9 Mehra and Jain JDT

2013

http://informahealthcare.com/drtISSN: 1061-186X (print), 1029-2330 (electronic)

J Drug Target, 2013; 21(8): 745–758! 2013 Informa UK Ltd. DOI: 10.3109/1061186X.2013.813028

ORIGINAL ARTICLE

Development, characterization and cancer targeting potential of surfaceengineered carbon nanotubes

Neelesh Kumar Mehra and N. K. Jain

Pharmaceutics Research Laboratory, Department of Pharmaceutical Sciences, Dr. H. S. Gour Central University, Sagar (M.P.), India

Abstract

The aim of the present study was to assess the in vitro and in vivo potential of doxorubicin-loaded, folic acid appended engineered multi-walled carbon nanotubes (DOX/FA-PEG-MWCNTs) for efficient tumor targeting. The loading efficiency was determined to be92.0� 0.92 (DOX/FA-PEG-MWCNTs) in phosphate buffer solution (pH 7.4) ascribed to p–pstacking interaction. The developed nanoconjugates were evaluated for in vitro DOX release,erythrocytes toxicity, ex vivo cytotoxicity and cell uptake studies on MCF-7 (breast cancer cellline). The DOX/FA-PEG-MWCNTs nanoconjugate affords higher efficacy in tumor growthsuppression due to its stealth nature and most preferentially taken up by the cultured MCF-7through caveolae-mediated endocytosis as compared to free DOX. The in vivo studies wereperformed to determine the pharmacokinetics, biodistribution and antitumor efficacy on tumorbearing female Sprague Dawley rats and improved pharmacokinetics confirm the functionof FA-PEG conjugated CNTs. The median survival time for tumor bearing rats treated withDOX/FA-PEG-MWCNTs (30 d) was extended very significantly as compared to free DOX(p50.001). The results concluded that developed water-soluble nano-conjugates mightemerge as ‘‘safe and effective’’ nano-medicine in cancer treatment by minimizing the sideeffects with and Generally Regarded as Safe prominence.

Keywords

Anti-tumor activity, carbon nanotubes,doxorubicin, drug targeting, folic acid,MCF-7 cells, pharmacokinetic

History

Received 12 March 2013Revised 5 June 2013Accepted 5 June 2013Published online 3 July 2013

Introduction

Cancer is amongst the top three killers in modern society, next

to heart and cerebrovascular diseases, tuberculosis and

acquired immune deficiency syndrome (AIDS). Breast

cancer is the most common and second leading cause of

cancer deaths today in women worldwide, both in the

developed and developing countries. Despite the high

incidence rates, in Western countries, 89% of women

diagnosed with breast cancer are still alive 5 year after

diagnosis, which is due to detection and treatment. In 2010,

nearly 1.5 million people were told ‘‘you have breast cancer’’.

It has been continuously rising due to the increase in life

expectancy, urbanization and adoption of western life styles.

The Canadian Cancer Society in 2011 reported that an

estimated 23 400 women were diagnosed and 5100 died with

breast cancer, moreover approximately 190 men were

also diagnosed and 55 died with breast cancer [1] (World

Health Organization fact sheet. Available from the URL

http://www.who.int/cancer/en/; http://www.worldwidebreast

cancer.com/learn/breast-cancer-statistics-worldwide/ Accessed

date 28th April 2013). Although the significant progress has

been made in the development of new, safe nanomedicines for

cancer therapy, yet we still strongly need a complete and

reliable cure of cancer. In the current scenario, carbon

nanotubes (CNTs) have attracted escalating attention and are

under investigation with surface modification with targeting

ligand to offer a sustained/controlled level of drug and to

accomplish cellular target with enhanced specificity. CNTs are

unique, three dimensional sp2 hybridized carbon nanomaterial

have attracted tremendous attention as valuable, promising,

alternative ‘‘safe and effective’’ nano-architecture to biomed-

ical applications due to their unique physicochemical proper-

ties such as biocompatibility, non-immunogenicity, high

loading efficiency, high aspect ratio, structural flexibility,

non-cytotoxic and non-biodegradable nature [2–10]. The

pristine CNTs (first generation) are not suitable for drug

delivery due to their hydrophobic nature, impurities and toxic

in nature. These major hurdles have been easily sorted out by

surface engineering with either covalent or non-covalent

modification strategies, thus surface engineered CNTs have

been designed and tested for targeted delivery by conjugating

targeting moieties and have proven non-cytotoxic to human

cells [2,5,11–18].

Doxorubicin (DOX) is a potent anthracycline

cytostatic antibiotic used in the treatment of several mali-

gnancies by intercalating with the DNA or DNA topoisom-

erase II [15,19,20]. Adriamycin� and Rubex� are two

Address for correspondence: Professor N. K. Jain, PharmaceuticsResearch Laboratory, Department of Pharmaceutical Sciences, Dr. H.S.Gour University, Sagar (M.P.) 470 003, India. Tel/Fax: +91-7582-265055. E-mail: [email protected], [email protected]

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commercially available intravenously administered injections.

Additionally, Myocet� (Enzon Pharmaceuticals, Piscataway,

NJ), a non-PEGylated doxorubicin formulation; and two other

approved PEGylated liposomal nanomedicine DOXIL�

(Centocor Ortho Biotech, Horsham, PA) and Caelyx�

(Schering-Plough, North Ryde, New South Wales, Australia)

are also available [15,21]. Doxorubicin has been easily

loaded/conjugated on to the engineered nanotubes surface

via p–p stacking and hydrophobic interactions allowed

pH-dependent release [9,15,20,22,23].

In the present investigation, folic acid (FA) used as a

targeting agent, participates in the biosynthesis of nucleotide

bases and available in pteroyl-L-glutamic acid, pteroyl-

L-glutamate and pteroylmonoglutamic acid forms. Folate

receptors (FRs) generally present in caveolae membrane

protein participates in cellular uptake via protocytosis mech-

anism by covalently linked to its activated g-carboxyl

functional group [6,24].

In the present study, DOX loaded folic acid-polyethylene

glycol-4000-bis amine- and folic acid-multi walled car-

bon nanotubes (DOX/FA-PEG-MWCNTs and DOX/FA-

MWCNTs, respectively) nanoconjugates were evaluated as

promising nano-architecture for site specific delivery with

improved therapeutic outcomes of DOX. These developed

nano-conjugates were evaluated for in vitro release as well as

ex vivo studies on MCF-7 cell line. The in vivo studies

were performed on female Sprague Dawley (SD) strain rats

wherein DOX/FA-PEG-MWCNTs nanoconjugates showed

improved pharmacokinetics, leading to higher cytotoxicity

on MCF-7 cells.

Materials and methods

Materials

Multi Walled Carbon Nanotubes (MWCNTs) produced by

chemical vapor deposition (CVD), with purity 99.3%, were

purchased from Sigma Aldrich Pvt. Ltd. (St. Louis, MO).

