Journal of Colloid and Interface Science 436 (2014) 234242

Contents lists available at ScienceDirect

Journal of Colloid and Interface Science

www.elsevier.com/locate/jcis

Quercetin conjugated superparamagnetic magnetite nanoparticles for

in-vitro analysis of breast cancer cell lines for chemotherapy applications
S. Rajesh Kumar a, S. Priyatharshni a, V.N. Babu b, D. Mangalaraj a, C. Viswanathan a, S. Kannan b,
N. Ponpandian a,
a
b

Department of Nanoscience and Technology, Bharathiar University, Coimbatore 641046, India

Proteomics and Molecular Cell Physiology Lab, Department of Zoology, Bharathiar University, Coimbatore 641046, India

a r t i c l e

i n f o

Article history:
Received 3 July 2014
Accepted 29 August 2014
Available online 16 September 2014
Keywords:
Magnetite
Nanoprism
Quercetin
Drug releasing
Anti-cancer

a b s t r a c t
The magnetic nanoparticles attract increasing interest due to their opportunities in cancer therapy and
used as drug carriers for several other diseases. The present study investigates the quercetin conjugated
superparamagnetic Fe3O4 nanoparticles for in-vitro analysis of breast cancer cell lines for chemotherapy.
A simple precipitation method was used to prepare the dextran coated Fe3O4 nanoparticles and the anticancer avonoid quercetin was conjugated on the surface via carboxylic/amine group using nanoprecipitation method. The structural, morphological and the magnetic properties of the prepared materials
were studied by using X-ray diffractometer (XRD), Fourier transformed infer-red spectrometer (FTIR),
transmission electron microscope (TEM) and vibrating sample magnetometer (VSM). The MTT (3-(4,5dimethylthiahiazol-2-yl)-2,5-diphenyl tetrazolium) assay of dextran coated Fe3O4 nanoparticles did not
exhibit notable toxicity against MCF7 cells, whereas the cytotoxicity of quercetin conjugated Fe3O4 nanoparticles increased signicantly in comparison with pure quercetin. The incubation of MCF-7 cells with
quercetin conjugated Fe3O4 nanoparticles (QCMNPs) shows signicant changes in cellular morphology
observed through uorescent microscopy. The results validate the prepared quercetin conjugated
Fe3O4 nanoparticles are promising anticancer agents for targeted drug delivery.
2014 Elsevier Inc. All rights reserved.

1. Introduction
The superparamagnetic nanoparticles with ultrane size, monodispersed shape, modied surfaces, colloidal stability, enhanced
magnetization, cellular uptake and bio-distribution are important
parameters for their use in biomedical applications [1]. These
properties enhance the usage of the magnetic nanoparticles for cell
separation, drug delivery, water purication, protein separation,
hyperthermia, diagnostics and MRI contrast agent etc. [2]. Most
of these applications require the surface modication for drug
loading or dynamic group of anchoring linkers in support of sustained drug release. The surface of the superparamagnetic Fe3O4
nanoparticles modied with PEG, PVP, PVA, peptides, carbohydrates, proteins, etc. facilitates the drug loading and controlled
release [3]. The surface functionalization with these polymers
controls the stability of the system through surface energy and also
reduces the aggregation during preparation of magnetic nanoparticles. The hydrophobic and neutrally charged natural polymeric
Corresponding author at: Department of Nanoscience and Technology, Bharathiar University, Coimbatore 641046, Tamil Nadu, India. Fax: +91 422 2422387.
E-mail address: ponpandian@buc.edu.in (N. Ponpandian).
http://dx.doi.org/10.1016/j.jcis.2014.08.064
0021-9797/ 2014 Elsevier Inc. All rights reserved.

carbohydrate dextran is used to modify the surface of the magnetite nanoparticles in the present study. It has very good biocompatibility, biodegradability and increased stability for the circulation
in the bloodstream for more time [4]. These dextran coated magnetite nanostructures can effectively used as MRI contrast agents, invivo cellular uptake in malignant neoplasms and transformation of
nanoparticles for targeted probes [58]. The dextran in the present
study might increase the bioactivity and enhances the efciency of
chemotherapy.
Cancer is one of the leading causes of death and the number of
death increases every day. The drug loaded nanostructures with
systematic administration to the targeted places for anticancer
activity in a controlled way is a challenging research [9]. Several
researchers already found that the commercial anticancer drugs
loaded on the magnetite for successful chemotherapeutic agents
[10]. These drugs have some practical difculties such as solubility,
low blood circulation, reduced drug clearance, side effects, etc. In
order to overcome these issues, an efcient drug delivery mechanism should be formulated based on the naturally available drugs
[11]. Quercetin (3,30 ,40 ,50 -7-penta-hydroxy avone) is a wellknown natural bioavonoid and is abundantly found in edible
fruits, vegetables and medicinal plants [12,13]. It has a wide range

