a modified dialysis
diffusion technique and was found to be significantly higher for
TPGS-MWCNTs than pristine MWCNTs at different ratio of the
MWCNTs: drugs. The starting ratio of MWCNTs: DOX was 1:0.5 (w/
w), which increased upto 1:4 (w/w) on varying DOX concentration.
The anthracycline antibiotic DOX was loaded by incubating with
pristine and TPGS-MWCNTs nanoconjugates as evidenced by red-
dish color forming “forest scrub” like complex [41]. DOX entrapment
was monitored by UV/Visible absorption to confirm indirectly, the
drug entrapment in the pristine and TPGS-MWCNTs, during which
the characteristic absorption peak of DOX at 480.2 nm was slightly
shifted, indicative of interaction between DOX and MWCNTs.
A significant improvement in the entrapment efficiency was
observed from 92.5 Æ 2.62 (pristine MWCNTs) to 97.2 Æ 2.50 (DOX/
Fig. 4. X-ray diffraction (XRD) analysis; (A) Pristine, (B) Oxidized, and (C) TPGS conjugated MWCNTs.
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TPGS-MWCNTs). Due to its aromatic structure the DOX tends to
strongly interact with sidewall and ends of the nanotubes through
pep stacking and hydrophobic interactions. In this, large p-conju-
gated structure could form pep stacking interaction with quinine
part of the DOX although amino (eNH2) and several hydroxyl (eOH)
groups are also present in the chemical structure of DOX. Thus, the e
OH and eCOOH functional groups of the nanotubes form strong
hydrogen bond with eOH and amino eNH2 groups in DOX. Another
possible reason for increased entrapment efficiency may be that the
cationically charged DOX molecules could easily get adsorbed with
lower potential of TPGS-MWCNTs than pristine MWCNTs via elec-
trostatic interaction as well as p-p stacking interaction. Very high
DOX loading efficiency mainly depends on the physical properties of
the nanotubes. Lu co-workers reported 96% DOX loading efficiency
(the weight percentage of initial DOX bound to MWCNTs) in folate
conjugated magnetic multi walled carbon nanotubes (FA-MN-
MWCNTs) due to strong pep stacking interactions [36]. The in vitro
release profile of any targeted drug delivery systems is a crucial
parameter for determining their overall clinical pharmaceutical ef-
ficacy. We investigated the cumulative DOX release from DOX/TPGS-
MWCNTs and DOX/MWCNTs nanoformulations at temperature 37 C
upto 200 h in the phosphate buffer solution (pH 7.4) and sodium
acetate buffer solution (pH 5.3), corresponding to physiological and
endosomal pH of the cancerous cells, respectively. The pH of the
cytosol is neutral to mildly alkaline (7.4e7.8), while lysosomal pH is
acidic (4e5.5) [26,27,34]. The obtained cumulative DOX release
pattern exhibited a non-linear release profile characterized by rela-
tively initial faster release followed by sustained and slower release
in later period. The DOX release was found to be 98.5 Æ 1.45,
38.9 Æ 0.85 and 88.3 Æ 1.57, and 19.2 Æ 1.86 for pristine MWCNTs and
TPGS-MWCNTs at pH 5.3 and 7.4, respectively in 24th h. The DOX
release at pH 5.3 from pristine MWCNTs was not detected after
24th h time point. The amount of drug released at 200th h at pH 7.4
was 64.3 Æ 2.39 and at pH 5.3 was 92.02 Æ 2.57% (Fig. 5). The DOX
release from surface engineered nanotubes critically depends on pH
and solubility of the drug in particular medium and follows the order
of release at all pH range:
DOX=TPGS À MWCNTs DOX=MWCNTs
ðSustained Release Faster ReleaseÞ
The pH-triggered DOX release pattern will be even more bene-
ficial if the functionalized-MWCNTs following the intracellular
internalization, trafficking to the lysosomes and lower pH value of
lysosomes will augment drug release inside the acidic microenvi-
ronment. At this low pH, the hydrophilicity of DOX increases due to
the protonation of the NH2 group native to its structure. The
increased hydrophilicity aids in overcoming the interaction (pep)
among the DOX and the functionalized MWCNTs while facilitating
its detachment from the nanotubes. The release of DOX from the
surface engineered MWCNTs is pH-triggered with slow and
controlled manner at different pH value [14,26,27,34,36,42].