Doxorubicin hydrochloride was received a benevolent gift

from M/s Sun Pharm Advanced Research Centre (SPARC),

Vadodara, Gujarat, India. PEG-4000 bis amine (Sigma

Aldrich Pvt. Ltd., St. Louis, MO), and Poly-tetrafluoroethy-

lene (PTFE) filters (0.22 mm pore size) were purchased from

Hangzhou Anow Microfiltration Co. Ltd., Hangzhou, China.

Dimethyl sulfoxide (DMSO), 1-ethyl-3-(3-dimethylaminopro-

pyl)carbodiimide (EDC) folic acid (FA)and dialysis mem-

brane (MWCO, 5–6 KDa) was purchased from HiMedia Pvt.

Ltd., Mumbai, India. All the reagents and solvents were used

as received.

MWCNTs preparation

Purification and chemical oxidation of procured pristine

MWCNTs was done by the following treatments with slight

modifications.

Microwave treatment. Briefly, procured pristine MWCNTs

(500 mg) were kept in a microwave oven at 400� 2 �C for

60 min.

Piranha treatment. Microwave-treated MWCNTs (500 mg)

were immersed with concentrated nitric acid and sulphuric

acid (HNO3: H2SO4:: 1:3) mixture in a flat bottom flask

(equipped with the reflux condenser and thermometer) with

continuous magnetic stirring (100 rpm; Remi, Mumbai, India)

at 120� 2 �C for 24 h. The dispersed MWCNTs were then

washed with deionized water, ultra centrifuged (Z36HK,

HERMLE LaborTchnik GmbH, Wehingen, Germany) at

20 000 rpm for 15 min and vacuum dried (Jyoti Scientific

Industries, Gwalior, India) [13,15,16].

Chemical treatment. Piranha-treated oxidized MWCNTs

(400 mg) were immersed in the mixture of ammonium

hydroxide (NH4OH) and hydrogen peroxide (H2O2) in 50:50

ratio in a round bottom flask at 80� 5 �C for 24 h. Then,

washed repeatedly to neutral pH, ultra centrifuged (Z36HK,

HERMLE LaborTchnik GmbH, Wehingen, Germany) at

20 000 rpm for 15 min and vacuum dried [25].

Determination of total acidic functional groups by Boehm

titration

The total acidic functional groups were quantitatively

determined by Boehm titration method [26]. Briefly, oxidized

MWCNTs were added into 0.05 N NaHCO3 and NaOH

solution (50:50) and magnetically stirred at 100 rpm for 48 h

to reach an equilibrium state, filtered, separate out and diluted

with deionized water. Dispersed MWCNTs (10 mL) was

mixed with 0.05 N HCL with heating for 30 min and titrated

with 0.05 N NaOH to determine the concentration of total

acidic functional groups present on ox-MWCNTs. Then,

ox-MWCNTs were further used for acylation and amidation

as previous reported by our group [27].

Folic acid (FA) conjugation to functionalized MWCNTs

Previously reported method with slight modifications [28]

was followed for amine-protection of FA and its further

ester activation. Briefly, folic acid (4 mM) was dissolved

in DMSO:DCM: (1:1 v/v) in a reaction vessel and t-BOC

(5 mM) was added with continuous magnetic stirring (Remi,

Mumbai, India) under dark at room temperature (RT) for 3 d.

Then g-carboxylic acid group of FA-t-BOC was activated

with NHS and DCC as coupling agents as shown in Scheme 1

[28]. The conjugation of FA was done in two types without

PEG and with PEG as spacer.

(i) Folic acid conjugation to surface engineered MWCNTs

(FA-MWCNTs)

The activated ester of folic acid (NHS-FA) (25 mg/mL)

was mixed with amine terminated MWCNTs in DMSO

(10 mg/mL) with continuous stirring (Remi, Mumbai,

India) at 200 rpm for 5 d at room temperature under dark

condition followed by addition of acetone to obtain yellow

precipitate and collected. The unconjugated FA was separated

out by dialysis tube (MWCO 5–6 KDa,) against deionized

water, collected, dried and characterized as represented in

Scheme 2 [1].

(ii) Folic acid conjugation through PEG spacer to surface

engineered MWCNTs (FA-PEG-MWCNTs)

The t-BOC-FA-NHS (32.5 mg; 74 mM) active ester was

mixed with PEG-4000 bis amine (Sigma Aldrich Pvt. Ltd., St.

Louis, MO) (450 mg; 112.5mM) in DMSO (5.0 mL) in the

presence of triethylamine (4.0 mL) with continuous magnetic

stirring at 100 rpm (Remi, Mumbai, India) for 24 h at room

temperature. The unconjugate PEG 4000-bis amine was

removed, filtered, dried under vacuum (Jyoti Scientific

746 N. K. Mehra & N. K. Jain J Drug Target, 2013; 21(8): 745–758

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Scheme 1. Synthesis and activation of folicacid [24].

Scheme 2. Synthesis of folic acid-MWCNTs nano-conjugate from NHS-folic acid conjugate.

DOI: 10.3109/1061186X.2013.813028 Development, characterization and cancer targeting potential 747

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Industries, Gwalior, India) to yield folate-conjugate (t-BOC-

FA-PEG-NH2) as pale yellow solid and detected using UV/

Vis spectrophotometer at lmax 363 nm (Shimadzu, 1601,

Kyoto, Japan) [24].

Carboxylated MWCNTs (33.36 mg) were dispersed

in DMSO and EDC dissolved in DMSO (6.41 mg/mL) was

added to it with continuous magnetic stirring (100 rpm; Remi,

Mumbai, India) for 6 h, followed by addition of t-BOC-FA-

PEG-NH2 (4.60 mg/mL). The reaction was continued under

vigorous stirring upto 5 d and remaining un-conjugated

FA-PEG-NH2 was removed by dialysis (MWCO, 5–6 KDa,

HiMedia, Mumbai, India); the product was collected, dried

and characterized by FTIR spectroscopy (Scheme 3)

[1,24,29]. The FTIR spectroscopy was performed by KBr

pellet method after absorption of small amount of FA-

MWCNTs and FA-PEG-MWCNTs (Perkin Elmer 783,

Pyrogen 1000 Spectrophotometer, Shelton, CT) and scanned

in the range from 4000 to 500 cm�1 [15].

Physicochemical characterization

Briefly, DOX (30 mg) in triethylamine (TEA) solution was

mixed with FA-MWCNTs and FA-PEG-MWCNTs (10 mg)

dispersions in phosphate buffer solution (PBS; pH 7.4) with

continuous magnetic stirring (Remi, Mumbai, India) up to 48 h

at room temperature in dark condition. Addition of TEA is a

very crucial step which converts salt form of DOX into its free

base to facilitate loading of the drug. Then free, unbound DOX

was removed through dialysis membrane (MWCO 5–6 KDa,

HiMedia, Mumbai, India) against deionized water, and prod-

ucts denoted (DOX/FA-MWCNTS and DOX/FA-PEG-

MWCNTS) were lyophilized (Heto dry winner, Denamrk,

Germany) and the amount of DOX was determined by UV/

visible spectrophotometrically at wavelength lmax 480.0 nm

(UV/Vis, 1601, Shimadzu, Kyoto, Japan). Where, standard

DOX solution (100mg/mL) was prepared for quantitative

analysis and loading efficiency was calculated as follows: [22].

