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

J Drug Target, Early Online: 1–12! 2013 Informa UK Ltd. DOI: 10.3109/1061186X.2013.778264

ORIGINAL ARTICLE

Gemcitabine-loaded smart carbon nanotubes for effective targetingto cancer cells

Ravendra Singh, Neelesh Kumar Mehra, Vikas Jain, and Narendra Kumar Jain

Department of Pharmaceutical Sciences, Pharmaceutics Research Laboratory, Dr H. S. Gour University, Sagar, Madhya Pradesh, India

Abstract

Carbon nanotubes (CNTs) are the three-dimensional sp2 hybridized nano-containers that haveattracted considerable interest in drug delivery by offering potential advantages such asbiocompatibility, non-immunogenicity, high loading efficiency, intrinsic stability and lowtoxicities. The aim of the present investigation was to assess the potential of gemcitabine-loaded folic acid (FA) conjugated multi-walled CNTs (GEM/FA-NT) for targeting to breast cancercells. Pristine MWCNTs was functionalized by FA followed by carboxylation, acylation andamidation and characterized by electron microscopy, FT-IR spectroscopy, X-ray diffraction,entrapment efficiency, cytotoxicity and in vivo studies. FDA-approved GEM was loaded to thepurified (GEM-NT) and GEM/FA-NT, and % entrapment efficiency was found to beapproximately 71.60� 0.25 and 79.60� 0.45, respectively. The developed formulation GEM/FA-NT was found to have significantly less hemolytic toxicity (8.23� 0.65) as compared to freeGEM (17.34� 0.56). The in vitro release was found to be in sustained pattern at the lysosomalpH, which depicts more cytotoxic response on human breast cancer cell line (MCF-7). It may beinterpreted that the GEM/FA-NT formulation is capable to carry drug and deliver it selectively atthe tumor site while minimizing side effects and thus holds promise in chemotherapy.

Keywords

Carbon nanotubes, folic acid,functionalization, gemcitabinehydrochloride, human breastcancer cells, pharmacokinetics

History

Received 18 December 2012Revised 12 February 2013Accepted 18 February 2013Published online 2 2 2

Introduction

In the current scenario, alleviation of diabetes, acquired

immune deficiency syndrome, tuberculosis and cancer are

still the foremost challenging task over worldwide. World

Health Organization (WHO) reported that cancer accounted

approximately 7.9 million deaths in the year 2007 and 458 000

death occurred due to breast cancer. Breast cancer is the most

common form of neoplasia in women accounting for almost a

third of all new cases of women’s cancer. In 2007, according to

the Centre for Control of Disease and Prevention, Division of

Cancer Prevention and Control, Atlanta reported that 40 598

women died only in the US and 202 964 diagnosed with breast

cancer. Deaths due to breast cancer are more prevalent in the

developed and lowest in less-developed countries around the

world. Recently in 2011, Canadian Cancer Society reported

that an estimated 23 400 women were diagnosed and 5100 died

from breast cancer, moreover approximately 190 men were also

diagnosed and 55 died from breast cancer [1–3]. Although

significant progress has been made in the field of cancer

therapy, yet we still strongly need reliable and complete cure of

cancer. Over the past decades medical and pharmaceutical

sciences have witnessed noteworthy advancement in the field

of drug development technologies in the cancer treatment [4–

7]. In the current scenario carbon nanotubes (CNTs) represent a

very promising, alternative, safe and efficacious delivery

system in the targeted drug delivery [8–18], due to their unique

physicochemical properties such as biocompatibility, non-

immunogenicity, high loading efficiency, aspect ratio, struc-

tural flexibility, non-cytotoxic and non-biodegradable nature

[9,11–16,19]. CNTs offer a new perception in nano-medicines,

however, functionalized CNTs can be degraded in the presence

of natural oxidative enzyme (horseradish peroxidise) [20,21].

CNTs are sp2 hybridized three-dimensional carbon nano-

material envisioned as seamless tubular cylinders rolled-up

graphite planes with open ends. However, CNTs are able to

carry the different cargo molecules and deliver them to their

requisite sites to make them as potential delivery nano-vectors

like proteins, nucleic acids, peptides and drugs [paclitaxel

(PTX), doxorubicin (DOX), cisplatin, methotrexate (MTX),

amphotericin B (AmB), epirubicin and daunorubicin (Dau)] in

biomedical applications [9,13–16,18,22–28].

Gemcitabine [20,20-difluoro-20-deoxycytidine, (dFdc)] is a

low molecular weight, cell cycle-dependent (S-phase) deox-

ycytidine analog of anti-metabolite, inhibiting cellular DNA

synthesis. It is a Food and Drug Administration (FDA)

approved frontline chemotherapeutic agent for the treatment

of metastatic pancreatic cancers [29–31].