Stability studies of the optimized pharmaceutical drug product
were performed as per ICH guidelines. The nanoformulations were
found to be the most stable at 5 Æ 3 C in dark. No turbidity was
seen in the formulation stored at 5Æ3 C and 25 Æ 2 C of MWCNTs
formulation in dark condition. However, after storage in light at
25 Æ 2 C, slight turbidity was observed in all the nanotubes for-
mulations, which might be due to aggregation of nanotubes when
stored both in light and dark condition at this temperature. At
40 Æ 2 C, all the formulations showed higher turbidity, which may
possibly be due to formation of larger aggregates (Table 2).
The drug leakage from the developed nanotubes formulation
was performed for a period of six months and found to be negligible
at 5Æ3 C. In general, all the developed nanotubes formulations
were found to be most stable at 5Æ3 C temperature in dark
(Table 2). The lesser drug leakage may be ascribed to the surface
engineering of the nanotubes, which provide stronger interaction
to the nanotubes. These developed nanotubes formulations are
capable to resist the accelerated stress condition and retain guest
moieties of the molecules even at the higher or elevated temper-
ature upto six months.
The hemolytic toxicity of pristine MWCNTs was enough to limit
its use as drug delivery system. The hemolytic toxicity of DOX,
pristine MWCNTs, DOX/MWCNTs, and DOX/TPGS-MWCNTs nano-
formulations was studied and compared to assess the effects on
erythrocytes (RBCs) in the blood on administration. The percent
hemolysis data of free DOX (15.7 Æ 0.42), pristine MWCNTs
(18.0 Æ 0.50), oxidized MWCNTs (15.5 Æ 0.55), DOX/MWCNTs
(14.6 Æ 0.28) and DOX/TPGS-MWCNTs (7.50 Æ 0.18) were
compared. Pristine MWCNTs showed (18.0 Æ 0.5) highest hemo-
lytic toxicity due to the presence of some metallic impurities.
However, DOX loaded TPGS-MWCNTs drastically reduced hemo-
lytic toxicity. It is clear from the hemolysis toxicity studies that the
degree of functionalization reduces the erythrocytes toxicity. These
results are in good agreement with the earlier report [5,14,17].
Fig. 5. Cumulative amount of DOX released from the DOX/MWCNTs and DOX/TPGS-MWCNTs nanoconjugates at 37 Æ 0.5 C in phosphate buffer solution (pH ¼ 5.3 and 7.4). (n ¼ 3).
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The Methylthiazole tetrazolium (MTT) cytotoxicity assay was
performed to determine the cancer targeting propensity of the DOX
laden developed optimized nanotubes formulation using MCF-7
(human breast cancer; derived from pleural perfusion) cell line
and compared with pristine MWCNTs and free DOX solution. The
cytotoxicity assay clearly demonstrated that upon increasing the
concentration from 0.001 to 100 mM the percent viability of the
cancerous cells was decreased owing to apoptosis by intercalating
DOX (anthracycline antibiotic) with DNA (Fig. 6; Supporting
Animation). Our cytotoxicity results are in accordance with the
previous published reports [14,24,41]. Similarly, Gu co-workers
reported the IC50 value for SWCNTs-HBA-DOX and SWCNTs-DOX
in HePG2 cells to be 4.8 and 7.4 mM, respectively [43].
In next cell culture studies, we determined the DNA content and
mechanism of cell death by cell-cycle phase distribution using flow
cytometry of the nanotubes formulation. The cell cycle analysis
could be recognized by the four distinct phases in a proliferating
cell population: G1-, S- (DNA synthesis phase), G2- and M-phase
(mitosis), while G2- and M-phase have an identical DNA content
and could not be discriminated on the basis of the DNA content
[44]. The apoptotic dead cells as well as fragmented nuclei were
showed in sub-G1 cells. Additionally, sub-G1 population also
indicated the apoptotic-associated chromatin degradation [29,45].