% Loading efficiency

¼Weight of loaded DOX�Weight of free DOX

Weight of loaded DOX� 100

Characterizations of engineered MWCNTs

The size and surface morphology were characterized by

Transmission Electron Microscopy (TEM; Morgani 268-D,

Fei, Holland) after drying on carbon-coated copper grid and

staining negatively by 1% PTA by metal shadowing tech-

nique. Similarly, the surface fracture was performed using

SEM (Philips XL 30 FEG FE SEM, New Jersey) of all

samples. The surface potential of functionalized MWCNTs

was determined by zeta potential (z) using Malvern Zetasizer

4, 10 (Malvern Instrument, Worcestershire, UK) [15,16].

pH-responsive in vitro release studies

The in vitro release of DOX from DOX/FA-MWCNTs and

DOX/FA-PEG-MWCNTs nanoconjugates was determined in

PBS (pH 7.4 and 5.3) as recipient media while maintaining

the physiological temperature 37� 0.5 �C throughout the

study using dialysis tube diffusion technique. The definite

amount (10 mL) of developed nanoconjugates (DOX/FA-

MWCNTs and DOX/FA-PEG-MWCNTs) free from any DOX

molecule was placed in the dialysis tube (MWCO 5–6 KDa,

HiMedia, Mumbai, India), hermetically tied at both ends and

immediately suspended in the receptor medium maintaining

Scheme 3. Synthesis of folic acid-PEG-MWCNTs nano-conjugate.


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strict sink conditions with constant stirring (100 RPM; Remi,

Mumbai, India). Samples were withdrawn at different time

points and determined by UV/Visible spectrophotometer at

lmax 480.0 nm (UV/Vis, Shimadzu 1601, Kyoto, Japan)

[1,15,19,24].

Hemolytic toxicity

Hemolytic toxicity was performed according to a previously

reported method with slight modifications [6,15,16,30].

Briefly, whole human blood was collected in Hi-clot vial

(HiMedia, Mumbai, India), centrifuged (3000 rpm; Remi,

Mumbai, India) for 15 min and red blood corpuscles (RBCs)

were separated out, washed, and resuspended in normal saline

solution (0.9% w/v) to obtain a suspension. The RBCs

suspension (1 mL) was mixed with the 0.9% w/v normal saline

(4.5 mL), free DOX, DOX/FA-MWCNTs and DOX/FA-PEG-

MWCNTs dispersions (0.5 mL) incubated to 60 min, and

allowed to interact. Then, appropriate dilutions have been

made and absorbance was taken at 480.0 nm (Shimadzu 1601,

Kyoto, Japan) considering 0.9% NaCl solution (normal saline)

and deionized water as nil and 100% hemolysis, respectively.

The percent hemolysis was calculated using the formula.

Hemolysis % ¼ ðAbs� Abs0ÞðAbs100 � Abs0Þ

� 100

where, Abs, Abs0 and Abs100 represent the absorbance of

samples, a solution of 0% hemolysis and a solution of 100%

hemolysis, respectively.

Cell culture studies

The MCF-7 (human breast cancer cell lines) cell line was

cultured in Dulbecco’s Modified Eagle Medium (DMEM;

HiMedia, Mumbai, India) supplemented with 10% heat-

inactivated fetal calf serum (FCS; HiMedia, Mumbai, India),

2 mM l-glutamine, 1% penicillin-streptomycin mixture

(Sigma, St Louis, MO) to discourage the growth of micro-

organism and maintained in a humidified atmosphere at 5%

CO2 at 37� 0.5 �C grown to 80% confluence in tissue culture

grade flasks and subcultured after discarding the used medium,

leaving the cells adhered to the bottom of the flask. These

adherent cells were further used for the determination of

cytotoxicity and induction of tumor in animals [1,31].

Cell viability assay

The cytotoxicity study was performed by the cleavage of

tetrazolium salt [{3-(4,5 dimethyl thiazole-2 yl)-2,5-diphenyl

tetrazolium bromide} (MTT)] to a blue formazan derivative

by living cells [6,9,15,23]. Exponentially grown cells were

seeded at 2� 105 cell/mL in different 96 well flat-bottomed

tissue culture plates (IIwaki, Glass, Tokyo, Japan). The cells

were separately treated with increasing concentration (1–

100mM) of DOX (Free DOX, DOX/FA-MWCNTs and DOX/

FA-PEG-MWCNTs) simultaneously under controlled envir-

onment for 24 h at 37� 0.5 �C in humidified atmosphere with

5% CO2. Subsequently, MTT solution (5 mg/mL) in PBS (pH

7.4) was added to each well and incubated for 4 h at

37� 0.5 �C, facilitating MTT to be reduced by viable cells

with the formation of purple formazan crystals. The formazan

crystals were dissolved in DMSO (100 mL) and the absorb-

ance was noted at 570 nm with the help of an ELISA plate

reader (Medispec Ins. Ltd, Mumbai, India) and the relative

(%) cell viability was calculated with the following formula:

Cell viability ð%Þ ¼ ½A�test

½A�control

� 100

where, [A]test is the absorbance of the test sample and

[A]control is the absorbance of control samples.

Hematological study

Hematological parameters such as erythrocytes (RBCs and

WBCs) and differential counts (monocytes, lymphocytes

and neutrophiles) were analyzed in female Sprague Dawley

strain rats having uniform weight and size. The four groups

comprised three rats in each group (n¼ 3) were divided

and free DOX, DOX/FA-MWCNTs, and DOX/FA-PEG-

MWCNTs dispersion containing 250 mg/mL equivalent to

DOX were administered intravenously into first, second and

third groups, respectively; and fourth group served as control;

all animals maintained on same regular diets upto 7 d. After

7 d blood samples were collected through retro-orbital plexus

from the animal eye and RBCs, WBCs and differential count

were determined [1,19].

Accelerated stability study

The DOX/FA-MWCNTs and DOX/FA-PEG-MWCNTs dis-

persions were stored in amber color and colorless vials

at 4� 0.5 �C, room temperature (25� 0.5 �C) and 35� 0.5 �Cup to 7 weeks in dark condition. The formulations were

analyzed initially and periodically every week upto 7 weeks

for any changes in color, precipitation, turbidity, crystalliza-

tion and consistency [6,15].

