Deferasirox-coated iron oxide nanoparticles as a potential cytotoxic agent

Article abstract of DOI:10.1039/c6md00293e

Two broad strategies for the use of iron chelators in cancer treatment have been explored. The first was using iron chelators to reduce the iron level in cancer cells. Cancer cells typically require more iron than normal cells to mediate their generally rapid DNA synthesis and growth. The second more recent strategy is using chelators that help the redox cycling of iron to generate cytotoxic ROS (reactive oxygen species) within tumors. Both methods are currently being pursued. Deferasirox is an FDA-approved medication used to reduce chronic iron overload. Because of the low toxicity and oral administration of deferasirox, the use of this drug in cancer therapy is a new approach. Therefore, a number of recent studies have examined the potential of deferasirox as an anticancer agent. According to these strategies, the present study has reported the new role of deferasirox (DFX) loaded on iron oxide nanoparticles (Fe₃O₄@DFX) as an anticancer agent. The cytotoxicity of Fe₃O₄@DFX was screened against MCF-7, HeLa, HT-29, K-562, Neuro-2a, and L-929 cell lines by MTT assay. The surface of magnetic nanoparticles (MNPs) was first coated with (3-aminopropyl) trimethoxysilane (APTMS) and was then linked with deferasirox via an amidation reaction between-NH₂ and-COOH to form well-dispersed surface-functionalized biocompatible MNPs. The obtained nanoparticles were thoroughly characterized by various spectroscopic and microscopic methods such as SEM, FT-IR, TGA and VSM. Because of the ability of magnetic nanoparticles in biomedical applications to be targeted toward the site of action using magnetic field gradients, we suggest that MNPs loaded with deferasirox could be considered as a potential formulation, which can simultaneously be used as a theranostic agent for both cancer treatment and imaging. The obtained results provide experimental evidence that magnetite nanoparticles loaded with deferasirox induce apoptosis in cancer cell lines. Moreover, flow cytometry results confirm that the investigated compound produces a high population of apoptotic cells (69.3%), 1.2 fold higher than cisplatin (58.1%) at the same concentration, and could induce apoptosis in human leukemia cell lines (K-562).