Address for correspondence: Prof. N. K. Jain, Department ofPharmaceutical Sciences, Pharmaceutics Research Laboratory,Dr H. S. Gour University, Sagar 470 003, Madhya Pradesh, India. Tel/fax: +91-7582-265055. E-mail: jnarendr@yahoo.co.in; neelesh81mph@gmail.com

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In the present study, folic acid (FA) was selected as a

targeting agent due to its high cell surface receptor binding-

affinity, which is over-expressed at cancerous sites predom-

inantly on breast, kidney, brain and lungs [32]. Folate receptor

(FRs), are 38 kDa glycosylphosphotidylinositol-linked mem-

brane glycoproteins that exist in three major forms: FR-a,

b and g to retain their binding affinity towards receptors.

FRs are generally located in caveolae that participates in the

cellular accumulation of folate via the protocytosis process

[32,33].

So, the present study was aimed at development, charac-

terization and assessment of the possible effectiveness and

prospective of a novel nanocarrier, i.e. surface-engineered

CNTs, gemcitabine loaded FA conjugated multi-walled CNTs

(GEM/FA-NT) for efficient selective delivery at tumor sites

with improved bioavailability of the drug. The developed

formulation was characterized for an entrapment efficiency,

in vitro release, human erythrocytes interaction, cytotoxicity

against human breast cancer (MCF-7) cells and in vivo

studies.

Materials and methods

Materials

Multi-walled CNTs (MWCNTs) produced by chemical vapor

deposition with purity (495 wt%), outer diameter� length

20–30 nm� 10–30 mm and metal content (0.7%) was obtained

as a gift sample from M/s Cheap Tubes Inc., Brattleboro,

USA. N-hydroxysccinimide (NHS) was purchased from

Sigma Aldrich, Munich, Germany and N,N-dicyclohexyl

carbodiimide (DCC) was purchased from HiMedia,

Mumbai, India. Dialysis membrane, molecular weight cut-

off (MWCO), 12–14 kDa, was purchased from HiMedia

Laboratories Pvt. Ltd. Poly-tetrafluoroethylene (PTFE) filters

(0.22mm pore size) were purchased from Hangzhou Anow

Microfiltration Co. Ltd, Hangzhou, China. All the reagents

and solvents were of analytical grade and deionized water was

used during experiments.

Purification of pristine MWCNTs

The pristine MWCNTs (200 mg) and concentrated hydro-

chloric acid (HCl) were magnetically agitated on a magnetic

stirrer (Remi, Mumbai, India) at 100 rpm for 5 h at room

temperature (RT), filtered through PTFE filter (Hangzhou

Anow Microfiltration Co. Ltd.) and dried in vacuum for 3 h

(Jyoti Scientific Industries, Gwalior, Madhya Pradesh, India)

[11,12,34].

Functionalization of purified MWCNTs

Generation of the carboxylic functional group. Carboxylic

acid (–COOH) groups were generated on the surfaces and

ends of the MWCNTs by oxidation in a round-bottom flask,

equipped with a reflux condenser, mechanical stirrer with

thermometer. Briefly, purified MWCNTs (100 mg) was

immersed in the concentrated HNO3:H2SO4 in 1:3 ratio

mixture with ultrasonication (Soniweld, Mumbai,

Maharashtra, India) for 3 h at 110� 2 �C, washed with

excess deionized water (100 times dilution) to reach the

neutral pH value, finally washed with methanol and dried

in a vacuum oven (Jyoti Scientific Industries) at 60� 0.5 �Cfor 24 h [11,12,24,35]. Then acylation and amidation of

carboxylated MWCNTs were done as previous reported

method [35].

Conjugation of FA to amine terminated MWCNTs

The NHS-FA-f-MOC was synthesized using (dicyclo hexyl

carbodiimide) DCC and (n-hydrosuccinimide) NHS in

equimolar concentration (1:1) for amine protection as previ-

ously reported method by our laboratory in case of dendrimer

[2,4,33] and schematic representation of f-MOC-FA and

activation is shown in Figure 1(a) and (b). Then activated

solution of amine protected active ester of FA (NHS-FA-f-

MOC) was dissolved in DMSO (25 mg/mL) and amine

terminated MWCNTs was dissolved in DMSO (10 mg/mL)

were mixed and magnetically stirred at 100 rpm (Remi) for 5 d

in dark condition at RT, followed by addition of acetone to

obtain yellow precipitate, filtered and vacuum dried [2,33].

The overall gemcitabine loaded FA conjugation to amine

terminated MWCNTs is shown in Figure 2.