In vivo studies

In vivo experimental studies were carried out on Albino rats

(female Sprague Dawley strain rats, 8–9 weeks old, weighing

120� 10 g) in accordance with standard institutional guiding

principles duly approved by the Committee for the Purpose of

Control and Supervision of Experiments on Animals

(CPCSEA) of Dr. Hari Singh Gour University, Sagar (M.P.),

India. Animals were housed in plastic cages and access to

water ad libitum by maintaining hygienic and ventilated cage

and fed a special low-folate diet (casein 100 g/kg, soya protein

100 g/kg, soyabean 70 g/kg, cellulose 47.5 g/kg. cornstarch

170 g/kg, sucrose 450 g/kg, mineral mix 45, folate-free

vitamin mix 12.68 g/kg, choline 1.5 g/kg, BHT 0.014 g/kg,

L-cystine 3.3) and acclimatized at temperature 25� 2 �C and

50–60% relative humidity under natural light/dark condition

prior to in vivo study [24].

Anti-tumor activity

The in vivo antitumor activity of the developed nanoconju-

gates was evaluated in Female Sprague Dawley (SD) rats. The

tumor model was generated by cultured, serum-free MCF-7

cells (2� 106 cells in 50 mL) using hypodermic needle into the

subcutaneous portion in the right shoulder of animals and

routinely monitored for tumor development by palpating the


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injected area with index finger and thumb. The tumor bearing

animals were randomly divided into four treatment groups

(Control, free DOX, DOX/FA-MWCNTs and DOX/FA-PEG-

MWCNTs) for treatment with 5 mg/kg body weight dose

equivalent to DOX. At predetermined time intervals tumor

volume was measured by measuring its dimension (major and

minor axis) using electronic digital caliper and computed

according to the formula: Tumor volume (mm3)¼Width�(length/2)2 up to 30 d. All animals were accommodated in

a pathogen-free laboratory environment during the studies.

Survival of the tumor bearing animals was also monitored

in the separate group up to 30 d [32–34].

Biodistribution study

Animals were divided into three groups and sterilized free

DOX, DOX/FA-MWCNTs and DOX/FA-PEG-MWCNTs

dispersion in normal saline (0.9% w/v) were administered

intravenously through caudal tail vein route (equivalent dose

of DOX¼ 5.0 mg/kg body weight) into animals.

Group I: DOX HCL served as control (free DOX).

Group II: DOX loaded FA-MWCNTs dispersion (DOX/

FA-MWCNTs).

Group III: DOX loaded FA-MWCNTs dispersion (DOX/

FA-PEG-MWCNTs).

Each group was administered the same i. v. dose of free

DOX, DOX/FA-MWCNTs and DOX/FA-PEG-MWCNTs

dispersions, animals carefully sacrificed by decapitation

method at time intervals of 1, 6, 12 and 24 h. Subsequently,

the different organs such as liver, spleen, kidney, heart and

tumor were carefully separated out, washed, weighed and

stored under freezed condition till used. Then contents treated

with 100mL of 10% TCA solution, vortexed (Superfit

vortexer, India) for 2 min, methanol was added and cen-

trifuged (3000 rpm, 10 min; Remi, Mumbai, India) and

supernatant was decanted into another vial and evaporated

to dryness at 60� 2 �C [19,33,35]. The dried residue was

collected in vials and analyzed for DOX content by HPLC

(Shimadzu, C18, Kyoto, Japan) methods reported by Agrawal

et al. [19] and Reddy and Murthy [35]. In which a mixture of

buffer pH 4.0/acetonitrile/methanol (60:24:16; v/v/v) used as

mobile phase with flow rate 1.2 mL/min at pressure of 102/

101 bars with adjusting 20 min runtime and peak at 480.2 nm

was consider with its retention time (RT) and area.

In vivo pharmacokinetics after intravenousadministration

The pharmacokinetics of DOX in plasma was measured

from plasma-concentration curve in healthy female Sprague

Dawley rats after intravenous injections (5.0 mg/kg body

weight dose) of developed formulations (free DOX, DOX/FA-

MWCNTs and DOX/FA-PEG-MWCNTs). The animals were

acclimatized at room temperature by maintaining the relative

humidity (RH) 55–60% under natural light/dark condition

prior to studies. The blood samples were collected from the

retro-orbital plexus of rat eyes under the mild anesthesia

into the Hi-Anticlot blood collecting vials (HiMedia,

Mumbai, India) at predetermined data points

(0.25, 0.5, 1, 2, 3, 6, 12, 18, 24 and 48 h) and centrifuged to

separate the RBCs, and serum, and supernatant (serum) was

collected. Then 100 mL of 10% w/v trichloro acetic acid in

methanol was added and vortexed (Superfit, Mumbai, India)

and ultracentrifuged (Z36HK, HERMLE LaborTchnik

GmbH, Wehingen, Germany) to obtained the clear super-

natant. The clear supernatant was collected in HPLC vials

(HiMedia, Mumbai, India) and analyzed by HPLC method.

The pharmacokinetic parameters such as peak plasma

concentration (Cmax) were calculated from the plasma con-

centration curve. The area under the curve (AUC0�t), area

under the first moment curve (AUMC), mean residence time

(MRT), plasma half-life (t1/2), apparent volume of distribution

at steady state (Vss) and at terminal phase (Vz) and half value

duration (HVD) were also calculated [19,33].

Pharmacokinetic data analysis

The pharmacokinetic data analysis of plasma concentration

time profile was conducted using the Kinetica 5.0 PK/PD

analysis software (Thermo Fischer Scientific, West Palm

Beach, FL) followed by non-compartment analysis.

Statistical analysis

The results are expressed as mean� standard deviation (�SD)

(n¼ 3) and statistical analysis was performed with Graph Pad

Instat Software (Version 3.00, Graph Pad Software, San Diego,

CA) by one-way ANOVA followed by the Tukey–Kramer

test for multiple comparisons. A probability p� 0.05 was

considered while significant and p� 0.001 was considered as

extremely significant.

Results and discussion

In the context of targeted drug delivery, first generation

(pristine) CNTs are not suitable due to inherent aqueous

insolubility and presence of impurities. The procured pristine

MWCNTs from Sigma Aldrich Pvt. Ltd. (St. Louis, MO) were

purified in a microwave oven and subsequently strong acid

treatment (H2SO4:HNO3) followed by NH4OH and H2O2 to

remove any metallic or amorphous impurities and also to

generate the carboxylic acid (–COOH) groups on to the

surfaces of MWCNTs.

The direct acid–base titration analysis was performed to

determine the total acidic functional groups present on

oxidized MWCNTS by Boehm titration method using

Table 1. Quantitative analysis of total functional group by Boehm titration on oxidized MWCNTs.

Samples TreatmentTotal functional

group (mmol–1/g)Carboxylic group

(mmol–1/g) Lengths (nm)

Pristine MWCNTs Microwave treated 10.2� 0.82 4.22� 0.55 960� 0.57Microwave-treated MWCNTs HNO3:H2SO4 treated 18.5� 0.20 10� 0.14 400� 0.85Carboxylated-MWCNTs H2O2/NaOH treated 24� 0.72 16� 0.33 80� 0.08

Values represent mean� SD (n¼ 3).


