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Anticancer (in vitro) and antimicrobial effect of gold

nanoparticles synthesized using Abelmoschus
esculentus (L.) pulp extract via a green route
Cite this: RSC Adv., 2014, 4, 37838
Published on 29 July 2014. Downloaded by Oakland University on 19/10/2014 06:30:03.

Md. Masud Rahaman Mollick,a Biplab Bhowmick,a Dibyendu Mondal,a

Dipanwita Maity,a Dipak Rana,b Sandeep Kumar Dash,c Sourav Chattopadhyay,c
Somenath Roy,c Joy Sarkar,d Krishnendu Acharya,d Mukut Chakrabortye
and Dipankar Chattopadhyay*a

Received 18th July 2014
Accepted 29th July 2014
DOI: 10.1039/c4ra07285e
www.rsc.org/advances
Green synthesis of gold nanoparticles (Au NPs) using Abelmoschus esculentus (L.) pulp extract has been
elaborately studied and reported here. The Au NPs have been characterized using several techniques.
Optical analysis indicates adequate stability of the synthesized Au NPs, while FTIR analyses the fact that
phytochemicals present in the Abelmoschus esculentus (L.) pulp extract play the key role in stabilizing
the Au NPs. Morphological study shows that the nanoparticles are mostly spherical in shape with an
average particle size of
14 nm, and these results are comparable with the particle size obtained from
XRD. The selected area electron diffraction pattern indicates the crystalline nature of the Au NPs, which
is further confirmed from XRD studies. The present study also demonstrates the in vitro efficacy of Au
NPs against Jurkat cells. Results show that the IC50 dose of Au NPs is capable of significantly elevating
intracellular reactive oxygen species and diminishing mitochondrial membrane potential, indicating the
effective involvement of apoptosis in cell death. Furthermore, the synthesized Au NPs show a sufficient
degree of antimicrobial activity against different types of bacteria. These results clearly show that the
Abelmoschus esculentus (L.) pulp synthesized Au NPs have excellent medicinal applications.

the shape and size of the nanoparticles as an effect of quantum

Introduction
Vast exploration and the rapid exploitation of nanotechnology
offers pathways for the development of new materials on the
nanometer scale, including nanoparticles. These are usually
dened as those particles where at least one dimension is less
than 100 nanometers (nm); the particles could even be zero
dimension as in the case of quantum dots. Now the question
arises: Why are these nanoparticles so interesting even if they
are quite challenging to handle and synthesize compared to
their macroscopic bulk counterparts?” The answer lies in their
distinctive features such as optical,1 antimicrobial,2,3 cancer
therapeutic4 and catalytic5 properties, which can be tuned with
a
Department of Polymer Science & Technology, University of Calcutta, 92 A. P. C. Road,
Kolkata 700009, India. E-mail: dipankar.chattopadhyay@gmail.com; Fax: +91 33
2351 9755; Tel: +91 33 2350 1397; +91 33 2350 6996; +91 33 2350 6387; +91 33
2350 8386
b
Department of Chemical and Biological Engineering, Industrial Membrane Research
Institute, University of Ottawa, 161 Louis Pasteur St., Ottawa, ON, KIN 6N5, Canada
c
Immunology and Microbiology Laboratory, Department of Human Physiology with
Community Health, Vidyasagar University, Midnapore 721 102, West Bengal, India
d
Department of Botany, Molecular and Applied Mycology and Plant Pathology
Laboratory, University of Calcutta, 35 Ballygunge Circular Rd., Kolkata 700 019, India
e
Department of Chemistry, West Bengal State University, Barasat, Kolkata 700 126,
India
connement of electrons. Nanoparticles play a pivotal role as
the ‘bridging element’ between bulk materials and atomic or
molecular structures. Metal nanoparticles with their unparal-
leled properties are extensively investigated due to their prom-
ising applications in diverse areas like electronics, coatings,
packaging, and biotechnology. Nondestructive attachment of
metal nanoparticles to single stranded DNA opens up the
prospect of medical diagnostic applications.6,7 Transparent
metallic nanoparticles are a promising vehicle in cosmetics,
coatings and packaging applications, whereas their tendency to
merge into a solid at lower temperature makes them an
important substance in electronic applications (e.g. capacitors).
Among the noble metal nanoparticles, gold has enormous
potential in applications such as DNA sequencing,8 wave-
guides,9 catalysis,10 and electron microscopy markers.11 Gold
nanoparticles (Au NPs), a highly versatile deep-tissue imaging
agent for various detection and imaging applications, show
their efficiency in clinical applications.12–15 Some interesting
shapes of Au NPs, such as nanotripods,16 nanowires and
nanosheets,17 nanoprisms,18 nanotriangles,19–21 nanotubes,22
and nanopyramids,23 have been synthesized through a chemical
reduction method. Different existing synthetic techniques for
metal nanoparticles, such as bottom-up and top-down
approaches or pyrolysis and wet-chemical approaches, have