S. Rajesh Kumar et al. / Journal of Colloid and Interface Science 436 (2014) 234242

of chemotherapeutic activities for many diseases such as anti-cancer, anti-inammatory, anti-viral and anti-oxidant [14]. However,
the strong anti-cancer therapeutic behavior is well exhibited in
colon, breast, ovarian and lung cancer cells [15,16]. Quercetin is
also used to treat malaria, HIV and cardiovascular diseases in addition to the cancer cells [1719]. The solubility in aqueous media,
permeability, oral bioavailability and biodegradation are poor for
the quercetin which limits its applications in pharmacology [20].
The quercetin is encapsulated on the dextran coated Fe3O4 nanocarriers is one of the potential techniques to circumvent the above
problems and it enhance the drugs bioavailability. The quercetin
entrapped polymeric micelles (PEG-OCL) has an excellent solubility and it inhibits the growth of cancer cells via inducing cell cycle
arrest in G2/M phase [21]. Similarly, the physicochemical properties of quercetin loaded Fe3O4 nanoparticles conrm their utility
in anticancer agent for drug delivery applications [22]. This motivates us to synthesize quercetin conjugated magnetite nanoparticles and study their in-vitro anticancer activity.
The monodispersed size and shape of the magnetic nanoparticles are inuenced by the method of synthesis. In general, the magnetite nanoparticles are prepared by solvothermal, microwave,
polyol, thermal decomposition methods using non-aqueous media.
The samples prepared with these methods are not suitable for biological applications due to their hydrophobic surface [23]. Thereby,
the simple precipitation method is adapted to synthesis the water
soluble, cost effective, eco-friendly and easily scalable magnetite
nanoparticles [24]. This method effectively controls the diameter
(1050 nm) of the particle and unique magnetic properties of magnetite nanoparticles by using the appropriate surface modication
[25]. Also, it has a limitation of non-uniform distribution of particle, agglomeration and less stability [26]. These issues will be
solved by adding urea during the preparation to control the
growth, shape, stability and prevent the agglomeration of Fe3O4
nanoparticles.
The present work explains the quercetin conjugated magnetite
nanoparticles prepared by simple precipitation method and studies their in-vitro anticancer activity in breast cancer cell lines.
The monodispersed nanoprism like morphology was prepared by
using urea as a stabilizing agent to avoid aggregation. The physicochemical properties conrm the successful conjugation of quercetin (drug) on the surface of Fe3O4 nanoparticles and the in-vitro cell
line studies conrm the inhibition of breast cancer cells. Based on
our literature survey, this is a preliminary and rst report on quercetin conjugated Fe3O4 nanoparticles for breast cancer cells and it
will be used for procient chemotherapy applications.

2. Experimental procedure
2.1. Synthesis of dextran coated magnetite (Fe3O4) nanoparticles
The simple precipitation method was employed to prepare
monodispersed nanoprism shaped Fe3O4 nanoparticles based on
the existing literature with some modications [27]. Ferric chloride
anhydrous (FeCl3), ferrous chloride (FeCl2), potassium hydroxide
pellets (KOH), urea, dextran, dimethyl sulfoxide (DMSO), ethanol
and deionized water (DI) were used as starting precursors. Quercetin dehydrate was used as an anticancer drug loaded on the magnetite nanoparticles via 1-ethyl-3[3-dimethylaminopropyl]
carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS).
All these chemicals were of analytical grade (AR) and used without
further purication. In a typical synthesis process, 5.40 g of FeCl3
anhydrous and 1.98 g of FeCl2 were dissolved in 200 ml of DI water
to form a pale yellow color solution. The required amount of KOH
and urea were dissolved in 100 ml of distilled water. These two
solutions were mixed well and allowed to stir for 30 min at room

235

temperature. Then, the required amount of dextran was dissolved

in DI water and slowly added to the mixture. The argon gas was
continuously bubbled in the solution to purge the suspended oxygen for avoiding the phase change and to improve the stability of
magnetite nanoparticles. This solution was kept at 8090 C for
90 min with constant stirring and cooled down naturally to room
temperature. The resultant precipitate was collected with the help
of a strong external magnet. The supernatant was washed several
times with DI water and ethanol under ultrasonication. This was
dried in hot air oven at 65 C overnight before further
characterization.
2.2. Synthesis of quercetin conjugated Fe3O4 nanoparticles (QCMNPs)
Quercetin conjugated magnetite nanoparticles were prepared
by nanoprecipitation method [28] with minor modications. In
general, the quercetin dihydrate is practically insoluble in water
and it is soluble in acetone. A required amount of quercetin dihydrate was dissolved in 3 ml of acetone. Then, 100 mg of dextran
coated magnetite nanoparticles was dissolved in 30 ml of dimethyl
sulfoxide (DMSO) and DI water under sonication. The pre-determined amount of N-hydroxy succinimide (NHS) and 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) was added to the
magnetite nanoparticles and allowed for ultrasonication for
30 min. Afterward, the quercetin solution was added to the above
mixture with continuous ultrasonication and allowed to stir for
24 h at room temperature. The pH of the solution was adjusted
to 10 by adding KOH solution. A resulting black color precipitate
was washed with distilled water to remove any unbound drug or
any other organic impurities. The resultant drug loaded magnetite
nanoparticles were collected using external magnet and dried
using lyophilizer/freeze drier. These drug conjugated magnetite
nanoparticles were stored below the room temperature for further
studies.
2.3. Determination of quercetin loading and encapsulation efciency
Initially, 5 mg of quercetin loaded magnetite nanoparticles was
dispersed in 10 ml of phosphate buffer solution (PBS) and centrifuged at 12,000 rpm for 30 min. The drug concentration in the
supernatant was determined by measuring the absorbance at
375 nm in a UVVisible spectrophotometer. The percentage of
QCMNPs was calculated as follows:

W0
 100%
W
W0
 100%
Encapsulation efficiency
W1

Loading efficiency

1
2

where W0 is the weight of quercetin on the Fe3O4 nanoparticles, W

is the weight of Fe3O4 nanoparticles and W1 is an amount of
quercetin added in the system.
2.4. In-vitro quercetin releasing studies
The releasing activity of quercetin from Fe3O4 nanoparticles
was investigated in the pH of 7.4 and 5.5. The drug conjugated
magnetite nanoparticles were suspended in PBS and transferred
to a dialysis bag. The bag was placed in the dialysis tubing
(MW3500, MD24, USA) and incubated in 250 ml of PBS (pH 7.4)
under shaking. The release study was performed at 37 0.5 C. At
pre-determined time intervals, 5 ml of the aqueous solution was
withdrawn and replenished with 5 ml of fresh buffer solution.
The amount of drug release was estimated by measuring the absorbance at 375 nm for quercetin (initial drug concentration in dialysis bag was 40 lg/ml) using UVVisible spectrophotometer. The
amount of drug release and the percentage of quercetin release

236

S. Rajesh Kumar et al. / Journal of Colloid and Interface Science 436 (2014) 234242

from Fe3O4 nanoparticles was measured for various time intervals.