The control cells which were not treated with the DOX, showed
27.99%, 29.17% and 42.84% population in the G1, G2 and S-phase,
respectively. According to Fig. 7, the percentage of cells in the G1
phase increased to G2 27.99%, 24.35%, 76.97% and 86.50% for con-
trol, DOX, DOX/MWCNTs and DOX/TPGS-MWCNTs, respectively
after 24 h exposure. The majority of the cancerous cells were
arrested in the G1 phase (86.50%), whereas only 5.35% in S-phase in
case of DOX/TPGS-MWCNTs nanoformulation. It is notably indi-
cated that the doxorubicin, especially DOX/TPGS-MWCNTs attack
and induce cell cycle arrest in the G1 phase by intercalating the
DNA synthesis and get accumulated in the nucleus with in short
span in high quantity.
The qualitative and quantitative cellular uptake of DOX was
studied (Fig. 8 FA, FB, FC, and FD, A, B, C and D). Fig. 8 FD shows
higher red fluorescence intensity as compared to other formulation
and control group. The doxorubicin has red auto-fluorescence as
observed in Fig. 8. Similarly, higher fluorescence intensity was
observed for DOX/TPGS-MWCNTs (78.72%), as compared to DOX/
MWCNTs (62.46%) and free DOX (58.15%) in R2 region. while con-
trol without DOX showed 69.16% fluorescence intensity in R1 re-
gion. The observed higher fluorescence intensity clearly suggests
the higher uptake of the DOX/TPGS-MWCNTs formulations,
possibly due to the receptor-mediated endocytosis as well nano-
needle specific mechanism (Fig. 8).
The overall pharmaceutical targeting efficacy of the DOX loaded
surface engineered CNTs nanoformulations were evaluated in Balb/
c mice. The plasma concentration profiles of the DOX after i.v.
administration of the free DOX, DOX/MWCNTs and DOX/TPGS-
MWCNTs (5.0 mg/kg body weight) and pharmacokinetic parame-
ters were determined using non-compartment modeling as sum-
marized in Table 3 (Fig. 9). The area under the curve (AUC0ÀN) and
area under the first moment curve (AUMC0ÀN) were calculated to
be 9.3292, 22.3127, 46.4690 and 22.0079, 149.8770 and 937.5830
for free DOX, DOX/MWCNTs and DOX/TPGS-MWCNTs, respectively.
The AUC(0ÀN) and AUMC(0ÀN) of DOX/TPGS-MWCNTs were
approximately 5.0 and 6.25 folds higher, respectively as compared
to free DOX. The elimination half-life (t1/2) of DOX/TPGS-MWCNTs,
DOX/MWCNTs and free DOX was found to be 12.1651, 4.7022 and
1.5648, while MRT was found to be 18.1848, 6.5381 and 2.3590,
respectively.
The average half-life (t1/2) of DOX/TPGS-MWCNTs (12.1651) was
2.58 and 7.8 times, while MRT was 2.78 and 7.7 times longer as
compared to DOX/MWCNTs and free DOX, respectively. The ob-
tained results are ascribed to biocompatibility of surface engi-
neered nanotubes upon TPGS conjugation allowing longer
residence time inside the body because TPGS render more hydro-
philicity and stealth character to nanotubes.
The PEGylated SWCNTs significantly increased the blood circu-
lation of the anticancer drug [46]. The obtained results also support
the extended residence time and sustained release profile of the
DOX loaded surface engineered nanotubes formulations in body as
compared to DOX solution and the maximum being for TPGS con-
jugated nanotubes formulations. It clearly evinces the improved
Table 2
Accelerated stability studies for the DOX/TPGS-MWCNTs formulations.