Entrapment efficiency

The entrapment efficiency of gemcitabine (GEM) of the

developed engineered MWCNTs formulations was deter-

mined through dialysis membrane following a previously

reported method [11,12,24], with slight modification. Briefly,

uniformly dispersed purified and FA-MWCNTs (2 min son-

ication; Soniweld) were incubated with GEM solution with

magnetic stirring (100 rpm; Remi) at 25� 0.5 �C for 24 h in

PBS (pH 7.4). The dispersion was dialyzed against deionized

water through dialysis membrane (MWCO, 12–14 kDa,

HiMedia) on magnetic stirring at RT for 15 min to separate

the unentrapped free GEM and lyophilized (Hetro Dry

Winner, Germany) to obtain the products (denoted as GEM-

NT and GEM/FA-NT formulation). The % entrapment

efficiency was determined spectrophotometrically (UV/Vis

1601, Shimadzu, Japan) at 268.5 nm (characteristic absorb-

ance of GEM) using following formula:

% Entrapment Efficiency

¼Weight of entrapped GEM in formulation

Weight of entrapped GEMþ free GEM� 100

Characterization of functionalized MWCNTs

Shape and surface morphology

Transmission electron microscope (TEM) photomicrographs

were taken at suitable magnification using Phillips CM 12

electron microscope, Eindhoven, the Netherlands. For surface

fracture morphology, the samples were mounted on metal

stubs, coated with gold under vacuum and then examined in a

scanning electron microscope (Phillips XL-30 FEG FE-SEM,

Eindhoven, the Netherlands).

Surface charge determination

The surface charge of purified MWCNTs and GEM/FA-NT

was determined by zeta potential (z) according to the

Helmholtz–Smoluchowski equation from their electrophoresis

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Figure 1. (a) Schematic representation of NHS-Folate conjugates (Intermediate). (b) Schematic representation of activated f-MOC FA.

DOI: 10.3109/1061186X.2013.778264 Gemcitabine-loaded nanotubes for cancer cells 3

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mobility using computerized Malvern Zetasizer (Malvern

Instruments, Worcestershire, UK).

FT-IR spectroscopy

The FTIR spectrum of GEM/FA-NT formulation was rec-

orded on a Perkin-Elmer Spectrum (Perkin-Elmer RX-1,

Shelton, CT) having the resolution of 1 cm�1 and scan range

from 4000 to 400 cm�1 by KBR pellet method.

X-ray diffraction analysis

X-ray diffraction (XRD) spectra were recorded by X-ray

diffractometer (PW 1710 Rigaku, San Jose, CA) by adjusting

X-ray power of 40 kV and 40 mA.

In vitro release study

The in vitro release of GEM from the GEM-NT and GEM/

FA-NT dispersion was performed by dialysis tube diffusion

technique. Briefly, the GEM-NT and GEM/FA-NT dispersion

equivalent to 5 mg of GEM were separately suspended in PBS

(pH 7.4 and 5.0) and placed in a dialysis membrane (MWCO,

12–14 kDa, Himedia) and dialyzed against release medium at

37� 0.5 �C with continuously stirring in a magnetic stirrer

(100 rpm; Remi). Aliquots were withdrawn at specific time

points while maintaining sink condition and assayed spectro-

photometrically (UV/Vis 1601, Shimadzu, Japan) at 268.5 nm

to quantify the drug release.

Hemolytic toxicity

Preparation of RBCs suspension

Whole human blood was collected in anti-clot blood collect-

ing vials (Himedia), centrifuged (3000 rpm; Remi) for 5 min

supernatant was removed and packed cell volume containing

RBCs was washed with normal saline (0.9% w/v) and again

centrifuged. This process was repeated until a clear, colorless

supernatant was obtained above the cell mass. The RBCs

were then separated and suspended in normal saline and

deionized water to get 10% hematocrit and 100% hemolysis,

respectively [11,12,36]. To assess the effect of chemical

functionalization on the hemolysis, it is far more essential to

perform hemolytic toxicity determination of plain formula-

tion, i.e. pristine, purified and FA-NT, without drug. Later,

GEM-loaded pristine (GEM-NT), purified (GEM-P-NT) and

FA-conjugated MWCNTs (GEM/FA-NT) were evaluated for

their hemolytic toxicity and results compared with free drug

solution.

Quantification of MWCNTs-induced release of hemoglobin

from human erythrocytes

RBCs suspension (1 mL) was placed in a series of

microcentrifuge tubes and normal saline (3.5 mL) was

added. Then plain and GEM/FA-NT (500mL) were added in

different tubes. Similar process was applied for purified and

FA-NT and their GEM-loaded counterparts. The tubes were

tightly closed and occasionally shaken during incubation

Figure 2. Schematic representation of GEM/FA-NT formulation.

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period of 1 h at room temperature and the microscopic

analysis (Leica, Wetzlar, Germeny) was performed. After 1 h

of incubation, sampling tubes were dipped in ice-cold water

and non-lysed RBCs were separated by centrifugation at

5000 rpm for 5 min. The supernatants were analyzed for

hemoglobin content, spectrophotometrically (UV/Vis-1601,

Shimadzu, Japan) at 268.5 nm against normal saline as blank

[37]. The percent hemolysis was determined using the

following equation:

% Hemolysis ¼ ABs

AB100

� �� 100,

where ABs and AB100 represent absorbance of sample and

control without formulation, respectively.