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0.05 N NaOH and observation are presented in Table 1 [26].

Microwave-treated MWCNTs gave 10.2� 0.82 mmol–1/g and

4.22� 0.55 mmol–1/g total functional groups and carboxylic

groups, respectively, whereas HNO3:H2SO4 and H2O2:NH4OH

treated MWCNTs gave 18.5� 0.20, 10� 0.14 mmol–1/g and

24� 0.72 and 16� 0.33 mmol–1/g total functional groups

and carboxylic groups, respectively. The determination of

free acidic functional groups by Boehm titration is based on the

fact that the 0.05 N NaOH neutralizes carboxylic, phenolic-

and lactone groups present on the oxidized MWCNTS,

whereas 0.05 N NaHCO3 neutralizes only the carboxylic acid

functional group [25–27]. Our results are in line with the

previously published reports [25–27], but MWCNTs treatment

by ammonium hydroxide (NH4OH) and hydrogen peroxide

(H2O2) (50:50) followed by initial microwave oven treatment is

a debut study. This combined treatment approach may

drastically increase the total concentration of acidic functional

groups (phenolic, lactone and carboxylic functional groups) on

nanotubes. Further, these ox-MWCNTs were subjected to

acylation and amidation process as reported previously [13].

FA was conjugated either without spacer or with PEG-bis-

4000 amine as spacer to NH2 terminated and carboxylated

MWCNTs, respectively. The FTIR spectra of FA-MWCNTs

and FA-PEG-MWCNTs are shown in Figure 1(A) and (B).

Figure 1(A) shows the peak of aromatic C–H bending

at 832 cm�1, esters unconjugated C¼O at 1243.2 cm�1,

aromatic C¼C bending and stretching at 1637.2 cm�1

suggesting the attachment of folic acid to the MWCNTs,

which contained aromatic rings. However, Figure 1(B) shows

the prominent peaks at 3436.7 cm�1, 2916.0 cm�1,

1652.0 cm�1, 1437.2 cm�1, 1315.1 cm�1, a strong and sharp

peak at 1025.0 cm�1 of C-O stretch ether linkage wherein

peak of C–O stretch of ether linkage was found to be strong

and sharp at 1025.0 cm�1 due to the polyether backbone of

PEG and remaining peaks of aromatic compounds indicated

the presence of folic acid (Supporting information Table S1).

The morphology and size of carboxylated and DOX/FA-

PEG-MWCNTs were characterized by Transmission Electron

Microscope (TEM) and are shown in Figure 2(A) and (B).

The TEM observations clearly depict that the CNTs are

tubular in shape with open ends and in nanometric size range.

Moreover the images suggest that there was no change in their

tubular structure even after conjugation of FA and PEG.

The surface charge of the pristine, ox�, DOX/FA-

MWCNTs and DOX/FA-PEG-MWCNTs nanoconjugates

was determined from their electrophoresis mobility at

acidic, neutral and alkaline pH by zeta potential (z) according

to the Helmholtz-Smoluchowski equation. The ox-MWCNTs

depicted the slightly negative zeta potential (�10 mV), which

could be due to the generation of acidic functional groups

during the oxidation. The free COOH– was ionized at alkaline

pH and thus negative zeta potential was observed. The

DOX/FA-PEG-MWCNTs nanoconjugate showed positive

zeta potential of þ5.0, þ3.8 and þ4.8 at acidic, neutral and

alkaline medium, respectively [13]. PEG being non-ionic

could decrease the zeta potential of the formulations due to its

presence on the surface of MWCNTs.

The anthracycline antibiotic DOX was physically loaded

by simple mixing in DOX/FA-PEG-MWCNTs and DOX/FA-

MWCNTs nanoconjugates as evidenced by reddish color.

The % loading efficiency (% LE) was calculated in PBS

(pH 7.4) at 480.0 nm using UV/Vis spectrophotometer

(Shimadzu, 1601, Kyoto, Japan) and found to be

90.2� 0.22 and 92� 0.92 (n¼ 3) for DOX/FA-MWCNTs

and DOX/FA-PEG-MWCNTs, respectively. UV/Vis spectro-

photometry data of MWCNTs formulations suggest that

DOX can easily adsorb on to the surface of MWCNTs

probably through strong p–p stacking interactions of quinine

part of DOX and CNTs and accordingly greatest loading was

found to be 92� 0.92 in DOX/FA-PEG-MWCNTs nano-

conjugte. Further, endohedral entrapment into the interior

cavity of nanotubes structure leading to higher entrapment is

also expected, however measurement technique is not

investigated yet. The observed data could possibly be

ascribed to loading of cationic DOX in and around PEG

based micro domains also via p–p stacking at pH 7.4

Figure 1. Fourier transform infra-red (FTIR)spectra of (A) FA-MWCNTs and(B) FA-PEG-MWCNTs nano-conjugates.


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(Scheme 4). The general loading efficiency (LE) of DOX in

different dispersion followed the order:

DOX=FA�MWCNTs! DOX=FA� PEG�MWCNTs

ðLeast crowding ends! greatest crowding endsÞ

The high loading efficiency of engineered nanotubes

makes it a better carrier with better stability of DOX complex

at normal pH and sustained release in acidic microenviron-

ments (lower pH). The sustained release behaviour of the

drug from the nanotubes at acidic pH is an important factor

in tumor specific targeted drug delivery. Recently, Huang

et al. reported approximately 91% DOX loading efficiency in

functionalized CNTs [22].

The in vitro release of DOX from DOX/FA-MWCNTs and

DOX/FA-PEG-MWCNTs dispersions was performed at pH

(7.4 and 5.3) through a dialysis membrane (MWCO 5–6 KDa,

HiMedia, Mumbai, India) at 37� 0.5 �C. The in vitro release

suggests sustained release at lysosomal pH (5.3) ascribed to

the greater hydrophilicity, and by cleavage of the interactions

between the DOX molecules and engineered CNTs. On

comparing the release of DOX/FA-PEG-MWCNTs to other

formulations, effect of PEG modification could be appre-

ciated and the order of release was as follows:

DOX=FA� PEG�MWCNTs! DOX=FA�MWCNTs

ðSustained Release! Faster ReleaseÞ

The initial burst release achieved due to diffusion or the

adsorbed DOX followed by the sustained released may

possibly suggest lesser exposure of loaded drug to external

microenvironment that could be due to greater steric

hindrance on ends and side walls, resulting in sustained

release pattern of the loaded drug following initial faster

release as shown in Figure 3(A) and (B). The DOX release

data best fits into the Higuchian release kinetic. Our in vitro

release data of DOX are in line with the previous reports

[8,9,22]. The in vitro DOX release pattern depends on several

factors like pH, surface charge characteristics, degradation

rate, particle size, rate of hydration and dehydration and

interaction force of DOX binding to the surface of nanotubes.