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several drawbacks, including surface imperfection, energy
consumption, use of toxic solvents, and hazardous by-products.
Synthesis of nanoparticles using microorganisms or different
parts of plants can potentially eliminate these problems by
making nanoparticles more biocompatible. Nanobiotechnology
is a eld where the nanoscale principle and techniques are used
to understand and transform bio systems (living and non-living)
and vice versa.24 While microorganisms such as fungi and
bacteria play signicant roles in metal nanoparticle synthesis,
the use of parts of plants or whole plants in similar synthesis
methodologies is a stimulating prospect that is currently under
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enormous investigation, having signicant potential utiliza-
tion. In this eld, the rst ever report of green synthesis of metal
nanoparticles was published by Jose-Yacaman and co-
workers.25,26 From an environmental viewpoint, this green
process meets the requirements of utilization of non-toxic,
environmentally benign, renewable materials towards nano-
particle synthesis. Nowadays, extensive studies on the synthesis
of gold and silver nanoparticles using different natural products
like lemongrass,20 Lactobacillus strains,27 Paederiafoetida L. leaf
extract,28 Shewanella algae,29 Rhodococcus sp.,30 Fusarium oxy-
sporum,31 neem,32 aloe vera,33 Cinnamomum camphora,34 are
reported in the literature.
Recently, metal nanoparticles exhibiting novel chemical and
physical properties due to their extremely small size and high
surface area to volume ratio have received tremendous atten-
tion. The antibacterial properties of such noble metal nano-
particles have been studied by a number of researchers.35,36 Gold
is known to have disinfecting effects and has been used in
applications ranging from traditional medicines to culinary
items. Moreover, several salts of gold, silver and their deriva-
tives are commercially manufactured as antimicrobial
agents.37,38 In small concentrations, gold is safe for human cells,
but lethal for bacteria and viruses.39–41 Recently, Mishra et al.42
tested the antibacterial action of gold nanoparticles against
different pathogenic bacteria. Worldwide statistical reports
show that cancer is the third leading cause of death (aer heart
disease and stroke) in developed countries and the second
leading cause of death (aer heart disease) in the United States,
according to http://www.cdc.gov. It has been reported that
worldwide, 10 million new cancer cases occurred, 6 million
deaths occurred, and 22 million people were living with cancer
in the year 2000.43 Recent advancements in nanotechnology
open a new area of interdisciplinary research for effective
implication of nano-size ranged materials for cancer diagnosis
and therapy.15 Au NPs, one of the most well studied materials,
exhibit unique physicochemical properties, including surface
plasmon resonance (SPR) and the ability to bind to amine and
thiol groups. These properties of Au NPs allow surface modi-
cation as well as vast use in biomedical applications.44 Previ-
ously few studies have been carried out to explore the
involvement of reactive oxygen species (ROS) in causing DNA
damage following exposure to Au NPs in different cell types.45,46
In this work, we report the simple one-step green approach
of synthesizing extracellular Au NPs using Abelmoschus escu-
lentus (L.) pulp extract. The phytochemicals present in the
Abelmoschus esculentus (L.) pulp extract are used as an effective
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reducing and capping agent. Abelmoschus esculentus (L.) pulp, a
purely natural product with high medicinal value, has drawn
our interest towards the green synthesis of metal nanoparticles,
i.e., Au NPs via an eco-friendly process. The Au NPs have been
characterized using several techniques like ultraviolet-visible
(UV-Vis) spectroscopy, dynamic light scattering (DLS), trans-
mission electron microscopy (TEM), X-ray diffraction (XRD) and
Fourier transform infrared (FTIR) spectroscopy studies. The
present study also shows the anti-proliferative activity of Au NPs
against the human T cell myeloma cell line (Jurkat). Further-
more, it is observed that the synthesized Au NPs show a suffi-
cient degree of antimicrobial activity against different types of
bacteria.
Experimental section
Materials
Analytical grade chloroauric acid was purchased from Sigma
Aldrich and used as received without further purication. The
Abelmoschus esculentus was collected from the local market of
our institute.
Culture media and chemicals
RPMI 1640, penicillin and streptomycin were procured from
Sigma Aldrich (St. Louis, MO). Fetal bovine serum (FBS) was
purchased from GIBCO/Invitrogen. 40 ,6-Diamidino-2-phenyl-
indole dihydrochloride (DAPI) was purchased from Sigma-
Aldrich. Commercially available dimethyl sulfoxide (DMSO),
sodium dodecyl sulphate (SDS), rhodamine 123, ethidium
bromide, acridine orange, 3-(4,5-dimethyl-2-thiazolyl)-2,5-
diphenyl-tetrazolium bromide (MTT) reagents were purchased
from Himedia, India. All other chemicals were from Merck Ltd.
and SRL Pvt. Ltd., Mumbai, and were of the highest purity grade
available. Solvents were dried according to standard procedure
and distilled prior to use.47
Synthesis of gold nanoparticles
The required amount of small slices of fresh Abelmoschus
esculentus (L.) pulp was taken into triple distilled water and le
for 2 h in order to maximize the diffusion of phytochemicals
present in the Abelmoschus esculentus (L.) pulp to the distilled
water. Aer that, the pulp was taken out from the medium and
the solution was subjected to centrifugation to remove any
impurities present in the stock. The supernatant was carefully
collected and used as the extract of Abelmoschus esculentus (L.)
pulp. The extract was mixed into an aqueous solution of 1 mM
chloroauric acid (4 : 1 v/v) at room temperature with continuous
stirring for approximately 6 h. Then, the colloidal solution was
le undisturbed for 18 h at room temperature. The formation of