The UVVis spectrophotometer (Hitachi, Japan) was used for this
analysis [29].
2.5. In-vitro cytotoxicity efcacy in MCF-7 cells
The cytotoxicity of the pristine and drug conjugated magnetite
nanoparticles was evaluated by MTT (3-(4,5-dimethylthiahiazol-2yl)-2,5-diphenyl tetrazolium) assay. The MCF-7 cells were rst
seeded into 96-well plates with a density of 1  104 cells per well
and incubated for 48 h. The MCF-7 cells were then treated for 24 h
with control, dextran coated, pure quercetin and quercetin conjugated Fe3O4 nanoparticles with the series of 1090 lg/ml at
37 C. The untreated MCF-7 cells served as the control. After incubation, 100 ll of MTT reagent (5 mg/ml) was added to each well.
The plate was then incubated at 37 C under 5% CO2 for 4 h. The
formazan crystals were formed and it was dissolved in 100 ll of
DMSO solution. Optical density (OD) was then taken at 620 nm
and the percentage viability of cells was calculated as the ratio of
absorbance of triplicate readings with control wells. The experiments were repeated three times and the mean viability of the
three standard deviation was determined.
2.6. Morphological observation
The MCF-7 cells were grown (1  104 cells/cover slip) and incubated with QCMNPs at their IC50 concentration and then they were
dissolved in methanol:acetic acid (3:1, v/v). The morphometric
studies were made by using cover slips and gently mounted on
the glass slides. Morphological changes of MCF-7 cells were analyzed with the Nikon (Japan) bright eld inverted light microscope
at 400x magnication.
2.7. Acridine orange (AO) and ethidium bromide (EtBr) staining
Approximately 5 ll of dye mixture (100 mg/ml AO and 100 mg/
ml EtBr) was mixed with 10 ml of cell suspension (1  104 cells/
ml) on a clean microscopic cover slip. After incubation for 35 min,
cells were visualized under uorescence microscope. The percentage of apoptotic cells was determined by the following formula [30].
Percentage of apoptotic cells

Total number of apoptotic cells

Total number of normal and apoptotic cells

2.10. Characterization techniques

XRD measurements were carried out on a PANalytical XPERT
PRO powder X-ray diffractometer by using monochromatic Cu
Ka1 (k = 1.54056 ) radiation. The surface chemistry and chemical
bonding of the nanoparticles were identied by Fourier transform
infrared (FTIR) spectra obtained from Nicolet 6700 (Thermo Scientic, USA) in transmission mode with samples in 1% KBr pellets,
operating from the wave numbers between 400 and 4000 cm1.
The zeta potential was measured by photon correlation spectroscopy (PCS) with a zeta potential analyzer add-on unit (Nicomp
Zetasizer 380ZLS, Urbana, IL, USA). The morphology of the magnetite nanostructures was characterized by transmission electron
microscope (Hitachi H600) operating at 80 kV. The magnetic properties of the samples were measured by using vibrating sample
magnetometer (VSM, EV X model) at room temperature.
3. Results and discussion
3.1. Structural analysis
The crystalline nature and phase analysis for the pristine, dextran coated and quercetin conjugated Fe3O4 nanoparticles has been
conrmed by XRD analysis as shown in Fig. 1(ac). All the diffraction peaks were very well matches with cubic inverse spinel structure of magnetite nanoparticles (JCPDS # 86-1354). The FWHM of
the major diffraction peak (3 1 1) was used to calculate the average
grain size. The estimated average grain sizes using Scherrer formula for the pristine, dextran coated and quercetin conjugated
magnetite nanoparticles were 15, 12 and 10 nm respectively. The
small decrease in the intensities of the diffraction peak for the dextran coated nanoparticles as shown in Fig. 1b may be due to the insitu addition of dextran on the surface of magnetite nanoparticles
and it also signicantly reduces the average crystalline size. The
intensity of the XRD pattern of QCMNPs was also reduces to 50%
as compared to pristine Fe3O4 nanoparticles as shown in Fig. 1c.
This may be due to the reduction in the grain size and amorphous
material (drugs) covers the surface of magnetite nanoparticles. No
other extra peaks were observed in the XRD pattern conrms the
high purity of the magnetite nanoparticles. The high intensity
and broadening of the peaks conrms the prepared magnetite
nanoparticles were good crystalline nature and signicant reduction in the grain size with the addition of surfactants.

 100
3

2.8. Fluorescence microscopic observation

The results were reported as mean SEM (standard error mean)

for the three independent experiments. The analysis of variance
test was used to estimate the difference between the untreated
control and test sample. P < 0.05 was considered to be statistically
signicant.

20

25

30

35

40

45

422

50

55

440

511

220

311

400

2.9. Statistical analysis

c
Intenisty (arb.units)

Hoechst 33258 is a blue uorescent dye having low cell toxicity

and sensitive to chromatin condition in cells, which can be used to
examine the nuclei change in apoptotic cells. The MCF-7 cells treated without magnetite nanoparticles were used as control. These
cells were pre-incubated with different concentration of QCMNPs
for 24 h and washed twice with PBS for 5 min. The treated cells
were stained with 0.5 ml of uorescent dye for 5 min. After washing with PBS for 5 times, the distributions of the cells were analyzed using uorescence microscopy [31].

60

65

70

2 (degrees)
Fig. 1. XRD pattern for the (a) pristine, (b) dextran coated and (c) quercetin
conjugated Fe3O4 nanoparticles.