Stability parameter DOX/TPGS-MWCNTs
Dark Light
T1 T2 T3 T1 T2 T3
Turbidity À À þþ þ þþ þþþ
Precipitation À À þþ þ þþ þþ
Change in color À À þ þ þ þþ
Crystallization À À þ þ þ þþ
Change in consistency À À þ þ þ þþ
Percent drug leakage
1 month 0.4 Æ 0.02 1.2 Æ 0.04 2.4 Æ 0.4 1.2 Æ 0.04 1.6 Æ 0.06 5.4 Æ 0.06
2 month 1.2 Æ 0.03 1.4 Æ 0.09 2.6 Æ 0.03 1.6 Æ 0.06 1.8 Æ 0.09 5.8 Æ 0.24
4 month 1.6 Æ 0.12 1.8 Æ 0.24 3.4 Æ 0.32 1.8 Æ 0.22 2.6 Æ 0.34 6.2 Æ 0.82
6 month 1.8 Æ 0.16 2.2 Æ 0.18 3.8 Æ 0.27 2.0 Æ 0.33 2.8 Æ 0.42 6.8 Æ 0.14
T1, T2 and T3 represent 5 Æ 3, 25 Æ 2, and 40 Æ 2
C temperatures, respectively.
All the values represented as mean Æ SD (n ¼ 3). “e, þ, þþ and þþþ” indicate no change, small change, considerable change and major change, respectively.
Fig. 6. Percent cell viability of MCF-7 cell after treatment with free DOX, DOX/
MWCNTs and DOX/TPGS-MWCNTs at 24 h (n ¼ 3).
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Fig. 7. DNA content and cell cycle analysis of the DOX and developed MWCNTs formulations on MCF-7 cell lines using flow cytometry. Cells were incubated with the formulation and analyzed by flow cytometry. Cell cycle results
displayed as a histogram. Dip G1: proportion of cells in G0/G1 phase; Dip G2: proportion of cells in G2 phase; Dip S: proportion of cells in S phase. Peaks corresponding to G1/G0, G2/M, and S phases of the cell cycle were indicated
where *p 0.05 vs. control.
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pharmacokinetics with better bioavailability and more prolonged
retention in systemic circulation than that obtained on adminis-
tration of drugs-MWCNTs and free drug solutions to mice.
A comparative tissue biodistribution study was performed to
assess the amount of DOX after i.v. administration of the developed
nanotubes formulations and highest DOX level was found in tumor,
liver and kidney in 24 h in case of DOX/TPGS-MWCNTs. The free
DOX accumulates progressively in liver, where upto 26.76 Æ 0.14%
of dose is localized after 1 h administration whereas only
16.43 Æ 0.056% of DOX was found in liver after 24 h. In case of DOX/
TPGS-MWCNTs amount of drug accumulated in liver of its initial
dose was found to be 38.72 Æ 0.54 and 28.88 Æ 0.98 at initial 1st
and 24th h (Fig. 10).
The amount of DOX in body depends upon its distribution,
metabolism and excretion. Initially the free DOX content in liver
was found to be highest and later it declined in case of free DOX
solution, which might be due to the rapid drug elimination from
liver i.e. prime site of its necessary action. The amount of DOX was
estimated in the tumor organs and was found to be 9.88 Æ 0.12,
12.75 Æ 0.11, 14.98 Æ 0.12 and 18.72 Æ 0.66 at 1, 6, 12, and 24 h,
respectively from the DOX/TPGS-MWCNTs formulations. The high
levels of the surface engineered MWCNTs were found at initial time
point of administered dose in kidney and the rapid decline in the
overall formulation thereafter indicating that most of the nano-
tubes were eliminated through the renal excretion route [14,47]. It
was inferred that TPGS appended nanotubes formulations were
highly accumulated in tumor-rich organs with sustained drug
release.