Au NPs was conrmed as observed by the colour change of this
colloidal solution from colourless to light violet.
Cell line culture and maintenance
Jurkat (human acute myeloid leukemia), K562 (human chronic
myeloid leukemia), and DL (Dalton's lymphoma) cell lines were
obtained from NCCS, Pune (India). These cell lines were
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cultivated and maintained in RPMI-1640 complete media sup-
plemented with 10% heat-inactivated fetal bovine serum (FBS),
100 U ml1 penicillin, 100 mg ml1 streptomycin and 4 mM
L-glutamine under 5% CO2 and 95% humidied atmosphere at
37  C in a CO2 incubator. Cells were used for different experi-
ments until the number of cells reached 1.0  106 cells per ml.
Isolation of peripheral blood lymphocytes
For collection of peripheral blood lymphocytes (PBL), blood
samples were collected from six healthy human volunteers by
venepuncture in 5 ml heparin-coated vacutainers (Becton,
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Dickinson and Company, India) according to the method of
Hudson and Hay.48 Five milliliters of blood were diluted 1 : 1
with PBS and layered onto a Histopaque 1077 (Sigma-Aldrich
Co. LLC, US) using a Pasteur pipette and centrifuged at 400 g
(1500 rpm) for 40 min at room temperature. The upper mono-
layer of buffy coat, i.e. lymphocytes, was transferred using a
clean Pasteur pipette to a clean centrifuge tube and washed
three times in balanced salt solution. The peripheral blood
lymphocytes (PBL) were re-suspended in RPMI complete media
supplemented with 10% FBS and incubated for a day at 37  C in
a 95% air/5% CO2 atmosphere in a CO2 incubator.
Drug preparation
One milligram per milliliter stock of Au NPs was prepared by
concentrating the solution containing the prepared Au NPs
using a rotary evaporator. The stock solution of Au NPs was then
serially diluted with RPMI media to prepare working
concentrations.
Experimental design
The PBL, Jurkat, K562 and DL cells were divided into 11 groups.
Each group contained 6 petri-dishes (2  105 cells in each). The
following groups were considered for the experiment and
cultured for 24 h:
Group I: Control i.e., Cells + culture media, Group II: Cells + 1
mg ml1 Au NPs in culture media, Group III: Cells + 5 mg ml1 Au
NPs in culture media, Group IV: Cells + 10 mg ml1 Au NPs in
culture media, Group V: Cells + 25 mg ml1 Au NPs in culture
media, Group VI: Cells + 50 mg ml1 Au NPs in culture media.
Aer the treatment schedule the cells were collected from
the petri dishes separately and centrifuged at 2200 rpm for 10
min at 4  C to separate the cells and supernatant.49 The cells
were washed twice with 50 mM PBS at pH 7.4. The intact cells
were used for mitochondrial membrane potential and ROS
measurement.
In vitro cell viability assay (MTT assay)
PBL, Jurkat, K562 and DL cells were maintained in RPMI-1640
supplemented with 10% FBS, penicillin (100 U ml1), and
streptomycin (100 mg ml1) in a humidied atmosphere of 5%
CO2 at 37  C. The cytotoxicity of synthesized Au NPs on PBL,
Jurkat, K562 and DL cells were determined by the MTT assay.50
The presence of any anticancer components in the Abelmoschus
esculentus (L.) pulp extract was examined by performing a cell
37840 | RSC Adv., 2014, 4, 37838–37848
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viability study on Jurkat cells by the MTT assay.50 Different
doses of extract (0–500 ml) were applied to determine any anti-
cancer role of the extract against Jurkat cells. The extract doses
selected for this study covered the total volume of Au NPs doses
applied. Cells (2  104 per well) were plated in 100 ml of medium
per well in 96 well plates. Aer 48 h of incubation, the cells
reached conuence, and then the cells were incubated in the
presence of various concentrations of sample in 0.1% DMSO for
48 h at 37  C. Aer removal of the sample solution and washing
with phosphate buffered saline (pH 7.4), 20 ml per well (5 mg
ml1) of 0.5% 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-tetrazo-
lium bromide (MTT) in phosphate-buffered saline solution were
added. Aer 4 h of incubation, 0.04 M HCl/isopropanol was
added to each well. Viable cells were determined by the absor-
bance at 570 nm with reference at 655 nm, and measurements
were performed 3 times. The optical density (OD) at 570 nm was
measured on an ELISA reader (Bio-Red, Japan) using wells
without the sample containing cells as blanks. All experiments
were performed in triplicate, and the effect of the Au NPs on the
proliferation of Jurkat cells was expressed as the % of cell
viability using the following formula:
% cell viability ¼ [ODsample  ODcontrol]  100/ODcontrol
Assay for antimicrobial activity of gold nanoparticles against
microorganisms
Gold nanoparticles in sterilized deionized water were tested for
their antibacterial activity by the agar diffusion method. Five
bacterial strains, Bacillus subtilis [MTCC 736], Bacillus cereus
[MTCC 306], Pseudomonas aeruginosa [MTCC 8158], Micrococcus
luteus [MTCC 1538] and Escherichia coli [MTCC 68] were used for
this analysis. These bacteria, grown in liquid nutrient agar
media (HiMedia Laboratories Pvt. Ltd., Mumbai, India) for 24 h
prior to the experiment, were seeded in agar plates by the pour
plate technique. A cup was made using a cork borer (10 mm
diameter) and was lled with the gold nanoparticle solution (0.2
mg ml1) and then incubated at 37  C for 24 h. Simultaneously,
the control sets (only plant extract and sterilized deionized
water respectively) were also examined against the same ve
bacterial strains, and every experiment was repeated three
times.
Characterization
The formation of Au NPs was monitored by measuring the UV-
Vis spectrum of the reaction medium in the wavelength range
from 300 to 700 nm. UV-Vis absorption spectrum of the sample
was performed using an Agilent 8453 Spectrophotometer, USA.
UV-Vis studies were performed using a quartz cell with a
thickness of 1 cm in the wavelength range of 190–1100 nm.
Dynamic light scattering (also known as photon correlation
spectroscopy or quasi-elastic light scattering) technique was