S. Rajesh Kumar et al. / Journal of Colloid and Interface Science 436 (2014) 234242

The Fourier transform infrared spectroscopy is an appropriate

technique to identify the chemical adsorption of functional group
on the magnetite nanoparticles. Fig. 2(ad) shows the FTIR spectra
for pristine, dextran coated, pure quercetin and quercetin conjugated Fe3O4 nanoparticles. The broad absorption peak at 585 cm-1
represents the FeAO bond of spinel structure validates the formation of magnetite nanoparticles as shown in Fig. 2a. The broad
peaks at 3450 and 1632 cm1 represent the OH stretching vibration of the hydroxyl molecules. The peak in Fig. 2b for the FeAO
bond was slightly shifted from 585 to 581 cm1 may be due to
the surface interaction between dextran and magnetite nanoparticles. The absorption bands at 1024 and 1166 cm1 represent the
characteristic stretching vibration of alcoholic hydroxyl CAOH
and bending vibration of CAH bond. These results strongly evidence that the dextran was successfully grafted onto the magnetite
nanoparticles. Fig. 2c shows the FTIR spectrum for the pure quercetin molecule and the major peaks were present in the range of
9001700 cm1. All the peaks represent the hydroxyl, carboxylic
and aromatic groups present in the quercetin molecule [12]. The
broad peaks at 3390 and 1664 cm1 were assigned to stretching
and bending vibration of hydroxyl group. Also, the prominent
peaks at 1028 and 945 cm1 represent the C@O stretching vibration and CAH bending vibration of aromatic group. All the quercetin peaks were very well matches with the existing literature
values [13,15]. The peak at 560 cm1 in Fig. 2d represents the shift
in FeAO bond compared to pristine nanoparticles due to encapsulation of quercetin molecules and it inuences the insignicant
change in electronic structure of Fe3O4 nanoparticles [28]. The
peak at 1655 cm1 represents the C@O stretching vibration of carboxylic group. The peak at 1453 cm1 attributed to the CAN
stretching vibration conrms the amide group present in the surface of the Fe3O4 nanoparticles with the addition of EDC and NHS
molecules. Therefore, the zero length linkers such as carbonyl
and amine groups were used to conjugate the quercetin on the
magnetite nanoparticles. The small peaks in the range of 2800
3000 cm1 attributes to the symmetric and asymmetric CH2
stretching vibration. The peaks at 945 and 1028 cm1 match very
well with pure quercetin peaks indicates the linkage between drug
and magnetite nanoparticles and it was highlighted in Fig. 2(c and
d). Thus, it is conrmed that these peaks represent the successful
conjugation of quercetin on the Fe3O4 nanoparticles.

237

3.2. Morphology and size distribution analysis

The morphology and size of the nal products were obtained by
recording the transmission electron microscopic images. The
obtained microscopic image for the pristine Fe3O4 nanoparticles
in Fig. 3a shows spherical aggregates with smooth surface in the
size of 10 nm. These spherical aggregates were obtained due to
the absence of driving force in the surface of the particles or more
plausible due to the inter-particle interaction [32]. The distance
between the two particles was below 10 nm and the particles
would form an aggregate [33]. It conrms the nal nanoparticles
were prone to get aggregated due to strong magnetic dipoledipole
interaction without using surfactant. The Fig. 3b shows that the
dextran coated Fe3O4 nanoparticles have monodispersed prism like
shape with a size of 20 nm and with signicantly reduced aggregation. The dextran was attached to the specic crystal planes which
inuences the oriented attachment to fabricate Fe3O4 nanoprism
[34]. The urea may control the aggregation and can be used as a
stabilizing agent to generate individual prism shape and to induce
the growth of Fe3O4 nanoparticles. It was conrmed that the surfactant dextran plays a dual role as controlling the shape of spherical aggregates and shielding of phase changes of Fe3O4
nanoparticles. Also, this biocompatible polymer is important to
provide a hydrophilic surface. This might improve the water dispersible and act as a capping agent to load the quercetin on the
surface. It also serves a signicant role in sustained drug release
in bio-molecules.
Fig. 4(a and b) shows the hydrodynamic size distribution of the
dextran coated Fe3O4 nanoparticles and quercetin conjugated
Fe3O4 nanoparticles determined by dynamic laser scattering
(DLS) method. The size distribution of the dextran coated Fe3O4
nanoparticles was found to be 63 nm whereas the quercetin conjugated Fe3O4 nanoparticles was 72 nm. The increase in the average
size is probably due to the effective conjugation of quercetin on the
dextran coated Fe3O4 nanoparticles via EDC/NHS reaction [35]. The
small increase in the hydrodynamic size conrms the excellent colloidal stability of the quercetin conjugated Fe3O4 nanoparticles.
The average hydrodynamic size determined from DLS is slightly
higher than the diameter observed from TEM. It is known that
the TEM demonstrate the images of dried particles whereas DLS
provides the data for the particles swollen in solution leads to
aggregation [36,37].
3.3. Surface charge analysis

d
1317
1655 1403 945
1453
1028

2910
2998
3442

560

Transmittance

1166
1024
894
790
581

3421

a
1624
4000

3500

3000

2500

2000

1500

585
1000

500

Wavenumber (cm-1)
Fig. 2. FTIR spectra for the (a) pristine, (b) dextran coated, (c) pure quercetin and
(d) quercetin conjugated Fe3O4 nanoparticles.