Ex vivo cytotoxicity study

The ex vivo cytotoxicity study was performed on human

breast cancer cell line (MCF-7) at Tata Memorial Hospital

and Research Centre, Mumbai, India. The MCF-7 cells was

grown as monolayer using Dulbecco’s MEM (DMEM;

HiMedia) supplemented with 10% fetal calf serum (Sigma,

St Louis, MO), penicillin (100 U/mL) and streptomycin

(100mg/mL) (Sigma) to discourage the growth of micro-

organisms and maintained in a humidified incubator at

37� 0.5 �C with 5% CO2. Sub-culturing was done twice a

week using 0.2% trypsin and 0.025% EDTA by replacing half

of the cells suspension with a fresh medium. Flat-bottom

tissue culture plates (Corning Incorporate, Corning, NY) with

96-wells (Iwaki Glass, Tokyo, Japan) were used. Adherent

cells were grown to 80% confluence and used for further

study [2]. The cleavage of tetrazolium salt [{3-(4,5dimethyl

thiazole-2 yl)-2,5-diphenyl tetrazolium bromide} (MTT)] to a

blue formazan derivative by living cells is clearly a very

effective principle on which the assay is based. The number of

cells was found to be proportional to the extent of formazan

production by the cells used [38,39]. The monolayer cell

culture was trypsinized and the cell count was adjusted to

1.0� 105 cells/mL using DMEM medium containing 10%

fetal calf serum. To each well of the 96-well microtitre plate,

0.1 mL of the diluted cell suspension (approximately 10 000

cells) was added. After 24 h, when a partial monolayer was

formed, the supernatant was removed, washed once with

medium and 100mL of free GEM, GEM-P-NT and GEM/FA-

NT formulations were added to the cells and incubated at

37� 0.5 �C for 3 h in 5% CO2 atmosphere and microscopic

examination was carried out every 24 h. After 72 h, the

formulations in the wells were discarded and 50 mL of MTT in

DMEM was added to each well with gentle shaking and

incubated for 3 h at 37 �C. Then supernatant was removed and

50 mL of propanol was added and the plates were again

gently shaken to solubilize the formed formazan. Complete

solubilization of formazan crystals was attained by repeated

pipetting of the solution. The plates were then read on a plate

reader (Molecular Devices, Bismarckring, Germany) and

the intensity of purple colour, which indicates the conversion

of MTT by redox activity of living cells, was measured

using microplate reader (Bio-Rad, Model 550-Microplate

Reader, Hercules, CA) at wavelength 268.5 nm [40–42].

Three replicates were read for each sample and mean value

was used as the final result. The relative (%) cell viability

related to control wells was calculated by equation:

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.

In vivo studies

The experimental protocol of in vivo studies was duly

approved by the Institutional Animal Ethics Committee of

Dr Hari Singh Gour Central University, Sagar, Madhya

Pradesh, India in accordance with the guidance of the

Committee for the Purpose of Control and Supervision of

Experiments on Animals (CPCSEA), Govt. of India. Albino

rats (Sprague-Dawley strain; 100� 10 g) either sex, fed with

commercial diet and allowed access to water ad libitum.

In vivo drug distribution study. Animals were divided into

three groups of three rats each and the formulations (Gem,

GEM-NT and GEM/FA-NT) dispersed in PBS (pH 7.4) were

intravenously administered via tail vein route.

Group I: Gemcitabine HCL (100 mg) served as control

Group II: GEM-loaded purified MWCNTs formulation

(GEM-NT)

Group III: GEM-loaded FA-MWCNTs (GEM/FA-NT)

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

GEM, GEM-NT and GEM/FA-NT formulations and the

animals were carefully sacrificed at intervals of 1, 6 and 24 h.

Subsequently, the different organs like liver, spleen, kidney

and lungs were carefully separated out, washed, weighed

and stored under frozen condition till used. These weighed

tissue samples were homogenized in PBS (pH 7.4),

vortexed (Superfit, Mumbai, India) and homogenates

were centrifuged; washed with normal saline and kept

aside for 30 min. The contents were treated with 10% TCA

solution, vortexed (Superfit), filtered (Millipore, Billerica,

MA) and centrifuged, the obtained clear supernatant was

analyzed the drug content from the calibration curve by the

reverse phase HPLC method. Reversed phase HPLC method

without any internal standard was used. Mixture of ammo-

nium acetate buffer: methanol (pH 6.8) (90:10 v/v) as mobile

phase was passed at a flow-rate of 1–5 mL/min by LC10 AT

pump on a 5 mm-Luna C18 column (Phenomenex, Torrance,

CA) with UV detection at 268 nm using photo diode array

detector (SPD-M10A) [43]. The results were expressed as

mean� SD and the statistical analysis was done by analysis of

variance (ANOVA). A probability level of p� 0.05 was

considered to be significant.