The obtained pharmacokinetic and tissue/organ biodistribution
data of the DOX laden TPGS MWCNTs formulations are in good
agreement with the previous published reports from our
[5,9,12,14,16,17,22] and laboratory [26,27,34,47,48]. The in vivo tu-
mor targeting efficacy was assayed on MCF-7 tumor bearing Balb/c
model and the starting tumor size was approximately 100 mm3
for
all dose receiving groups of the developed nanoconjugates as well
as normal saline and control group. The tumor volume (mm3
) was
107.4 Æ 3.5,103.8 Æ 3.9, 99.2 Æ 3.4 and 89.5 Æ 2.84 in case of normal
saline, free DOX, DOX/MWCNTs and DOX/TPGS-MWCNTs at 5th
day. The size of the tumor volume (mm3
) was reduced to
85.9 Æ 2.92 and 45.6 Æ 2.35 in 30th days after treatment with DOX/
MWCNTs and DOX/TPGS-MWCNTs formulations, respectively
(Fig. 11).
The percentage body weight changes of the MCF-7 bearing Balb/
c mice after intravenous injection of the DOX loaded formulations
was calculated upto 30 days. It clearly suggests that the developed
formulations does not affect the body weight of the Balb/c mice,
while in case of the normal saline treated group the loss of body
weight was observed.
KaplaneMeier survival curves based on survival time were
plotted for different groups of animals using Log-rank test. The
curves suggested that the median survival time for tumor bearing
mice treated with DOX/TPGS-MWCNTs (44 days; p 0.001) was
Fig. 8. Qualitative and Quantitative cellular uptake of the DOX in MCF-7 cell: (FA A) Control, (FB B) Free DOX solution, (FC C) DOX/MWCNTs, and (FD D) DOX/TPGS-MWCNTs
formulations (where p 0.001 vs control).
Table 3
Pharmacokinetic parameters of free DOX and DOX loaded MWCNTs formulations.
Parameters Cmax (mg/mL) HVD (h) AUC(0Àt) (mg/mL h) AUC(0ÀN) (mg/mL h) AUMC(0Àt) (mg/mL h2
) AUMC(0ÀN) (mg/mL h2
) t1/2 (h) MRT (h)
Free DOX 6.11 0.36564 9.3066 9.3292 21.6860 22.0079 1.5648 2.3590
DOX/MWCNTs 6.06 0.8329 22.3127 22.9233 131.0810 149.8770 4.7022 6.5381
DOX/TPGS-MWCNTs 6.52 1.4356 46.4690 51.5587 603.9520 937.5830 12.1651 18.1848
Probability p 0.05; standard deviation 5%.
Abbreviations: Cmax, peak plasma concentration; t1/2, elimination half life; MRT, mean residence time; AUC(0ÀN), area under plasma drug concentration over time curve; HVD,
half value duration.
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extended compared to DOX/MWCNTs (23 days), free DOX (18 days)
and control group (12 days) (Fig. 11).
These results further confirmed that the higher tumor treatment
potential possessed by the surface engineered MWCNTs; resulted
in longer survival span for tumor bearing mice. The longest survival
span (44 days; p 0.001) was observed in case of DOX/TPGS-
MWCNTs. The obtained results were similar to tumor inhibition
growth studies. The median survival span for doxorubicin loaded
nanotubes formulations was found in the following order:
DOX=TPGS À MWCNTs DOX À MWCNTs free DOX
The DOX/TPGS-MWCNTs (targeted stealth; long circulatory na-
ture) were found to be more active than pristine MWCNTs and drug
solution with a significant reduction in tumor growth. Although the
free DOX was initially efficient in suppressing further tumor growth
but the inhibition activity did not last long. The median survival of
DOX loaded angiopep conjugated MWCNTs (DOX-O-MWCNTs-
ANG) was observed 43 days while DOX-O-MWCNTs-PEG showed
only 36 days [30].
Hematological parameters (RBCs, WBCs and differential counts)
were determined to investigate the relative effects of the MWCNTs
formulations (DOX/MWCNTs, DOX/TPGS-MWCNTs) and compared
with free DOX on the different components of blood. Blood samples
were analyzed for RBCs, WBCs and differential counts are shown in
Table 4.