Zeta potential measurement was utilized to study the surface

charge of the magnetite nanoparticles. Generally, it was used to
study the physical stability, cellular uptake, bio-distribution and
interaction of bio-molecules. The surface charges of the dextran
coated and quercetin loaded Fe3O4 nanoparticles were shown in
Fig. 5(a and b). The potential value of the dextran coated Fe3O4 nanoparticles has negative surface charge of 25.6 mV due to the deprotonated surface. The quercetin conjugated Fe3O4 nanoparticles
illustrate the positive surface charge of 6.14 mV due to strong electrostatic interaction between drug and magnetite protonation of the
surface groups via carboxyl molecules and the presence of anionic
ions from the drug molecules. This positively charged surface
quickly interacts with the negatively charged cell surface and provides efcient delivery of the quercetin drug. Also, it may enhance
the deep penetration into tumor cells to attain a homogeneous allocation of drugs [38]. Therefore, the positive surface charges have
more cellular uptake and interaction than negatively charged
Fe3O4 nanoparticles. The result conrms the increased efciency of
the anticancer drug by using positive charged nanoparticles. The
reason for the change in surface charge from negative to positive
that may be due to more drug residuals was binding in the surface

238

S. Rajesh Kumar et al. / Journal of Colloid and Interface Science 436 (2014) 234242

50 nm

50 nm

Fig. 3. TEM images of the (a) pristine and (b) dextran coated Fe3O4 nanoparticles.

Fig. 4. DLS graph for the (a) dextran coated Fe3O4 nanoparticles and (b) quercetin conjugated Fe3O4 nanoparticles.

600000

a
b

Total Counts

500000

400000

300000

200000

100000

0
-200

-150

-100

-50

50

100

150

200

Zeta Potential (mV)

Fig. 5. Zeta potential graph of (a) dextran coated and (b) quercetin conjugated
Fe3O4 nanoparticles.

of the Fe3O4 nanoparticles. The change in the surface charge conrms the successful conjugation of organics or drugs materials on
the surface of the magnetite nanoparticles. These properties may
inuence the change in surface reactivity of breast cancer cell lines
due to change in radical species and oxidation sites.
3.4. Magnetic properties
The magnetic properties of the pristine, dextran coated and
quercetin conjugated Fe3O4 nanoparticles were studied by measur-

ing the hysteresis loop at room temperature by using vibrating

sample magnetometer. The hysteresis loop for all these samples
is shown in Fig. 6(ac). It conrms that the obtained magnetite
nanoparticles have superparamagnetic behavior at room temperature with negligible hysteresis loop. This superparamagnetic
behavior which occurs due to the thermal energy overcomes the
anisotropic barrier and randomizes the magnetic moment. This
behavior is very much essential for the biomedical applications
due to the zero magnetization when the applied magnetic eld is
removed. The saturation magnetization value of pristine, dextran
coated and quercetin conjugated Fe3O4 nanoparticles was found
as 41, 30 and 26 emu/g respectively. The saturation magnetization
of the pristine nanoparticles was less compared with the bulk
value of 90 emu/g due to the surface spin canting and smaller particle size effects [39]. The decrease in the saturation magnetization
of the other samples may be due to the effect of higher density of
polymer or quercetin on the surface of magnetite nanoparticles in
addition to the above reasons [40]. This polymer molecule produces a magnetic dead layer on the surface of the magnetite nanoparticles that quenches the surface magnetic moments [41]. The
result clearly demonstrates the chemisorptions of non-magnetic
layers decreases the magnetization and conrms the surface
entrapment of the Fe3O4 nanoparticles.
3.5. Drug loading and releasing proles
The drug loading and encapsulation efciency were found to be
6.08 0.3 and 81.6% respectively. Generally, the drug releasing
activity depends on size, surface characteristics, internal force
between drug and nanoparticles, rate of hydration and dehydration
of polymers. The in-vitro drug release behavior of quercetin loaded

239

S. Rajesh Kumar et al. / Journal of Colloid and Interface Science 436 (2014) 234242

not easy to break the bond for the release of drug. Also, the water
molecules may form a barrier for hydrophobic molecules of quercetin to decrease the degradation by Fe3O4 nanoparticles [42].
However, on decreasing the pH to 5.5, the solution environment
changed and the quercetin was quickly released from the Fe3O4
nanoparticles. The higher percentage of release was obtained due
to the deprotonation of carboxylic and amine groups linkage
between dextran and quercetin molecules. The in-vitro drug
release is not only dependent on the pH of magnetite nanoparticles
but also dependent on the physicochemical properties of the dextran stabilizer.

50

a
b
c

Magnetization (emu/gm)

40
30
20
10
0
-10
-20
-30

3.6. In-vitro anticancer activity

-40

The control, dextran coated, pure quercetin and quercetin conjugated Fe3O4 nanoparticles were incubated individually with
MCF-7 cells to study the inhibition of the cell growth rate using
MTT assay. The corresponding cell viability graph for different concentrations was shown in Fig. 8(ad). The absorption percentage of
treated cells compared to control cells was used to calculate the
percentage of cell viability. In the case of dextran coated magnetite
nanoparticles, the cell viability was obtained at 7890% in a broad
concentration range of 1100 lg/ml for 24 h compared with control cells. This clearly demonstrated that the magnetite carrier
anchored with dextran had good biocompatibility due to strong
electrostatic attraction [9]. The small increase in cytotoxicity might
be due to the exposure of magnetite nanoparticles to the cells.
These magnetite particles interact with the plasma membrane over
a period of time and causes cell lysis to induce cell shortage as
compared to control [43]. Signicantly, the QCMNPs exhibits a cell
viability of below 25% at the same concentration which could be
much higher compared to pure quercetin (50%). The toxicity of
the breast cancer cells might happen due to the damaged DNA
via drug release due to magnetic oxidation. This may be attributed
to the strong binding between the MCF-7 cells and quercetin molecules due to the partial release of quercetin from the magnetite
nanoparticles. The efcient drug release was also conrmed from
drug releasing proles at different pH conditions. This conrms
the high therapeutic effect of the drug quercetin which greatly
enhances the anticancer activity.
Together with the cytotoxicity assay, the studies on cell morphology and the integrity of the membrane were used to estimate
the toxicity or biocompatibility. This information is of great

-50
-25

-20

-15

-10

-5

10

15

20

25

Applied Field (KOe)

Fig. 6. Room temperature hysteresis loops for the (a) pristine, (b) dextran coated
and (c) quercetin conjugated Fe3O4 nanoparticles.