Pharmacokinetic studies

The bioavailability of free GEM and GEM/FA-NT in Albino

rats (Sprague-Dawley strain) was determined from plasma-

concentration curve. The free GEM and GEM/FA-NT

formulations were administered and the blood samples were

collected from retro-orbital plexus of rat eyes at different time

points 0.5, 1, 2, 3, 6, 12, 18 and 24 h and centrifuged at

3000 rpm for 10 min (Remi) to separate RBCs and serum. The

upper supernatant (serum) was collected with the help of

DOI: 10.3109/1061186X.2013.778264 Gemcitabine-loaded nanotubes for cancer cells 5

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micropipette and GEM was estimated. The therapeutic plasma

drug concentration profile was obtained and data used to

determine the different pharmacokinetic parameters such as

peak plasma concentration (Cmax) and time taken to reach

Cmax, i.e. Tmax from the plasma concentration curve. The area

under the curve (AUC0-t) was calculated by trapezoidal rule

followed by non-compartment analysis by Kinetica 5.0 PK/PD

analysis software (Thermo Fischer Scientific, West Palm

Beach, FL). The area under the first moment curve (AUMC),

mean residence time (MRT), plasma half-life (t1/2) and half

value duration (HVD) were also calculated.

Stability studies

Physical changes at exaggerated condition

GEM/FA-NT formulation was kept in tightly closed amber

colored and colorless vials at (4� 0.5 �C), room temperature

(25� 0.5 �C) and 55� 0.5 �C in controlled oven and moni-

tored initially and periodically every week up to 5 weeks for

changes in precipitation, turbidity, crystallization, color and

consistency. The obtained data was used to assess any

physical or chemical degradation at accelerated storage

conditions [41].

Residual drug content

The drug content was measured after exposure to accelerated

condition, using a dialysis sac. The GEM/FA-NT formulation

was placed in the dialysis sac and analyzed periodically every

week up to 5 weeks, taking initial drug content as 100%. The

samples were diluted with methanol and analyzed for drug

through HPLC analysis [43]. The percent residual drug

content at scheduled time point was determined and assessed

the effect of accelerated storage condition.

Statistical analysis

Statistical analysis was performed with Graph Pad Instat

Software (Version 3.00, Graph Pad Software, San Diego, CA)

by one-way ANOVA followed by Tukey–Kramer test for

multiple comparisons. A probability p� 0.05 was considered

statistically significant. The pharmacokinetic data was

determined followed by non-compartment analysis using

trapezoidal rule.

Results and discussion

Gemcitabine is a FDA approved cell cycle dependent anti-

metabolite inhibiting the cellular DNA synthesis.

Functionalized CNTs are more biocompatible and nontoxic

at the cellular level which may be used in targeted drug

delivery. Initially, pristine MWCNTs was purified to remove

any type of impurities like amorphous carbon and metallic

with acid treatment (HCl) [34]. Purified MWCNTs was

further oxidized with the strong acid treatment (Piranha

solution) for the preparation of different conjugates. The

purified MWCNTs was treated with strong acid mixture

(H2SO4:HNO3) in 3:1 ratio to generate the carboxylic

functional group on surface of MWCNTs. During oxidative

treatment not only carboxylic functional groups but lactone

and phenolic functional groups are also generated with few

defects sites. Recently, Yudianti et al. [44] quantified the

carboxyl and acidic functional groups on the carboxylated

MWCNTs by Boehm titration analysis and reported the total

acidic functional group to be 43.9 mmol/g. After carboxyl-

ation of MWCNTs, acylation and amine modifications were

carried out following the well reported methods [35]. Finally,

FA conjugation was performed on the amine modified

MWCNTs using EDC and NHS and characterized [2,33].

FTIR spectroscopy was performed by KBr pellet method

to assess the presence of different functional groups over their

surface. The pristine MWCNTs showed a peak at 3387 cm�1

that can be ascribed to broad O–H stretching owing to bound

moisture. Peaks at 2955.04, 2893.32, 2337.80 and

1651.12 cm�1 suggesting the CNTs backbone and other

undefined peaks present in spectrum may be attributed to

the presence of amorphous carbon, catalytic and metallic

impurities. The IR spectrum of purified MWCNTs exhibits

peaks at 2355.4, 1631.9, 1020.6 and 3443.8 cm�1 that could

be ascribed to the stretching of MWCNTs backbone, C¼C

stretching, O–H in plane bending and broad O–H stretching,

respectively (spectra not shown). Moreover, GEM/FA-NT

showed peaks at 3286.81 cm�1 (symmetrical N–H stretching),

1612.54 cm�1, (NH2 stretching) to confirm the presence of

primary NH2 group. Peaks at 1705.13 cm�1 (aromatic C¼C

stretching), 1519.96 cm�1 (N–H bending; scissoring) and

1411.94 cm�1 (O–H deformation of phenyl skeleton), suggest

the attachment of FA to amine-terminated MWCNTs con-

taining aromatic rings (Figure 3). Our results are in line with

the previous reports [35,45,46].

The surface morphology of purified and GEM/FA-NT was

studied by electron microscopy (SEM and TEM) as shown in

Figures 4 and 5, respectively. Electron microscopy revealed that

the GEM/FA-NT was in nanometric size range with open tubu-

lar structure. Even after conjugation of FA and loading of GEM

topography of MWCNTs did not change significantly with no

aggregation or bundling. Functionalization drastically reduces

the bundling and aggregation and cut the CNTs into the shorter

tubes upon treatment with different acidic treatment. The mean

particles size of the developed nanotubes formulation was

calculated to be 140� 1.10 (n¼ 3).