used for determining the particle size distribution and the
nature of the particle's motion in the medium. The light scat-
tering technique of the synthesized gold nanoparticles was
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observed using Malvern Zetasizer Version 6.20. Furthermore,
the size, shape, and morphology of the resultant nanoparticles
were conrmed by TEM. A specimen for the TEM sample was
made by casting a drop of suspension on a carbon-coated
copper grid, and the excess solution was removed by tissue
paper and allowed to air dry at room temperature overnight.
The TEM study was performed on a HRTEM, JEOL JEM 2010 at
an accelerating voltage of 200 kV and tted with a CCD camera.
The crystal structure of the produced Au NPs was determined
and conrmed by XRD analysis. The sample for XRD analysis
was prepared by depositing the centrifuged sample on a
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microscopic glass slide and air-dried overnight. The diffracto-
gram was recorded on a PANalytical, XPERT-PRO diffractometer
using CuKa (l ¼ 1.54060 Å) as an X-ray source. Fourier trans-
form infrared (FTIR) spectroscopy measurements were per-
formed using a Perkin Elmer Spectrum Express Version 1.03.00.
The scanning data was obtained from the average of 47
scans in the range between 4000 and 400 cm1 with a resolution
of 4 cm1.
For anticancer activity
Calculation of IC50 value. The concentration required for a
50% inhibition of viability (IC50) was determined graphically.
Multiple linear regressions were used to compare data using the
Statistica version 5.0 (Statso, India) soware package.
Intracellular ROS measurement. ROS measurement was
performed using 20 ,70 -dichlorodihydrouorescein diacetate
(H2DCFDA) according to the method of Roy et al.51 In brief,
Jurkat cell lines were treated with Au NPs at 8.17 mg ml1 for 24
h. Aer treatment, schedule cells were washed with culture
media, followed by incubation with 1 mg ml1 H2DCFDA for 30
min at 37  C. Then, the cells were washed three times with fresh
culture media. DCF uorescence was determined at 485 nm
excitation and 520 nm emission using ow cytometry, by setting
appropriate lters. The cell population in M2 phase was noted
as the ROS positive cells, where the viable/ROS negative cells
were denoted as M1.
Measurement of mitochondrial membrane potential (DJm)
The alteration of mitochondrial membrane potential by the
spectro-uorometric method was done according to our
previous reported method.50 In brief, Jurkat cell lines (2  104
per well) were treated with Au NPs at 8.17 mg ml1 for 24 h. Aer
treatment, schedule cells were washed with culture media fol-
lowed by incubation with 1.5 mM Rhodamine 123 for 10 min at
37  C in a humidied incubator. Then, the cells were washed
three times with culture media. The cellular uorescence
intensity of Rh 123 was monitored for 2 min using a Hitachi
F-7000 uorescence spectrophotometer. Cellular mitochondrial
membrane potential was expressed as a percentage of the
control cells at an excitation wavelength of 493 nm and an
emission wavelength of 522 nm. Both excitation and emission
slit widths were set to 5.0.
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Cellular morphology analysis by acridine orange (AO)–
ethidium bromide (EtBr) double staining
To conrm the probable pathway of cell death, we analyzed the
cells by the EtBr-AO double staining method. A number of 2 
104 Jurkat cells were seeded into each well of a 6-well plate and
incubated for 24 h at 37  C in a humidied 5% CO2 atmosphere.
Au NPs at 8.17 mg ml1 were then added to the well for 24 h.
Aer incubation, cells were washed once with phosphate buffer
saline (PBS). Ten microlitres of the cells were then placed on a
glass slide and mixed with 10 ml acridine orange (50 mg ml1)
and ethidium bromide (50 mg ml1). The cells were viewed
under a uorescence microscope (NIKON ECLIPSE LV100POL)
with 400 magnication.52
Assessment of nuclear morphological changes by 40 ,6-
diamidino-2-phenylindole dihydrochloride (DAPI) staining
For DAPI staining, all the test cells were seeded into six well
plates. A number of 2  105 cells per ml were treated with or
without Au NPs (0 and 8.17 mg ml1) for 24 h and were then
isolated for DAPI staining according to the method of Lin et al.53
with some modication. Aer treatment, the cells were xed
with 2.5% glutaraldehyde for 15 min, permeabilized with 0.1%
Triton X-100 and stained with 1 mg ml1 DAPI for 5 min at 37  C.
The cells were then washed with PBS and examined by uo-
rescence microscopy (NIKON ECLIPSE LV100POL).
Caspase-3 activity
Involvement of apoptosis was measured by measuring caspase 3
levels using ELISA according to the manufacturer's instructions
(eBioscience, India). In brief, Jurkat cell lines were treated with
Au NPs at 8.17 mg ml1 for 24 h and aer treatment schedule 10
ml of Jurkat cell extracts were taken and used as samples to
examine caspase 3 activity at the wavelength of 450 nm.
Statistical analysis
All the parameters were repeated at least three times. The data
was expressed as mean  SEM, n ¼ 06. Comparisons between
the means of control and treated groups were made by one-way
ANOVA test (using a statistical package, Origin 6.1, North-
ampton, MA) with multiple comparison t tests, p < 0.05 as a
limit of signicance.
Results and discussion
UV-Vis absorption spectroscopy analysis
The UV-Vis spectra of as-synthesized Au nanoparticles were
acquired to investigate the optical properties. The surface
plasmon resonance (SPR) absorption band arises due to the
coherent oscillation of the electrons in the conduction band of
the nanoparticles induced by the electromagnetic eld. The
distinct colour of colloidal gold nanoparticle solution arises
from their surface property known as SPR, attributed to the
nanodimensions of gold. It is reported that gold nanoparticles
exhibit a violet colour in aqueous solution due to the excitation
of surface plasmon vibrations in the metal nanoparticles.54
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Fig. 2 UV-Vis absorption spectra of Au NPs stabilized with Abelmo-