Fe3O4 nanoparticles was studied in PBS at pH 7.4 with the temperature of 37 C to maintain the experimental condition similar to
body uids. Signicantly, the dextran can regulate the release of
quercetin from the Fe3O4 nanoparticles in intracellular and extracellular environments as shown in Fig. 7(a and b). The two stage
release prole was exhibited by the loaded Fe3O4 nanoparticles
i.e. initial burst release at pH 7.4 and further fast release rate at
pH 5.5 respectively. Generally, the drug release rate was initially
fast and becoming slower as time prolonged. Initially, 25% of drug
was released gradually in 4 days with the pH 7.4 and decreasing
the pH to 5.5, the same percentage was obtained for 2 days. The
quercetin release was gradually increased and reached a maximum
value of 69 and 81.6% in 16 days for the two different pH. This
higher drug releasing rate may be due to the feeble interaction
between the Fe3O4 nanoparticles and quercetin molecules. Already
available report for the maximum release of quercetin was 14.5%
after 96 h due to strong interaction between the nanoparticles
and drug molecules [21]. The maximum percentage of quercetin
release was obtained under acidic conditions in the present study.
In the weak basic condition (pH-7.4), the carboxylic bond between
the dextran and Fe3O4 nanoparticles was tightly bound and it was

120

90

pH 7.4
pH 5.5

100

70

% of cell viability

Cumulative Release (%)

80

60
50
40

80

60

40

30
20

20
10

0
0

0
0

10

12

14

16

15

30

45

60

75

90

Concentration (g/ml)

Time (days)
Fig. 7. Drug release prole of quercetin conjugated Fe3O4 nanoparticles and the
values are mean SD (n = 3).

Fig. 8. Cytotoxicity of (a) control, (b) dextran coated, (c) free quercetin and (d)
quercetin conjugated Fe3O4 nanoparticles at different concentrations and the values
are mean SD (n = 3).

240

S. Rajesh Kumar et al. / Journal of Colloid and Interface Science 436 (2014) 234242

relevance to estimate their interactions and it is highly dependent

on the surface characteristics, size and shape of the nanoparticles.
Fig. 9(ac) shows the optical microscopic images of control and
cells treated with QCMNPs with different concentrations. The large
number of cells became rounded and shrunken which implies the
adherence between the breast cancer cells and magnetite nanoparticles. There was a very high reduction in cell count due to the
release and interaction of the drug quercetin. The obtained results
show that the treatment with 73 lg/ml of QCMNPs shows more
survival rate when compared to cells treated with 31 lg/ml. The
cells were stained with AO and EB dyes. The apoptotic cells were
observed by red nucleus due to the DNA binding capacity of EB
and the viable cells had a green nuclear uorescence as shown in
Fig. 9(df). The cells show green uorescence when the AO can
penetrate the normal cell membrane. The orange color cells conrm apoptosis in breast cancer cells. The apoptotic bodies were
formed as a result of nuclear shrinkage and blebbing. The necrotic
cells were also observed due to loss of membrane integrity which
further inhibited the growth of the breast cancer cells [30].
The uorescent microscopic study for the MCF-7 breast cancer
cells treated with QCMNPs exhibited a bright blue color emission
(DAPI) as shown in Fig. 9(gi). The uorescent image shows that
the nuclear fragmentation and chromatin condensation of the cancerous cells which leads to the apoptosis while the normal cells
exhibit round intact nucleus. When the concentration of QCMNPs
increases, the bright blue color contrast also enhanced due to the
increase in apoptosis cells or cell death. The quercetin showed an
induced apoptosis and halted the cell cycle mechanism due to
strong electrostatic attraction between them [44]. This further conrmed the conjugation of quercetin in the nanoparticles which can
be used as potential anticancer agent against breast cancer cells.

Control

3.7. Proposed mechanism

The schematic illustration of the reaction mechanisms for the
overall synthesis process of the pristine and quercetin loaded
Fe3O4 nanoparticles were shown in Fig. 10. The average crystalline
size of the prepared pristine and dextran coated magnetite nanoparticles were less than 20 nm. The XRD conrms the phase purity
and the cubic inverse spinel structure of the magnetite nanoparticles. The in-situ coating of dextran molecules on magnetite could
signicantly improve the formation of agglomeration free nanoprism shape from agglomerated spherical particles (pristine nanoparticles) due to strong driving repulsive force and also provides
high degree of biocompatibility. In addition, the biocompatible
dextran coating enhances the blood circulation time and thus making it a promising candidate for targeted delivery applications. The
FTIR analysis conrms the hydrophobic quercetin molecules were
conjugated with dextran coated magnetite nanoparticles via the
addition of water soluble NHS and EDC. The prepared nanoparticles
have superparamagnetic behavior with a positive surface charge to
improve the suspension stability. The saturation magnetization
was decreased due to the conjugation of drug molecules and it
induces the non-collinear spins at the particle surfaces compared
with pristine nanoparticles.
The positive surface charge was obtained due to the anionic surface of quercetin molecules which enhances the cell interaction
due to strong electrostatic force. So, the quercetin drug releasing
mechanism could be dependent on the different pH conditions.
In the basic conditions, the drug release rate is much less compared
with the acidic environment. However, acidic pH could have more
plausible effects to interact with cancerous cells than normal cells.
Hence, the present study shows that the drug release of 81.6% due

31 g/ml

73 g/ml

DAPI

AO/EtBr

Cell Morphology

Fig. 9. Phase contrast microscopic images of cellular morphology, AO/EtBr staining and DAPI of (A,D,G) control, (B,E,H) 31 lg/ml, (C,F,I) 73 lg/ml of quercetin loaded Fe3O4
treated with MCF-7 cells.