Zeta potential of purified and GEM/FA-NTs was deter-

mined according to the Helmholtz–Smoluchowski equation

from their electrophoresis mobility and was found to be �1.32

and þ9.53 mV, respectively. The positive charge of GEM/FA-

NT indicating the cationic-charged gemcitabine can readily

get entrapped into the nanotubes as well as adsorbed onto the

surfaces with lower surface potentials through electrostatic

interactions as well as �–� stacking interactions.

XRD is a valuable tool for characterizing the CNTs and

surface modifications with different chemical reagents. XRD

analysis of the purified and GEM/FA-NT formulation was

shown in Figure 6. XRD analysis clearly depicted that there

was no change in the seamless tubular structure and was

similar with pristine and purified nanotubes.

The in vitro release study was performed in PBS (pH 5.0

and 7.4) upto 144 h under strict sink condition. The release of

GEM was found to be in sustained pattern followed after

initial burst release. The sustained release pattern at pH 5.0

corresponding to lysosomal pH was due to the ionization of

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GEM, which repels the drug molecules form each other by

breaking the �–� interaction. Figure 7 clearly shows that the

GEM/FA-NT released GEM for a longer duration as

compared with purified, which possibly suggest the lower

exposure of loaded drug into the external environment that

could be ascribed to the greater steric hindrance of available

major and minor grooves on ends and side walls. The GEM

release from the GEM/FA-NT followed the Higuchian release

kinetic. The % hemolytic toxicity of the developed formula-

tions was found to be: GEM (17.34� 0.56), raw MWCNTs

(12.76� 0.76), purified MWCNTs (11.87� 0.96), FA-NT

(12.62� 0.34), GEM/MWCNTs (16.98� 0.42), GEM-NT

(15.45� 0.75) and GEM/FA-NT (8.23� 0.65) (n¼ 3).

Hemolytic toxicity attributes the interaction of human

erythrocytes with CNTs formulations and free GEM and

was found to be maximum (17.34� 0.56) in free GEM as

compared to other nanotubes formulations. The developed

GEM/FA-NT formulation significantly reduced the toxicity

due to the better dispersion or decreasing the bundling and

aggregation. Upon functionalization, hydrophilicity of CNTs

dramatically increases that reduces the extent of interaction

with RBCs resulting into the less hemolytic toxicity of

Figure 3. Fourier transform infrared spectra. (A) Activated ester of FA, (B) amine functionalized oxidised MWCNTs and (C) FA conjugated, amineoxidised MWCNTs.

DOI: 10.3109/1061186X.2013.778264 Gemcitabine-loaded nanotubes for cancer cells 7

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GEM/FA-NT (8.23� 0.65) as compared to free GEM

(17.34� 0.56) or other formulations (Figure 8).

The cytotoxicity study was performed employing MCF-7

(human breast adenocarcinoma cancer cell lines) cell line

which is non-invasive having estrogens positive receptor

(ERs) in the cell cytoplasm, and results are shown in Figure 9.

The MTT (3 -(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-

lium bromide) assay is a simple non-radioactive colorimetric

assay to measure the mitochondrial function of the cells in

terms of cell proliferation or viability after treatment with

the developed formulations, resulting into the metabolic

dysfunctions. It is clearly observed from the cytotoxicity

result that upon increasing the concentration of formulation

the % cell viability dramatically decreases. Interestingly, we

found the concentration-dependent cytotoxicity of the GEM/

FA-NT formulation possibly due to the tubular nanoneedle

shape structure, which easily penetrate into cellular mem-

branes through caveolae membrane proteins. The GEM/FA-

NT formulation may enter into the cancerous cell, through

caveolae mediated endocytosis mechanism, followed by FRs

over-expression in breast cancer cells which mainly partici-

pated in the cellular accumulation. From the cytotoxicity

results it can be concluded that the GEM/FA-NT causes

relatively effective apoptosis as compared with free GEM.

The in vivo studies were carried out to assess to explore the

presence of macromolecules showing the retention in sys-

temic circulation of engineered CNTs formulation as

compared with free GEM in PBS (pH 7.4). The group was

divided on the basis of the developed MWCNTs formulations,

i.e. free GEM, GEM-NT and GEM/FA-NT as presented in

Figure 10. Tissue biodistribution study was conducted to

evaluate the drug delivery at the different sites of interest like

liver, spleen, kidneys and lungs. The free GEM was primarily

accumulated progressively in liver 25.49� 0.54 mg/g 1 h post

injection. A concordant result was reported by Singh et al.

[47]. After 6 h, only 6.13� 0.23 mg/g of drug concentration

was found in liver, whereas spleen, kidney and lungs were

found to have drug levels upto 2.49� 0.09, 4.23� 0.44 and

3.82� 0.15 mg/g, respectively. When GEM-NT was

Figure 4. TEM nano-graphs of (a) purified MWCNTs and (b) GEM-FA-NT.