The initial fast release was attributed to the rapid swelling

of DOX associated with diffusion, another reason being the

chemical interaction through hydrogen bonding between

DOX and nanotubes surface leading to sustain release pattern

[22]. Zhang et al. similarly reported pH-triggered drug release

response from the modified nanotubes under normal physio-

logical conditions and release at reduced pH typical of micro-

environments of intracellular lysosomes or endosome or

cancerous tissue [23]. It is clearly depicted that engineered

CNTs may show the pH-responsive DOX release.

The % hemolysis data of free DOX (15.7� 0.5), pristine

MWCNTs (18.0� 0.5), DOX/FA-MWCNTs (12.5� 0.5)

and DOX/FA-PEG-MWCNTs (9.0� 0.5) were compared.

Pristine MWCNTs shows highest (18.0� 0.5) while

Figure 2. Photomicrographs of (A and C)carboxylated MWCNTs and (B and D)FA-PEG-MWCNTs.


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Scheme 4. Development of DOX loaded FAconjugated PEG-MWCNTs nanoformulation. Folic acid

PEG-bis amine

Engineered CNTs

OMe O

O

OOH

OH

OHC

O

O

OCH3

CH2

H

OH

Figure 3. Cumulative DOX release (%) from the DOX/FA-MWCNTs and DOX/FA-PEG-MWCNTs nanoconjugates at 37� 0.5 �C in phosphate buffersolution (pH¼ 5.3 and 7.4). Where, (A) represents the DOX released upto 200 h and (B) 12 h (n¼ 3).


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DOX/FA-PEG-MWCNTs shows minimum (9.0� 0.5) hemo-

lytic toxicity. The hemolytic toxicity of pristine MWCNTs

was enough to limit its use as drug delivery system. Pristine

MWCNTs shows (18.0� 0.5) highest hemolytic toxicity due

to the presence of some metallic impurities; however on

functionalization it was reduced to 12.5� 0.5 in case of DOX/

FA-MWCNTs. Recently, Sachar and Saxena reported that

pristine and acid-treated CNTs were toxic to mouse blood-

derived erythrocytes in vitro as well as in vivo [36]. It is well

reported that the pristine MWCNTs (first generation CNTs)

are not suitable for drug delivery, but their compatibility may

be improved through functionalization. However, our hemo-

lytic toxicity results clearly suggest that functionalization

or PEGylation considerably reduced the hemolysis upto

9.0� 0.5 possibly due to non availability of any free

positively charged functional moieties. PEGylation make

nanotubes more biocompatible module in comparison to

pristine and acid-treated ones.

Hematological parameters (RBCs, WBCs and differential

counts) were determined to assess the relative effect of

MWCNTs formulations (DOX/FA-MWCNTs and DOX/FA-

PEG-MWCNTs) compared to free DOX. Blood samples were

analyzed for RBCs, WBCs and differential counts (Table 2).

RBCs and WBCs counts of DOX/FA-PEG-MWCNTs for-

mulations were calculated to be 9.0� 0.3� 106/mL and

10.6� 0.3� 103/mL, respectively. Differential counts were

found to be very similar with control group. These data

clearly suggest that the RBCs count (9.0� 106/mL) is very

similar to control group (9.2� 106/mL) in case of DOX/FA-

PEG-MWCNTs-treated animals, which was decreased sig-

nificantly in case of FA-MWCNTs (7.2� 106/mL). Similarly,

WBC counts of free DOX (9.6� 103/mL) and DOX/FA-

MWCNTs (10.4� 103/mL) were slightly increased compared

to the normal values. In addition, in DOX/FA-PEG-MWCNTs

formulation (10.6� 103/mL) relatively increases the WBC

count as compared to DOX/FA-MWCNTs, closer to the

control group (10.8� 103/mL). However differential count i.e.

leucocytes, monocytes and lymphocytes was found almost

similar in case of DOX/FA-PEG-MWCNTs nanoconjugates

to normal values. Hematological study in case of MWCNTs

(free DOX, DOX/FA-MWCNTs and DOX/FA-PEG-

MWCNTs) is a debut report from our group and the results

are in accordance with earlier report on the dendrimeric

formulations from our own Laboratory [1,19]. Recently,

Meng et al. indicated that the DPX-FA-CHI-SWCNTs have

a lower systemic toxicity as compared to free DOX, at

equivalent doses. They also suggested that the FA-CHI-

SWCNTs are enriched in the tumorous cells and reduce the

toxicity to liver [20].

The MTT assay was performed to measure the extent of

cell viability of free DOX, DOX/FA-MWCNTs and DOX/FA-

PEG-MWCNTs nanoconjugates to assess the potential antic-

ancer activity on cultured MCF-7 (human breast cancer) cell

line. Cell viability was determined by performing MTT assay

after treatment with MWCNTs formulations at 24 h with

increasing concentration ranges from 0.001 to 100 mM. The

MTT assay data clearly suggest increased cytotoxicity by

reducing the viability of cancerous cells due to apoptosis

by intercalating DOX with DNA in case of DOX/FA-PEG-

MWCNTs formulation as compared to free DOX. The

increased cytotoxic response may possibly be due to caveolae

mediated endocytosis, and specific uptake by cancerous cells

causing dose-dependent cytotoxic response. Our cell cytotox-

icity assay results are similar to Lu et al. [37]. The IC50 value

of DOX-FA-MN-MWCNTs was found approximately 15 mg/

mL as compared to free DOX (50mg/mL) suggesting efficient

delivery of DOX to the cell nucleus area due to the higher

internalization through receptor-binding endocytosis mech-

anism. It conform the biocompatibility of DOX-FA-MN-

MWCNTs in a broad concentration range on both normal cell

line (3T3) and U 87 cells [37]. Caveolae are pinocytic vesicles

(50–80 nm) coated with a self-assembly of caveolin, an

integral membrane protein with high affinity for cholesterol

[38]. DOX/FA-PEG-MWCNTs conjugate was found to be

more cytotoxic as compared to DOX/FA-MWCNTs and free

DOX on MCF-7 cell line with reduced half-maximum

inhibitor concentration (IC50) value as shown in Figure 4.

Liu et al. reported the IC50 of DOX loaded PL-SWCNTs

approximately 8 mM, by transporting inside cancerous cells as

nanotubes transporters via endocytosis [9]. Recently, Gu et al.

[8] reported the IC50 value for SWCNTs-HBA-DOX and

SWCNTs-DOX in HePG2 cells to be 4.8 and 7.4mM,

respectively [8].