Fig. 1 UV-Vis absorption spectra of Au NPs synthesized by treating
schus esculentus (L.) pulp extract (a) after reaction termination; (b)
0.001 M aqueous chloroauric acid solution with Abelmoschus escu-
after storage for 5 months at room temperature.
lentus (L.) pulp extract at room temperature.

When Abelmoschus esculentus (L.) pulp extract is mixed into an

aqueous solution of the gold ion complex, the solution starts to
change colour from colourless to light violet due to the reduc-
tion of gold ions present in the Abelmoschus esculentus (L.) pulp
extract, which indicates the formation of Au NPs. Fig. 1 shows
the UV-Vis spectra of the synthesized Au NPs which give a sharp
absorbance peak at around 538 nm at room temperature. The
peak originates from the excitation of electrons in the conduc-
tion band of Au NPs induced by the electromagnetic eld.

Analysis of the stability of nanoparticles

The stability of synthesized metal nanoparticles is strictly
dependent on the degree of aggregation, which can be effec-
tively reected by changes in absorption characteristics, espe-
cially the displacement of the SPR band for gold nanoparticles
in the UV-Vis spectrum.55 It is quite obvious that the strong
aggregation of metal nanoparticles indicates the red shi of the
SPR band observed in the UV-Vis spectra. In this work, we have
investigated the stability of gold nanoparticles synthesized
using Abelmoschus esculentus (L.) pulp extract aer a 5 month
storage period at room temperature, and the UV-Vis spectra
(Fig. 2) give the SPR band at 538 nm without any signicant
change in absorbance intensity. The result indicates that the as-
prepared gold nanoparticles are very stable at room tempera-
ture for several months with negligible aggregation.
Dynamic light scattering (DLS) measurement
The dynamic light scattering (DLS) technique is used to deter-
mine the particle size and particle size distribution prole of
small particles in suspension. The average mean size of the gold
nanoparticles is 23.27 nm, as shown in Fig. 3.
Transmission electron microscopy (TEM) measurement
A high-resolution transmission electron microscope (HRTEM)
was used for morphological analysis of the as-synthesized Au
Fig. 3 DLS showing particle size distribution of gold nanoparticles
prepared by treating 0.001 M aqueous chloroauric acid solution with
Abelmoschus esculentus (L.) pulp extract at room temperature.
NPs. It is clear from Fig. 4a that the synthesized Au NPs are
mostly spherical in nature and a small number of hexagonal
and trigonal nanoparticles are also present. Fig. 4b depicts the
particle size histogram of the nanoparticles and it can be seen
from the histogram that the gold nanoparticles have sizes of 4–
32 nm and possess an average size of
13.96 nm. A small
difference is observed in the diameter value obtained from TEM
and DLS measurement mainly due to the process involved in
the sample preparation. The particle size determined by TEM
represents the actual diameter of the nanoparticles as it is
measured at the dry state of the sample, whereas the size
measured by the laser light scattering method (DLS) is a
hydrodynamic diameter (hydrated state); therefore, the nano-
particles will have a larger hydrodynamic volume due to solvent
effects in the hydrated state.56 The crystallinity of the Au NPs is
further evidenced by the selected area electron diffraction

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Fig. 5 XRD patterns showing peaks corresponding to the diffraction

from (111), (200), (220), and (311) planes of fcc lattice of Au NPs.

where d is the average crystallite size, l is the wavelength of

radiation, and b is the full width at the half maximum at
diffraction angle q. The calculated crystallite size for Au NPs is
found to be
14.38 nm using this equation, which is close to the
average particle size of
13.96 nm obtained from TEM analysis.