Figure 5. SEM nano-graphs of (a) purified MWCNTs and (b) GEM-FA-NT.

Figure 6. XRD of purified and GEM/FA-NT.

8 R. Singh et al. J Drug Target, Early Online: 1–12

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administered, the drug concentration was mainly found in

liver 10.95� 0.17 mg/g and kidney 6.54� 0.23 mg/g, 1 h post

injection, however, appreciably lesser amounts were present

in spleen and lungs. The purified MWCNTs which are devoid

of free terminal functional groups, readily goes into the liver.

According to previously published report, after i.v. injection,

CNTs are mainly excreted by urine and feces [47]. The GEM-

NT is mainly accumulated in liver and drug concentration was

found to be 10.95� 2.17 mg/g 6 h post injection. The appre-

ciable amount of drug (6.54� 0.23 mg/g) from the developed

CNTs formulation was found in kidney. No significant

accumulation of GEM-NT was observed in spleen and

lungs. As the folate conjugation was carried out on the

amine modified MWCNTs, the dispersibility, hydrophilicity

of MWCNTs were enhanced significantly as revealed by

dispersion and loading experimentation. In general, GEM/FA-

NT formulation was administered by i.v. route; the concen-

tration of drug was comparatively lesser in all the tissues than

the free GEM and GEM-NT in the initial 1 h, slight increase

in 6 h and reduction due to the clearance in 12 h post injection.

Figure 7. Cumulative percentage of Gemcitabine release from purified and GEM/FA-NT formulations in PBS at different pH, i.e. pH 5.0 and 7.4 at37� 0.5 �C up to 144 h and data presented is mean� SD (n¼ 3).

Figure 8. % Hemolytic toxicity of free GEM and different functionalized MWCNTs formulations. # Mean� SD (n¼ 3).

Figure 9. Percent cell viability of free GEM,pristine MWCNTs, GEM-NT and GEM/FA-NT formulations (n¼ 3).

DOI: 10.3109/1061186X.2013.778264 Gemcitabine-loaded nanotubes for cancer cells 9

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This outcome could be attributed to the fact that GEM/FA-NT

released appreciable drug in the systemic circulation than

in any other tissue. The MWCNTs are non-biodegradable

in nature and known to be excreted by biliary pathway

from the liver through the bile duct, intestine, which ends up

in feces [47].

Pharmacokinetic study was performed to determine the

different kinetic parameters with the free GEM by measuring

the plasma concentration upto 24 h followed by i.v. admin-

istration of GEM/FA-NT formulation dispersed in 100 mL of

saline solution (100mg/mL nanotubes concentration) through

tail vein route. From the blood-plasma concentration level

studies, pharmacokinetics parameters like MRT, AUC,

AUMC, HVD were determined. However the free GEM

solution was rapidly cleared from the blood within initial 12 h.

Further, the AUC(0–t), AUC(0–1), AUMC(0–t) and AUMC(0–1)

values were calculated to be 14.467, 14.469, 29.751, 29.771,

20.2375, 20.4242, 61.27, 63.990 and 39.645, 41.446, 283.585,

341.587 for free GEM, GEM-NT and GEM/FA-NT, respect-

ively. The AUMC(0–t) and AUMC(0–1) were found to be

approximately 10 times higher as compared to free GEM. The

long circulatory pattern of functionalized nanotubes in body

compartment upon i.v. administration due to their greater

dispersibility is clearly observed. The half-life (t1/2) of free

GEM (0.76668), GEM-NT (1.7809) and GEM/FA-NT

(5.6770) suggested the sustained release and prolonged

circulation time of developed nanotubes formulation.

Gemcitabine has a very short-life (0.76668 h) which is a

major limitation, and enhanced approximately seven time

through nano-carrier system. Moreover, the extent of drug

release was determined by the HVD as the time that plasma

concentration of GEM was above one-half of Cmax and MRT,

determined by linear interpolation was found to be 1.443 h

(free GEM), 1.5733 (GEM-NT) and 4.977 hr (GEM/FA-NT).

The obtained pharmacokinetic data demonstrates that GEM/

FA-NT markedly improved the bioavailability and signifi-

cantly improved the systemic circulation and also highlights

the efficacy in terms of sustained release pattern for temporal

or spatial distribution of GEM. The various calculated

pharmacokinetics parameters are summarized in Table 1,

which suggested that surface-engineered CNTs are alterna-

tive, safe and effective delivery system. The pharmacokinetics

results show the relevant increase in residence time and half-

life as envisioned in biomedical sciences. Cherukari et al. [48]

reported the low acute toxicity and long circulation of

deaggregated SWCNTs by low dose of nanotubes. Liu and co-

workers [26,27] reported that the PEG functionalized

SWCNTs enabled the longest blood circulation up-to 1 d,

relatively low uptake in the RES system, and near-complete

clearance from the main organs in approximately 2 months.