241

S. Rajesh Kumar et al. / Journal of Colloid and Interface Science 436 (2014) 234242

Fe3O4

OH

COOH

OH

HO

O
OH n

Dextran

O CH2
OH

HOOC

OH m

COOH
OH
Quercetin

OH

Fe3O4

QCMNPs

NHS/EDC

O CH2

Endosome
ROS

Nucleus
Drug
Releasing
Mitochondria

OH

HO

GADD m -RNA

Biodegradation

OH
OH

Caspase 3

DNA
Fragmentation

Apoptosis

C
O

COOH
OH
Fe3O4
COOH
OH

QCMNPs

QCMNPs interact
with cancer cells

Fig. 10. Schematic illustration for the proposed formation mechanism of dextran and quercetin conjugated Fe3O4 nanoparticles and its anticancer activity.

to the increasing circulation time can be attributed solely to the

magnetite nanoparticles. This higher drug release percentage
might be due to the formation of ester bond between drug and
dextran coated magnetite nanoparticles as shown in Fig. 10. The
ester bond can be easily broken in biological environment due to
enhanced nucleophilic reaction.
The dextran coated magnetite nanoparticles did not show any
induced cytotoxicity due to the biocompatible coating and the
absence of anticancer drugs which shows cytocompatible to
MCF-7 cells. In the case of QCMNPs, the cytotoxicity gradually
increased to 75% when increasing the concentration from 10 to
100 lg/ml. This is due to the rapid release rate of quercetin owing
to the breakage of ester bond from Fe3O4 nanoparticles and interacts with particular cancer cells in acidic environment. Thus, it was
conrmed that the loss of cell viability is due to the enormous
release rate of the quercetin from Fe3O4 nanoparticles. The results
conrm that the Fe3O4 nanoparticles could be considered as a sustained drug delivery system with quercetin with improved bioavailability for normal cells but toxic to the breast cancer cells.
The contrast in the cell images observed from optical microscope
further conrms the nuclear fragmentation, cell shrinkage and
membrane blebbing that undergo apoptosis in MCF-7 cells. The
DAPI image shows the bright blue contrast which implies the fragmentation of cells in 73 lg/ml. So, higher concentrations of the
QCMNPs induce rapid apoptosis due to good internalization and
specic binding of drug molecules compared to lower concentrations [45]. All these results clearly conrm the potential anticancer
activity of quercetin loaded Fe3O4 nanoparticles against breast cancer cells due to their fast release of drug in the acidic environment
with different concentrations based on the apoptosis mechanism.
4. Conclusions
In conclusion, quercetin was chosen as a potent anticancer drug
and it was conjugated onto the dextran coated magnetite nanoparticles by nanoprecipitation method. The successful conjugation of

quercetin on the surface of Fe3O4 nanoparticles was conrmed by

FTIR analysis. The monodispersed prism like shape with superparamagnetic behavior was conrmed from TEM and VSM analysis. The
controlled release of quercetin from the nanoparticles was
observed by varying the pH. The release rate of quercetin was more
rapid in the acidic condition than the basic environment and there
was an increase in tumoricidal action. Also, the QCMNPs could
induce apoptosis in MCF-7 cells in a dose dependent manner.
The uniform size, shape, and surface properties of the quercetin
conjugated magnetite nanoparticles signicantly enhance the anticancer activity in breast cancer cells. The present study proposes
that magnetite nanoparticle based drug conjugation may be an
effective way to deliver an anticancer drug to the targeted cancer
cells and also enhances the possible usage in biological
applications.
Acknowledgments
The authors would like to thank DST-SERB, Government of India
for the nancial support under FAST TRACK Young Scientist
Scheme. Also, the author SRK would like to thank DST-PURSE programme, Government of India for providing nancial support to
carry out this work successfully.
References
[1] A.Z.M. Badruddoza, L. Junwen, K. Hidajat, M.S. Uddin, Colloids Surf. B: Biointerf.
92 (2012) 223.
[2] A. Joshi, S. Solanki, R. Chaudhari, D. Bahadur, M. Aslam, R. Srivastava, Acta
Biomater. 7 (2011) 3955.
[3] L.H. Reddy, J.L. Arias, J. Nicolas, P. Couvreur, Chem. Rev. 112 (2012) 5818.
[4] G. Liu, R.Y. Hong, L. Guo, G.H. Liu, B. Feng, Y.G. Li, Colloids Surf. A: Physicochem.
Eng. Aspects 380 (2011) 327.
[5] B.R. Jarrett, M. Frendo, J. Vogan, A.Y. Louie, Nanotechnology 18 (2007) 035603.
[6] R. Weissleder, A. Bogdanov, M. Papisov, Magn. Reson. Q 8 (1992) 55.
[7] A. Moore, E. Marecos, A. Bogdanov, R. Weissleder, Radiology 214 (2000) 568.
[8] D. Thorek, A. Chen, J. Czupryna, A. Tsourkas, Ann. Biomed. Eng. 34 (2006) 23.
[9] J.M. Shen, T. Yin, X.Z. Tian, F.Y. Gao, S. Xu, ACS Appl. Mater. Interf. 5 (2013)
7014.