Fourier transform infrared (FTIR) spectroscopy analysis

Fig. 4 (a) HRTEM images of Au NPs; (b) particle size distribution
histogram of Au NPs determined from HRTEM micrograph prepared at
room temperature; (c) the selected area electron diffraction (SAED)
image of Au NPs; (d) EDX spectrum of AuNP from HRTEM.
(SAED) pattern with bright circular spots, which is shown in
Fig. 4c. The elemental composition analysis by energy disper-
sive spectroscopy (EDS) indicates the presence of gold atoms.
The result shows a strong optical absorption peak at 2.3 keV,
which is typical for the absorption of metallic Au NPs shown in
Fig. 4d.
The different phytochemicals such as vitamins and proteins
present in Abelmoschus esculentus (L.) pulp extract play an
important role in the reduction of metal ions and stabilization
of the nanoparticles. The FTIR spectra for Abelmoschus escu-
lentus (L.) pulp extract reveal several characteristic bands as
given in Fig. 6a. The peak at 3402 cm1 corresponds almost
entirely to the N–H stretching vibrations of the peptide link-
ages.57,58 The peak at 2934.5 cm1 belongs to the stretching
vibration of methyl groups.59 The peaks at 1741.1, 1427, 1631,
and 1079.7 cm1 are attributed to C]O stretching vibrations of
cyclic esters from the vitamins,60,61 germinal methyl groups,62
amide I groups of proteins,58 and the bending vibration of

X-ray diffraction (XRD) measurement

Fig. 5 shows the XRD patterns of air-dried Au NPs synthesized
from aqueous Abelmoschus esculentus (L.) pulp extract. A
number of Bragg reections can be seen with 2q values of
38.33 , 44.51 , 64.76 , and 77.8 , corresponding to the lattice
planes, which may be indexed to the (111), (200), (220), and
(311) facets of gold, respectively. Thus, the XRD pattern illus-
trates that the formed gold nanoparticles are crystalline in
nature. The broadened peaks (full width at half maximum)
indicate that the crystallite size of the gold nanoparticles lies in
the range of nanometers. This is further determined by the
calculation of average crystallite size from the Scherrer formula:
0:9l
d¼
b cos q Fig. 6 FTIR spectra of (a) pure Abelmoschus esculentus (L.) pulp
extract and (b) extract stabilized Au NPs.

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C–OH groups and the anti-symmetric stretching band of C–O–C
groups of polysaccharides and/or chlorophyll,63 respectively.
The FTIR spectra for gold nanoparticles synthesized from
Abelmoschus esculentus (L.) pulp extract is represented in Fig. 6b.
The reasonable shis in the peak positions from 3402, 2934.5,
1741.1, 1427, 1631 and 1079.7 cm1 to 3411.89, 2921.32, 1730.9,
1635 and 1019.18 cm1, respectively, of the synthesized Au NPs
indicates that the different phytochemicals present in the
Abelmoschus esculentus (L.) pulp extract are involved in reducing
and stabilizing the nanoparticles.
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In vitro cell proliferation assay
PBL, Jurkat, K562 and DL cells were exposed to Au NPs at
concentrations of 0, 1, 5, 10, 25 and 50 mg ml1 for 24 h, and
cytotoxicity was determined using the MTT assay (Fig. 7a). The
results show that Au NPs up to the concentration of 50 mg/ml
Fig. 7 (a) In vitro cell proliferation assay of Au NPs treated PBL, Jurkat,
K562, and DL cells. (b) Examination of cytotoxic effects of Abelmo-
schus esculentus (L.) pulp extract against Jurkat cells. Values are
expressed as mean  SEM of three experiments; * and # indicate
significant differences (p < 0.05) compared to the control group.
37844 | RSC Adv., 2014, 4, 37838–37848
View Article Online
Paper
produce a signicant reduction in the viability of Jurkat cells (P
> 0.05 for each). The reduction in viability of Au NP-treated
cancer cells occurs in a dose-dependent fashion. The Au NP-
exposed Jurkat cell viability signicantly decreased by 45.1%,
48.6%, 81.3% and 87.2% at 5, 10, 25 and 50 mg ml1 doses. In
the cases of K562 cells and DL cells, viability was signicantly
decreased by 38.38% and 50.165% and by 28.51% and 48.165%
at 25 and 50 mg ml1 doses, respectively. On the other hand, no
signicant reduction of PBL viability was noted with doses of Au
NPs up to 25 mg ml1. With a 50 mg ml1 dose of Au NPs, PBLs
viability was lost by 38.27%. This suggested that at higher doses
(>25 mg ml1) of Au NPs the cell specic toxicity is lost. From the
above result, it is clearly revealed that Au NPs show maximal
anticancer activity on Jurkat cells. Therefore, the remaining
experiments were performed using these cells only. On the
other hand, it was also noted that no signicant loss of cell
viability was observed for any dose of pulp extract used in the
study (Fig. 7b). These ndings clearly demonstrate the absence
of any anticancer components in the extract and the killing of
cancer cells can denitely be attributed to the Au NPs only.
The IC50 value of Au NPs against Jurkat cells was determined
using the Statistica version 5.0 (Statso, India) soware
package. From this statistical calculation, it was found that the
IC50 values of the Au NPs are 8.17 mg ml1 and 68.18 mg ml1 for
Jurkat cells and PBLs, respectively. Because a 8.17 mg ml1
concentration of Au NPs is found to be the IC50 and at this dose
no signicant loss of cell viability is observed for PBLs; there-
fore, further experiments were carried out using this concen-
tration to see the effect of Au NPs against the leukemic cell line
in vitro.
Cellular reactive oxygen species (ROS) level
Jurkat cells exposed to Au NPs for 24 h showed increased
reactive oxygen species (ROS) formation as evidenced by the
increase in uorescence intensity of the DCF. We analyzed the
results obtained by FACS by M2 and M1 percentage uorescence
variation. M1 was placed around the nonstressed Jurkat cells
(viable). M2 was placed to the right of M1 to designate positive
events. The % gated for M2 was 6.26% (viable Jurkat cells) and
81.50% (Jurkat cells treated with Au NPs). The results indicate
that a signicant (p < 0.05) elevation of ROS level is noted due to
Au NP treatment (Fig. 8a and b). The uorescence images
(Fig. 8c and d) are highly correlated to the above results.
ROS are molecules and ions containing unpaired valence
shell electrons, and being free radicals, they are highly active
and have an important role in cell signaling, leading to oxida-
tive cell damage.64 In physiologically active systems, ROS are
produced intrinsically or extrinsically within the cell. Intracel-
lular ROS generation occurs by a mitochondrial respiratory
chain reaction and membrane-bound superoxide-generating
enzyme i.e., nicotinamide adenine dinucleotide phosphate
(NADPH) oxidase mediated reactions. Moreover, reduction of
oxygen may either lead to H2O2 or OH_ via dismutation and
metal-catalyzed Fenton-like reactions, respectively.65–67 In our
study, we found that Au NPs disrupted mitochondrial
membrane integrity, as well as metabolic activity in Jurkat cells,
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Paper RSC Advances