The pharmacokinetics of doxorubicin, DOX/FA-MWCNTs

and DOX/FA-PEG-MWCNTs were investigated in the blood

samples using HPLC technique (Shimadzu, C18, Kyoto,

Japan). Figure 5 represents the plasma concentration time

profile after single i.v. administration of free DOX, DOX/FA-

MWCNTs and DOX/FA-PEG-MWCNTs in female Sprague

Dawley rats. The detailed pharmacokinetic parameters were

calculated from the blood-plasma concentration curve such as

mean residence time (MRT), area under the curve (AUC),

area under the mean curve (AUMC), half value duration

(HVD), clearance, volume of distribution at steady state (Vss)

which are summarized in Table 3. The area under the

curve (AUC0–1) and area under the first moment curve

(AUMC0–1) were calculated to be 9.5717, 25.9492,

60.1756 and 23.5777, 191.828, 1261.38 for free DOX,

Table 2. Hematological study of free DOX and DOX loaded MWCNTs formulations.

Differential count (�103/mL)

Formulations RBC count (�106/mL) WBC count (�103/mL) Monocytes Lymphocytes Neutrophils

Control 9.2� 0.4 10.8� 0.4 1.4� 0.6 7.9� 0.4 1.6� 0.3Free DOX 6.4� 0.3 9.6� 0.4 0.9� 0.3 6.1� 0.4 1.0� 0.3DOX/FA-MWCNTs 7.2� 0.4 10.4� 0.3 1.7� 0.5 7.0� 0.3 1.2� 0.3DOX/FA-PEG-MWCNTs 9.0� 0.3 10.6� 0.3 1.1� 0.3 7.8� 0.8 1.4� 0.5

Values represent mean� SD (n¼ 3).


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DOX/FA-MWCNTs and DOX/FA-PEG-MWCNTs, respect-

ively. The AUC0–1) and AUMC(0–1) of DOX/FA-PEG-

MWCNTs were approximately 6-fold and 53-fold higher as

compared to free DOX, respectively. The elimination half-life

(t1/2) of DOX/FA-PEG-MWCNTs, DOX/FA-MWCNTs and

free DOX was found to be 14.956, 4.8432 and 1.8846 while

MRT was found to be 20.9616, 7.3924 and 2.4632, respect-

ively. In contrast with t1/2 of DOX/FA-PEG-MWCNTs

(14.956) were 3 and 8 times (p50.005), while MRT was 3

and 8 times longer as compared to DOX/FA-MWCNTs and

free DOX, respectively. The prolonged t1/2 clearly depicted

the DOX/FA-PEG-MWCNTs in the systemic circulation.

The obtained results are ascribed to biocompatibility of

engineered nanotubes upon PEGylation to reside it for longer

time inside the body. Our pharmacokinetics data clearly

suggest the improved bioavailability of DOX as compared to

free DOX, which make nanotubes a most promising alterna-

tive, smart nanobiomedicine in targeted drug delivery. Liu

et al. reported long-term fate of PEG functionalized SWCNTs

by intravenous administration in animals and found longest

blood circulation upto 1 d and near-complete clearance of

SWCNTs from the main organs approximately in 2 months

[38]. Cherukari et al. reported the low acute toxicity and

long circulation of disaggregated SWCNTs by low dose of

nanotubes [39]. The intrinsic stability and structural flexibil-

ity of surface engineered CNTs may enhances the circulation

time as well as the bioavailability of drug molecules [40,41].

Huang et al. only described a new family of folate-decorated

and carbon nanotubes mediated delivery system encapsulating

doxorubicin for controlled release [22].

Recently, Jain and co-investigators reported Amphotericin

B loaded mannosylated MWCNTs (AmBitubes) was released

in a controlled manner at different pH environment with

increased cell uptake and higher disposition in macrophages

rich organs using J774 cell line indicating the site-specific

drug delivery. Authors suggested that the AmBitubes could

be employed as efficient nano-carrier for anti-leishmanial

therapy [16].

The anti-tumor activity of free DOX, DOX/FA-MWCNTs

and DOX/FA-PEG-MWCNTs nanoconjugates was studied in

tumor bearing female Sprague Dawley strain rats and tumor

growth inhibition rate in terms of mean tumor volume (mm3)

as represented in Figure 6. The results suggested that DOX/

FA-PEG-MWCNTs nanoconjugate reduced extremely signifi-

cant tumor volume compared to free DOX (p50.001) after

tumor implantation. Survival of tumor bearing SD rats after

treatments with the nanotubes conjugates are represented in

Kaplan Meier survival curves, which suggested that the

median survival time with DOX/FA-PEG-MWCNTs-treated

animals (30 d) significantly (p� 0.001) as compared to free

DOX and control group due to their biocompatible and long

circulatory nature. Liu et al., 2008, reported that no obvious

toxicity or negative health effects observed over 3 months by

injected i.v. PEGylated SWCNTs and no mortality or loss of

body weight were seen in any mice [33]. Recently, Ji et al.

developed a new type of targeted drug delivery system

(TDDS) using chitosan modified SWCNTs for controlled

release of DOX by constructing folic acid (FA) modified

chitosan encapsulating doxorubicin (DOX/FA/CHI/SWCNTs)

wherein FA was bound to the outer CHI layer and effectively

Table 3. Pharmacokinetic parameters of free DOX, DOX/FA- MWCNTs and DOX/FA-PEG-MWCNTs dispersion.

ParametersCmax

(mg/mL)HVD(h)

AUC(0–t)

(mg.h/mL)AUC(0–1)

(mg.h/mL)AUMC(0–t)

(mg. hr2/mL)AUMC(0–1)

(mg.h2/mL)t1/2

(h)MRT(h) Clz Vz Vss

Free DOX 6.11 0.3564 9.0445 9.5717 17.926 23.5777 1.8846 2.4632 10.4474 28.4065 25.7346DOX/FA-MWCNTs 6.06 0.8329 25.0625 25.9492 164.351 191.828 4.8432 7.3924 3.8536 26.9279 28.4881DOX/FA-PEG-MWCNTs 6.50 1.7482 53.6275 60.1756 805.781 1261.38 14.956 20.9616 1.6618 35.8567 34.8341

Probability p50.001; standard deviation55%.Cmax¼ peak plasma concentration; Tmax¼ time taken to reach Cmax; t1/2¼ elimination half life; MRT¼mean residence time; AUC(0–1)¼ area under

plasma drug concentration over time curve; HVD¼ half value duration; Clz¼ clearance; Vz¼Volume of distribution; Vss¼Volume of distributionat steady state.

Mean� SD (n¼ 3).

Figure 4. Percent cell viability of MCF-7 cell after treated with freeDOX, DOX/FA-MWCNTs and DOX/FA-PEG-MWCNTs at 24 h (n¼ 3).

Figure 5. Serum concentration of DOX obtained from free DOX, DOX/FA-MWCNTs and DOX/FA-PEG-MWCNTs at different data points.Mean� SD (n¼ 6; p� 0.001).


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depressed the growth of liver cancer in nude mice using the

Hepatocellular Carcinoma Cancer Cell line (HCC SMMC-

7721). The DOX/FA/CHI/SWCNTs exhibits superior pharma-

ceutical efficiency as compared to free DOX on HCC SMMC-

7721 cells. No significant difference in the measurement of

relative tumor volumes was found, thus suggesting further

research to explore the pharmaceutical targeting efficiency to

kill the cancer cell more effectively [32].