As the pristine CNTs are toxic and not suitable for targeted

delivery system, thus functionalization minimizes/reduces the

toxicities. The various toxicological parameters suggested that

the surface-engineered CNTs are not toxic [12,26,27,48].

The stability data of the GEM/FA-NT formulation was

evaluated by varying the condition, i.e. at temperature

(4� 0.5, 25� 0.5 and 35� 0.5 �C) after storage in dark

(amber-colored bottle) and light (colorless bottles) every

week upto 5 weeks, and found to be most stable in dark at

4� 0.5 �C (Table 2). Effect of storage condition was studied

by analyzing the change in residual drug content. Initial drug

content was assumed to be 100%. Study revealed significant

loss of drug, i.e. 10–20% and 5–10% in case of purified

MWCNTs stored at 35� 0.5 �C and 25� 0.5 �C, respectively,

Figure 10. Time based organ distribution of gemcitabine hydrochloride (GEM) in represents mean % initial dose of GEM found in various organs aftera definite period of time (n¼ 3).

Table 1. Pharmacokinetic parameters of free GEM, GEM-NT andGEM/FA-NT.

Parameters Free GEM GEM-NT GEM/FA-NT

Cmax (mg/mL) 6.22 6.02 5.40HVD (hr) 1.443 1.5733 4.977AUC(0–t) (mg/ml/min) 14.467 20.237 39.645AUC(0–1) (mg/ml/min) 14.469 20.424 41.446AUMC(0–t) (mg/ml/min2) 29.751 61.270 283.585AUMC(0–1) (mg/ml/min2) 29.771 63.990 341.587t1/2 (h) 0.766* 1.7809 5.677*MRT (h) 2.057 3.1330 8.241

*p� 0.05 with respect to free drug, according to Student’s unpairedt-test.

10 R. Singh et al. J Drug Target, Early Online: 1–12

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as compared to very little loss (2–5%) at 4� 0.5 �C (Table 3).

In case of folate-conjugated formulation, drug loss was found

to be less under all storage conditions.

Conclusions

CNTs have emerged as new platform in the use of numerous

nanotechnological biomedical applications including the

targeted drug delivery due to its unique outstanding proper-

ties. Functionalized CNTs provide the greater stability as well

as loading efficiency as compared to existing nano-carriers

like liposomes, dendrimers and nanoparticles. The entire

focus of this study was to assess the targeting potential of

GEM/FA-NT formulation on to the breast cancer cell line.

The developed formulation possesses the greater entrapment

efficiency, better stability and improved bioavailability and

pharmacokinetics of the free drugs. To the best of our

knowledge, this is a debut study report wherein gemcitabine

has been used as a model drug in case of functionalized

MWCNTs to assess their targeting capability by improved

bioavailability and MRT, which may emerge as a novel

strategy in targeted drug delivery. Till date only few studies

have been published in CNT-mediated drug delivery in

chemotherapy, like doxorubicin HCl, Cisplatin, epirubicin,

amphotericin B, paclitaxel, daunroubicin and methotrexate. It

may be concluded that the functionalized CNTs may open

new vista in biomedical application including the chemother-

apy for the complete cure of human breast cancer in the

coming years.

Acknowledgements

The authors are grateful to M/s Khandelwal Laboratory Pvt.

Ltd, Mumbai, India for the gift sample of gemcitabine; the

National Institute of Pharmaceutical Education and Research

(NIPER), Mohali, Chandigarh for the Zeta potential study and

the All India Institute of Medical Sciences (AIIMS), New

Delhi for Electron Microscopy facility.

Declaration of interest

The authors report no conflict of interest.

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Table 2. Physical changes in GEM/FA-NT formulation at exaggerated condition.

After 5 weeks

Dark Light

Parameter 4� 0.5 �C 25� 0.5 �C 55� 0.5 �C 4� 0.5 �C � 0.5 �C 55� 0.5 �C

Turbidity � � � � þ þPrecipitation � � � � � �Colour change � � � � � �Change in consistency � � þþ � þ þþ

�: no change.þ: small change.þþ: enough change.

Table 3. Residual drug content (%) of GEM/FA-NT formulation.*

% Residual drug (�SD*) after week

Formulations Temp. (�C) 1 2 3 4 5

GEM-NT 55 91� 1.4 87� 1.1 84� 0.8 79� 1.2 77� 1.825 95� 1.4 91� 1.6 88� 2.8 83� 2.2 80� 2.5

4 96� 2.3 92� 1.3 89� 2.7 86� 3.2 84� 2.8GEM/FA-NT 55 97� 2.2 94� 2.6 92� 1.1 90� 0.5 87� 1.4

25 98� 0.2 97� 2.5 95� 1.3 94� 1.6 92� 1.94 99� 1.7 98� 0.5 96� 1.5 95� 0.1 94� 2.4

*Mean� SD (n¼ 3).

DOI: 10.3109/1061186X.2013.778264 Gemcitabine-loaded nanotubes for cancer cells 11

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