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Fig. 9 Measurement of mitochondrial membrane potential (MMP) Au

Fig. 8 Measurement of ROS levels in Au NPs pre-treated Jurkat cells
by FACS analysis. DCF fluorescence intensity was expressed in term of
ROS production. The cell population of M2 phase was noted as the
ROS positive cells, whereas M1 denotes the viable/ROS negative cells.
Here (a): untreated Jurkat Cells, (b): Jurkat cells treated with 8.17 mg
ml1 Au NPs. Fig. (c and d): Qualitative characterization of reactive
oxygen species formation by H2DCFDA staining using fluorescence
microscopy. Here, (c): untreated Jurkat cells, (d): Jurkat cells treated
with 8.17 mg ml1 Au NPs.
conrmed by MTT assay and mitochondrial membrane poten-
tial measurements. Thus, in our present study, the probable
cause of ROS generation is the impairment of mitochondrial
electron transport chains and depletion of antioxidant defense
systems (glutathione cycle). The involvement of ROS plays a
great role in different types of anti-cancer research. Not only
that, the involvement of ROS can be considered as an effective
contribution of apoptosis in the etiology of cell death.68 Elevated
levels of ROS stimulate the release of different pro-inamma-
tory markers, including TNF-a. This TNF-a activates
nuclear factor-kB (NF-kB) and c-Jun N-terminal kinase (c-Jun
NH2-terminal kinase, JNK), and ultimately leads to cell death by
apoptotic and necrotic pathways.69
NPs in pre-treated Jurkat cells. Values are expressed as mean  SEM of
three experiments; * indicates a significant difference (p < 0.05)
compared to the control group.
EtBr-AO double staining cell morphological analysis
The results obtained from the EtBr-AO double staining are
shown in Fig. 10. This staining revels that the viable cells with
intact DNA and nuclei have a round and green nucleus. Early
apoptotic cells have fragmented DNA, which appears as several
green-colored nuclei. Late apoptotic and necrotic cell DNA is
fragmented and stained orange and red.52 From the data, it is
clear that Au NPs are able to decrease the number of viable cells
tremendously. Most of the cells exhibit typical characteristics of
apoptotic cells like plasma membrane blebbing and formation
of apoptotic bodies. The number of cells stained orange
increases signicantly. A very small number of cells are stained
red. These results indicate that most of the cells are not
undergoing necrosis and cell death occurs primarily through
apoptosis.

Alteration of mitochondrial membrane potential (MMP)

In our study, the mitochondrial membrane potential (MMP)
was estimated in terms of rhodamine (Rh) 123 uorescence
intensity. Mitochondria are known to be involved in the process
of programmed cell death; thus, we investigated the changes in
mitochondrial membrane potentials in terms of Rh 123 uo-
rescence intensity. The results showed that the percentage of
MMP decreased signicantly (P < 0.05) in the Jurkat cell line
with the increase of Au NP concentration, and reached 55.2%
when treated with an IC50 dose (Fig. 9). The impairment of MMP
in nanoparticle-treated Jurkat cells may be due to the mal- Fig. 10 Apoptotic morphology study of Jurkat cells treated with Au
NPs. Cells were treated with EtBr-AO and visualized under a fluores-
function of ATP synthesis and maintenance of ATP levels,
cence microscope. Here, (a): untreated Jurkat cells, (b): Jurkat cells
leading to either apoptosis or necrosis. Cellular apoptosis does treated with Au NPs. Yellow arrows indicate viable cells, white arrows
not depend only on depleted ATP synthesis; however, a certain indicate early apoptotic cells, blue arrows indicate late apoptotic cells
drop in ATP levels may lead to apoptosis-induced cell death.70 and red arrows indicate necrotic cells.