Organ distribution study was undertaken to assess the

amount of DOX that reaches in to different organs such as

liver, spleen, kidney, lungs and tumor on Sprague Dawley rats

(Figure 7). In case of DOX/FA-PEG-MWCNTs formulation

Figure 6. Tumor regression analysis afterintravenous administered of free DOX, DOX/FA-MWCNTs and DOX/FA-PEG-MWCNTsnanoconjugates (dose 5 mg/kg) as shown inthe schematic (right). The DOX/FA-PEG-MWCNTs-treated group shows significant(p� 0.05) suppression of tumor growthcompared with the other groups (n¼ 3).In Kaplan–Meier survival curve analysis oftumor bearing female SD, treated with freeDOX, DOX/FA-MWCNTs and DOX/FA-PEG-MWCNTs nanoconjugates at dose 5 mg/kg equivalent to DOX. Each data pointrepresents mean� SEM (n¼ 3).

Figure 7. Biodistribution patterns of free DOX, DOX/FA-MWCNTs and DOX/FA-PEG-MWCNTs nanoconjugates in different tissues and tumor.Values represent as mean� SD (n¼ 3). * Significant; ** More significant; *** Extremely significant.


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high uptake of DOX was observed in tumor, liver, kidney and

spleen at time intervals upto 24 h. The high levels of DOX

was found after 1 h of administered dose in liver, tumor and

kidney and rapid decline in the overall formulation, thereafter

indicating that most of MWCNTs were eliminated through the

renal excretion route. The amount of DOX was found to be

remarkably increased at tumor site with time in case of DOX/

FA-PEG-MWCNTs formulation due to receptor-mediated

endocytosis (RME) mechanism. Our results are in accordance

with previous Leading Opinion by Meng et al. [20] in

targeting doxorubicin to tumors using raw and treated carbon

nanotubes. In vitro drug release data suggested initial rapid

release followed by gradual slow release, similar pattern was

observed in in vivo study. The variation in quantity of drug

estimated in vivo is due to biological effects on the bioactive

that predominate its biodistribution pattern. Biodistribution

study data suggested that the DOX/FA-PEG-MWCNTs

nanoconjugate could deliver drug selectively at the tumor

cells.

Recently, our laboratory developed and characterized

the dexamethasone conjugated MWCNTs for controlled

DOX delivery with reduced toxicity using ‘‘A-549’’

lung epithelial cancer cell line where the DOX loaded

DEX-MWCNTs showed less hemolytic and more cytotoxic

as compared to free DOX [15]. Our ex vivo and in vivo

results are in accordance with the previous published

reports [42,43].

Stability of the formulations (DOX, DOX/FA-MWCNTs

and DOX/FA-PEG-MWCNTs) was studied at different con-

ditions of temperature (4� 0.5 �C, 25� 0.5� �C and 50 �C)

after keeping in dark (amber color bottle) and light (colorless

vials) up to 7 weeks [15]. The formulations were found to be

most stable in dark at 4� 0.5 �C (Table 4). Stability of the

DOX/FA-PEG-MWCNTs formulations was observed at dif-

ferent conditions of temperature (4� 0.5 �C, 25� 0.5� �Cand 50 �C) after keeping in dark (amber color bottle) and light

(colorless vials) and evaluated every week upto 7 weeks.

Among all formulations DOX/FA-PEG-MWCNTs was found

to be most stable in dark at RT. In terms of stability profile

f-MWCNTs could possibly present themselves as a most

stable system due to p–p stacking interaction in all tempera-

ture ranges and environment required for biological applica-

tions. Thus we conclude that the DOX/FA-PEG-MWCNTs

formulation is more stable than other MWCNTs formulation

at 4� 0.5 �C, in dark suggesting that the developed nanotubes

formulation may be suitably stored in amber color bottle or

vials at a cool place.

Conclusions

To best of our knowledge, this is the complete study

report with evidence of improved selective treatment of

cancer using DOX/FA-PEG-MWCNTs formulations most

suitable as controlled and targeted drug delivery. The results

suggested that the DOX/FA-PEG-MWCNTs formulation

showed the better targeting response using MCF-7 breast

cancer cell line through cavaeolin-mediated endocytosis

mechanism. From the outcomes of our present research

studies, it can be concluded that the DOX loaded surface

modified MWCNTs showed better in vitro, ex vivo and

biocompatibility profile as compared to other nano-carriers

depicting higher loading (92.0� 0.92) and sustained release

profile especially at acidic microenvironment corresponding

to conditions existing at cancerous tissues/sites. The improved

kinetics of nanotubes formulation upon PEGylation such

as MRT, t1/2, HVD and AUMC(0–1) 20.9616, 14.956, 1.7582

and 1261.38, respectively for DOX/FA-PEG-MWCNTs as

compared to free DOX may be considered significantly

effective for intravenous administration. However, folate

conjugation makes it more targetable approach precluding

the non-target sites such as existing nanoparticles, liposomes

and dendrimers [1,24,44–46]. Thus optimal therapeutic

response and improved bioavailability may be achieved with

minimized side effects associated with the carrier and

anticancer drug.

Acknowledgements

The authors gratefully acknowledge M/s Sun Pharmaceutical

Advanced Research Center (SPARC), Vadodara, India, for

a gift sample of Doxorubicin hydrochloride, All India

Institute of Medical Sciences (AIIMS), New Delhi, India,

for Transmission Electron Microscopy (TEM) and Scanning

Electron Microscopy (SEM). One of the authors (Neelesh

Kumar Mehra) is thankful to the University Grants

Commission (UGC), New Delhi, India for providing the

Junior Research Fellowship (JRF) during the tenure of these

studies.

Declaration of interest

The authors report no conflict of interest.

Table 4. Accelerated stability testing of nanotubes formulations.

Dark (�C) Light (�C)

Parameters Formulations 4� 0.5 25� 0.5 35� 0.5 4� 0.5 25� 0.5 35� 0.5

Turbidity DOX/FA-MWCNTs – – þþ þ þþ þþþDOX/FA-PEG-MWCNTs – – þþ þ þþ þþþ

Precipitation DOX/FA-MWCNTs – – þ – þ þþDOX/FA-PEG-MWCNTs – – þ – þ þþ

Change in colour DOX/FA-MWCNTs – þ þ – þ þþDOX/FA-PEG-MWCNTs – þ þ – þ þþ

Crystallization DOX/FA-MWCNTs – – þ – þ þDOX/FA-PEG-MWCNTs – – þ – þ þ

Change in consistency DOX/FA-MWCNTs – þ þþ – þ þþDOX/FA-PEG-MWCNTs – þ þþ – þ þþ

(–) no change; (þ) small change; (þþ) considerable change; (þþþ) enough change.


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9 Mehra and Jain JDT

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