The RBCs and WBCs counts of free DOX, MWCNTs, DOX/
MWCNTs, and DOX/TPGS-MWCNTs formulations were found to be
7.8 Æ 0.34 Â 106
/mL, 7.5 Æ 0.56 Â 106
/mL, 7.7 Æ 0.88 Â 106
/mL,
9.1 Æ 0.22 Â 106
/mL and 9.5 Æ 0.46 Â 106
/mL, 8.6 Æ 0.62 Â 106
/mL,
8.8 Æ 0.87 Â 106
/mL and 10.6 Æ 0.50 Â 106
/mL, respectively. The
differential counts were found to be very similar to control group.
The hemoglobin (Hb) and HCT counts of the DOX/TPGS-MWCNTs
were found to be 12.2 Æ 0.22 (g/dl) and 34.8 Æ 0.54 and were
almost similar with the control group 12.4 Æ 0.33 (g/dl) and
35.5 Æ 0.65, respectively.
These obtained data from the serum biochemical parameters
clearly suggest that the RBCs, WBCs and differential count of DOX/
TPGS-MWCNTs were almost similar with the control and normal
saline groups. Similarly, the differential counts i.e. leucocytes,
Fig. 10. Biodistribution of DOX after intravenous administration of DOX solution, DOX/MWCNTs and DOX/TPGS-MWCNTs formulation in tumor bearing Balb/c mice at a single dose
(5.0 mg/kg body weight). *p 0.05; **p 0.01; ns: not significant Vs. Free DOX. (Values represented as means Æ SD; n ¼ 3).
0
1
2
3
4
5
6
7
8
0 10 20 30 40 50
Concentration(µg/mL)
Time (hr)
Free DOX DOX-MWCNTs DOX/TPGS-MWCNTs
Fig. 9. Serum concentration of DOX from free DOX, DOX/MWCNTs and DOX/TPGS-MWCNTs at different data points. Mean Æ SD (n ¼ 3; p 0.001).
N.K. Mehra et al. / Biomaterials 35 (2014) 4573e4588 4585

15. Author's personal copy
monocytes and lymphocytes of DOX/TPGS-MWCNTs were almost
similar to normal values. The hematological values clearly indicate
the superior non-toxic behavior of the TPGS appended than the
pristine MWCNTs and free DOX.
The DOX solution induced toxicity was observed in mice as re-
flected by the increased activity of SGPT (35.2 Æ 2.6 IU/L), ALP
(69.8 Æ 2.3 IU/L) and total bilirubin concentration (0.62 Æ 0.18 mg/
100 mL). Upon entrapment of DOX in the pristine MWCNTs a small
increase in the activity of SGPT (25.6 Æ 1.9 IU/L), ALP (61.2 Æ 2.4 IU/
L) and total bilirubin concentration (0.46 Æ 0.020 mg/100 mL) was
observed. The surface engineering of MWCNTs with subsequential
step and conjugation of the TPGS have much reduced the in vivo
toxicities of the drug loaded formulations.
4. Conclusion
The surface engineered carbon nanotubes have been continu-
ously attracting escalating attention in targeted drug delivery. In
the present studies, TPGS appended stealth surface engineered
MWCNTs nanoformulation was found to be significant in tumor
growth suppression as compared to non-targeted CNTs and free
DOX solution. The quantitative biodistribution studies further
corroborated the anti-tumor activities, survival span wherein
higher amount of DOX was found from MWCNTs at the site of
cancerous tissue. The pharmacokinetics studies clearly revealed
that the developed MWCNTs formulations are long-circulating
(stealth). Significant reduction in subsequent growth in tumor
**
120
100
80
60
40
20
0
0 10 20
Time (days)
30 40 50
Percentsurvival
Normal saline
Free DOX
DOX-MWCNTs
DOX/TPGS-MWCNTs
0
20
40
60
80
100
120
140
160
0 5 10 15 20 25 30
TumorVolume(mm3)
Days after treatment
Normal saline Free DOX DOX/MWCNTs DOX/TPGS-MWCNTs
Fig. 11. KaplaneMeier survival curves of MCF-7 bearing Balb/c mice and analyzed by Log-rank (Mental-Cox) test with normal saline group as control. *p 0.05; **p 0.01; ns: no
significant difference (Upper). Tumor regression analysis after intravenous administration of free DOX, DOX/MWCNTs and DOX/TPGS-MWCNTs nanoconjugates (5.0 mg/kg body
weight dose). The DOX/TPGS-MWCNTs treated group showed significant (P 0.05) suppression of tumor growth compared with the other groups (lower) (n ¼ 3).