This journal is © The Royal Society of Chemistry 2014 RSC Adv., 2014, 4, 37838–37848 | 37845
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RSC Advances Paper

Induction of caspase-3 activity and chromosome

condensation by Au NPs
Caspase-3 plays a regulatory role in apoptotic cell death, which
is induced aer exposure to Au NPs (Fig. 11a). When Jurkat cells
were treated with IC50 concentrations of Au NPs for 24 h, cas-
pase-3 activity increased signicantly compared to untreated
cells. In addition to caspase-3 activity, chromatin condensation
was also examined by DAPI staining. When Jurkat cells were
treated with the above concentrations of Au NPs for 24 h,
chromatin condensation and fragmentation was observed in
the treated group (Fig. 11b and c). Caspase-3 activation and
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chromatin condensation/fragmentation in Jurkat cells suggest

that Au NPs causes cell death by an apoptotic process.71

Antimicrobial activity
Fig. 12 Histogram showing the radial diameters of the inhibitory
The Au NP solution exhibits excellent antibacterial activity zones by Au NPs at the same concentration (0.2 mg ml1) against
against the bacteria Bacillus subtilis, Bacillus cereus, Escherichia different microorganisms.
coli, Micrococcus luteus and Pseudomonas aeruginosa, showing a
clearing zone around the holes with bacterial growth on petri
plates by the cup plate method. The radial diameter of the
inhibiting zones of Bacillus subtilis, Bacillus cereus, Escherichia
coli, Micrococcus luteus and Pseudomonas aeruginosa are 26, 24,
15, 35 and 21 mm, respectively. The Au NPs at a concentration
of 0.2 mg ml1 show a range of specicity toward their anti-
microbial activity (Fig. 12). Fig. 13 shows the clear inhibition
zone made by Au NP solution against the strain of Bacillus
subtilis only. The clear zone indicates bacterial growth

Fig. 13 Antibacterial activity of Au NPs assayed by the agar diffusion

method in petri plates. Au NPs poured in the circular well showed a
zone of inhibition against Bacillus subtilis. The cavity of the Petri plate
was filled with Au NP solution (0.2 mg ml1).

restriction by diffused Au NP solution. At the same time, the

control sets did not show any inhibition. The results are the
mean of three separate experiments, each in triplicate. We have
already discussed earlier in this manuscript that several gold
containing salts show good antimicrobial activity. A high
concentration of gold is harmful to both the consumer and the
microbes; therefore, a smaller concentration (nano-range) is
much more applicable for therapeutic purposes. This design of
Au NP synthesis has great potential due to its antibacterial
activity.

Fig. 11 Increase of chromosome condensation and caspase-3 activity

in Jurkat cells after exposure of Au NPs for 24 h. (a) Caspase-3 activity; Conclusions
(b) control; (c) exposed to 8.17 mg ml1 Au NPs. Values are expressed as
mean  SEM of three experiments; * indicates a significant difference We have described an eco-friendly and cost-effective protocol
(p < 0.05) compared to the control group. for synthesizing Au NPs using Abelmoschus esculentus (L.) pulp

37846 | RSC Adv., 2014, 4, 37838–37848 This journal is © The Royal Society of Chemistry 2014

Paper
extract at room temperature. This extracellular synthesis of Au
NPs makes these nanoparticles a potential candidate in various
biomedical applications. It may be inferred that the phyto-
chemicals present in the Abelmoschus esculentus (L.) pulp extract
are responsible for capping the Au NPs as evidenced from FTIR
studies. The Au NPs have an average size of
14 nm and show
sufficient stability and crystallinity. The results addressed
herein disclose the anticancer activity of green synthesized Au
NPs. It is reasonable to infer that the developed Au NPs show
potent anti-proliferative efficacy against the Jurkat cell line.
Elevation of ROS and disruption of mitochondrial membrane
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potential in Au NP-exposed Jurkat cells suggests the possible
contribution of apoptosis in the etiology of cell death. The
involvement of apoptosis rather than necrosis was conrmed by
the EtBr-AO double staining method. These Au NPs have great
promise as antimicrobial agents. The application of Au NPs
based on these ndings may lead to valuable discoveries in
various applications such as medical devices and antimicrobial
agents.
Acknowledgements
The authors Md. M. R. Mollick gratefully acknowledge DST,
Govt. of India, for providing fellowship under INSPIRE fellow-
ship. B. Bhowmick wishes to thank the Council of Scientic &
Industrial Research (CSIR) Project [Vide letter no. 02 (0077)/12/
EMR-II dated 01.11.2012] and D. Mondal likes to thank CSIR,
New Delhi, for his fellowship. We also acknowledge the Center
for Research in Nanoscience and Nanotechnology (CRNN),
University of Calcutta, for instrumental facilities.
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