242

S. Rajesh Kumar et al. / Journal of Colloid and Interface Science 436 (2014) 234242

[10] G. Ding, Y. Guo, Y. Lv, X. Liu, L. Xu, X. Zhang, Colloids Surf. B: Biointerf. 91
(2012) 68.
[11] T.D. Schladt, K. Schneider, H. Schild, W. Tremel, Dalton Trans. 40 (2011) 6315.
[12] A. Kumari, S.K. Yadav, Y.B. Pakade, B. Singh, S.C. Yadav, Colloids Surf. B:
Biointerf. 80 (2010) 184.
[13] T.H. Wu, F.L. Yen, L.T. Lin, T.R. Tsai, C.C. Lin, T.M. Cham, Int. J. Pharm. 346
(2008) 160.
[14] S. Chakraborty, S. Stalin, N. Das, S.T. Choudhury, S. Ghosh, S. Swarnakar,
Biomaterials 33 (2012) 2991.
[15] M. Kakran, N.G. Sahoo, L. Li, Colloids Surf. B: Biointerf. 88 (2011) 121.
[16] P. Wang, D. Heber, S.M. Henning, Food Funct. 3 (2012) 635.
[17] Y. Gao, Y. Wang, Y. Ma, A. Yu, F. Cai, W. Shao, G. Zhai, Colloids Surf. B: Biointerf.
71 (2009) 306.
[18] H. Patir, S.K.S. Sarada, S. Singh, T. Mathew, B. Singh, A. Bansal, Free Rad. Biol.
Med. 53 (2012) 659.
[19] M.Y. Wong, G.N.C. Chiu, Nanomed.: Nanotechnol., Biol., Med. 7 (2011) 834.
[20] A.R. Patel, P.C.M. Heussen, J. Hazekamp, E. Drost, K.P. Velikov, Food Chem. 133
(2012) 423.
[21] R. Khonkarn, S. Mankhetkorn, W.E. Hennink, S. Okonogi, Euro. J. Pharm.
Biopharm. 79 (2011) 268.
[22] A.C.H. Barreto, V.R. Santiago, S.E. Mazzetto, J.C. Denardin, R. Lavin, Giuseppe
Mele, M.E.N.P. Ribeiro, Icaro G.P. Vieira, Tamara Goncalves, N.M.P.S. Ricardo,
P.B.A. Fechine, J. Nanopart. Res. 13 (2011) 6545.
[23] H.L. Ding, Y.X. Zhang, S. Wang, J.M. Xu, S.C. Xu, G.H. Li, Chem. Mater. 24 (2012)
4572.
[24] C. Pereira, A.M. Pereira, C. Fernandes, M. Rocha, R. Mendes, M.P.F. Garca, A.
Guedes, P.B. Tavares, J.M. Greneche, J.P. Araujo, C. Freire, Chem. Mater. 24
(2012) 1496.
[25] D. Baba, Y. Seiko, T. Nakanishia, H. Zhang, A. Arakaki, T. Matsunaga, T. Osaka,
Colloids Surf. B: Biointerf. 95 (2012) 254.
[26] H. Qu, H. Ma, A. Riviere, W. Zhoub, C.J. OConnor, J. Mater. Chem. 22 (2012)
3311.

[27] M. Mahmoudia, A. Simchi, M. Imani, M.A. Shokrgozar, A.S. Milani, U.O. Hfeli,
P. Stroeve, Colloids Surf. B: Biointerf. 75 (2010) 300.
[28] H. Sun, X. Jiao, Y. Han, Z. Jiang, D. Chen, Eur. J. Inorg. Chem. 1 (2013) 109.
[29] M.L. Barreto, M.G. Teixeira, F.I. Bastos, R.A. Ximenes, R.B. Barata, L.C. Rodrigues,
The Lancet 377 (2011) 1877.
[30] R. Vivek, R. Thangam, K. Muthuchelian, P. Gunasekaran, K. Kaveri, S. Kannan,
Process Biochem. 47 (2012) 2405.
[31] V.N. Babu, S. Kannan, Int. J. Biol. Macromol. 51 (2012) 1103.
[32] B. Chang, X. Zhang, J. Guo, Y. Sun, H. Tang, Q. Ren, W. Yang, J. Colloid Interf. Sci.
377 (2012) 64.
[33] S. Kumar, C. Ravikumar, R. Bandyopadhyaya, Langmuir 26 (2010) 18320.
[34] X. Wang, J. Qiu, J. Qu, Z. Wang, D. Su, RSC Adv. 2 (2012) 4329.
[35] R. Qiao, Q. Jia, S. Huwel, R. Xia, T. Liu, F. Gao, H.J. Galla, M. Gao, ACS Nano 6
(2012) 3304.
[36] J. Li, P. Yao, Langmuir 25 (2009) 6385.
[37] B. Sivakumar, R.G. Aswathy, Y. Nagaoka, M. Suzuki, T. Fukuda, Y. Yoshida, T.
Maekawa, D.N. Sakthikumar, Langmuir 29 (2013) 3453.
[38] F.M. Kievit, F.Y. Wang, C. Fang, H. Mok, K. Wang, J.R. Silber, R.G. Ellenbogen, M.
Zhang, J. Contr. Release 152 (2011) 76.
[39] D.H. Zhang, G.D. Li, J.X. Li, J.S. Chen, Chem. Commun. 29 (2008) 3414.
[40] W. Fua, H. Yang, H. Bala, S. Liu, M. Li, G. Zou, Mater. Chem. Phys. 100 (2006)
246.
[41] B. Aslibeiki, P. Kameli, I. Manouchehri, H. Salamati, Curr. Appl. Phys. 12 (2012)
812.
[42] D. Li, J. Tang, J. Guo, S. Wang, D. Chaudhary, C. Wang, Chem. Eur. J. 18 (2012)
16517.
[43] A.B. Salunkhe, V.M. Khot, N.D. Thorat, M.R. Phadatare, C.I. Sathish, D.S.
Dhawaleb, S.H. Pawar, Appl. Surf. Sci. 264 (2013) 598.
[44] P. Knekt, R. Jarvinen, R. Seppanen, M. Hellovaara, L. Teppo, E. Pukkala, A.
Aromaa, Am. J. Epidemiol. 146 (1997) 223.
[45] S.C. Wuang, K.G. Neoh, E.T. Kang, D.W. Pack, D.E. Leckband, J. Mater. Chem. 17
(2007) 3354.