Table 4
Serum biochemical parameters of Balb/C mice treated with free DOX, MWCNTs, DOX/MWCNTs and DOX/TPGS-MWCNTs formulation after 7 days.
Group RBCs (Â106
/ml) WBCs (Â106
/mL) Differential counts (Â103
/mL) Hb (g/dl) HCT
Monocytes Lmphocytes Neutrophils
Control 9.2 Æ 0.40 10.8 Æ 0.40 1.4 Æ 0.60 7.9 Æ 0.42 1.6 Æ 0.42 12.4 Æ 0.33 35.5 Æ 0.65
Normal saline 8.4 Æ 0.32 9.6 Æ 0.42 0.9 Æ 0.34 6.1 Æ 0.44 1.0 Æ 0.66 10.5 Æ 0.22 34.4 Æ 0.25
Free DOX 7.8 Æ 0.34 9.5 Æ 0.46 0.9 Æ 0.33 6.1 Æ 0.42 1.1 Æ 0.82 10.6 Æ 0.32 33.8 Æ 0.62
MWCNTs 7.5 Æ 0.56 8.6 Æ 0.62 0.7 Æ 0.76 5.9 Æ 0.88 0.9 Æ 0.85 9.8 Æ 0.94 33.6 Æ 0.12
DOX/MWCNTs 7.7 Æ 0.88 8.8 Æ 0.87 0.9 Æ 0.90 6.1 Æ 0.62 1.1 Æ 0.48 10.2 Æ 0.66 34.0 Æ 0.23
DOX/TPGS-MWCNTs 9.1 Æ 0.22 10.6 Æ 0.50 1.2 Æ 0.65 7.8 Æ 0.92 1.5 Æ 0.4 12.2 Æ 0.22 34.8 Æ 0.54
All values are expressed as mean Æ SD. No. of animals per time points were three (n ¼ 3); WBCs, white blood corpuscles; RBCs, red blood corpuscles; Hb, haemolglobin; HCT,
haematocrit.
N.K. Mehra et al. / Biomaterials 35 (2014) 4573e45884586

16. Author's personal copy
was probably due to ligand-driven internalization of the surface
engineered nanotubes targeted drug delivery system at the target
site/tissues, which was accompanied by slow and sustained release
of the drug. The tumor growth inhibition study clearly indicated
that inclusion of the pH-responsive characteristics increases the
overall pharmaceutical targeting efficiency of the targeted nano-
tubes formulations. To the best of our knowledge, the present one is
a debut study report and suggest that DOX/TPGS-MWCNTs is a
promising and efficient targeted drug delivery system in the
treatment of cancer.
Declaration of interest
The authors report no conflict of interest.
Acknowledgments
One of author Neelesh Kumar Mehra is highly thankful to Uni-
versity Grants Commission (UGC), New Delhi for providing the
Senior Research Fellowship during the tenure of the studies. The
author also acknowledge Dr. Ranveer Kumar Department of Phys-
ics, Dr. H. S. Gour University, Sagar for Raman spectroscopy; Central
Instruments Facilities (CIF), National Institute of Pharmaceutical
Education and Research (NIPER), Mohali, Chandigarh; Central Drug
Research Institute (CDRI), Lucknow; All India Institute of Medicine
and Sciences (AIIMS), New Delhi (TEM); Diya Laboratory, Mumbai.
Authors are also thankful to National Centre for Cell Sciences
(NCCS), Pune for providing the cell lines.
Appendix A. Supplementary data
Supplementary data related to this article can be found online at
http://dx.doi.org/10.1016/j.biomaterials.2014.02.022.
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