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RESEARCH ARTICLE
Deferasirox-coated iron oxide nanoparticles as a
potential cytotoxic agent†
Cite this: DOI: 10.1039/c6md00293e
a
a
*
Faezeh Taghavi, Amir Sh. Saljooghi,
Mostafa Gholizadeha and Mohammad Ramezanib
Two broad strategies for the use of iron chelators in cancer treatment have been explored. The first was
using iron chelators to reduce the iron level in cancer cells. Cancer cells typically require more iron than
normal cells to mediate their generally rapid DNA synthesis and growth. The second more recent strategy
is using chelators that help the redox cycling of iron to generate cytotoxic ROS (reactive oxygen species)
within tumors. Both methods are currently being pursued. Deferasirox is an FDA-approved medication
used to reduce chronic iron overload. Because of the low toxicity and oral administration of deferasirox,
the use of this drug in cancer therapy is a new approach. Therefore, a number of recent studies have ex-
amined the potential of deferasirox as an anticancer agent. According to these strategies, the present study
has reported the new role of deferasirox (DFX) loaded on iron oxide nanoparticles (Fe3O4@DFX) as an anti-
cancer agent. The cytotoxicity of Fe3O4@DFX was screened against MCF-7, HeLa, HT-29, K-562, Neuro-
2a, and L-929 cell lines by MTT assay. The surface of magnetic nanoparticles (MNPs) was first coated with
(3-aminopropyl) trimethoxysilane (APTMS) and was then linked with deferasirox via an amidation reaction
between –NH2 and –COOH to form well-dispersed surface-functionalized biocompatible MNPs. The
obtained nanoparticles were thoroughly characterized by various spectroscopic and microscopic methods
such as SEM, FT-IR, TGA and VSM. Because of the ability of magnetic nanoparticles in biomedical applica-
tions to be targeted toward the site of action using magnetic field gradients, we suggest that MNPs loaded
with deferasirox could be considered as a potential formulation, which can simultaneously be used as a
theranostic agent for both cancer treatment and imaging. The obtained results provide experimental evi-
dence that magnetite nanoparticles loaded with deferasirox induce apoptosis in cancer cell lines. Moreover,
flow cytometry results confirm that the investigated compound produces a high population of apoptotic
cells (69.3%), 1.2 fold higher than cisplatin (58.1%) at the same concentration, and could induce apoptosis
in human leukemia cell lines (K-562).
Received 29th May 2016,
Accepted 18th August 2016
DOI: 10.1039/c6md00293e
www.rsc.org/medchemcomm
role in drug delivery to target tissues and to enhance stability
against degradation by enzymes.7 The superparamagnetic
1. Introduction
Recent research and development has focused on the increas-
ingly growing area of nanotechnology in modern science.
Nanomaterials, with special chemical and physical properties,
have many advantages.1 Magnetic nanomaterials, with their
unusual characteristics, are applied to cell separation,2 immu-
noassay, magnetic resonance imaging (MRI),3 drug and gene
delivery,4 minimally invasive surgery,5 hyperthermia, and arti-
ficial muscle applications.6 Also, nanoparticles have a main
nanoparticle, as one of these nanoparticles, can be manipu-
lated by an external magnetic field, which directs it to the tar-
get tissue.8 Superparamagnetic nanoparticles, due to their
unique mesoscopic physical, chemical, thermal, and mechan-
ical properties, offer a high potential for several biomedical
applications,9 including: (a) cellular therapy, such as cell la-
belling and targeting, and as a suitable tool to perform cell
biology studies involving the isolation and purification of cell
populations;10 (b) tissue repair; (c) magnetic field-guided car-
riers for localizing drugs or radioactive therapies;11 (e) tumor
hyperthermia.12 Drugs, proteins, enzymes, antibodies, and
nucleotides are directed to an organ, tissue, or tumor by
linking them to magnetic nanoparticles.13,14 The diffusion co-
efficient and the biodegradation rate are two main factors in
the release mechanism of drugs that govern the drug release
a Department of Chemistry, Ferdowsi University of Mashhad, P. O. Box 91775-
1436, Mashhad, Iran. E-mail: saljooghi@um.ac.ir; Tel: +98 513880 5527
b Pharmaceutical Research Center, School of Pharmacy, Mashhad University of
Medical Sciences, Mashhad, Iran
† The authors declare no competing interests.
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rate. The binding of drugs to the nanoparticles was investi-
gated, either in the preparation stage of nanoparticles or by
adsorption of drugs on the nanoparticles.7
nals are quoted as s (singlet), br (broad), d (doublet) and m
(multiplet). FT-IR spectra were recorded on an AVATAR-370-
FTIR Thermo Nicolet instrument. All mass spectra were
scanned on a Varian Mat CH-7 instrument at 70 eV. Reac-
tions were monitored by TLC using silica gel plates and the
products were identified by comparison of their spectra and
physical data with those of authentic samples. Melting points
were measured on an Electrothermal 9100 apparatus. Scan-
ning electron microscopy (SEM) images were taken on a Zeiss
LEO 1450 VP/35Kv instrument, Germany. Thermogravimetric
analysis (TGA) was carried out using thermogravimetric ana-
lyzer TGA-50 (Shimadzu Japan) instruments. Elemental analy-
sis was carried out using a CHNS (O) Analyzer Model FLASH
EA 1112 series made by Thermo Finnigan, Italy. Magnetiza-
tion values were obtained by using a vibrating sample magne-
tometer (VSM), 7400 Lake Shore, America.
The
compound,
(3-aminopropyl)
trimethoxysilane
(APTMS), is widely used to promote the interfacial behaviour
of inorganic oxides, including silica,15,16 ceramics,17 titania,18
and magnetic iron oxide nanoparticles (MNPs).19 Also,
APTMS has been especially attractive for biological applica-
tions, such as drug delivery, and is widely used because of its
amino functional group.20–23
This work describes APTMS-modified MNPs as a novel
support
for
4-[3,5-bisIJ2-hydroxyphenyl)-1,2,4-triazol-1-yl]-
benzoic acid (deferasirox) loading for anticancer studies. The
deferasirox was covalently bound to APTMS assembled on the
MNPs surface through an amidation reaction between the
carboxylic acid end groups on deferasirox and the pendant
amine group on the capping linker. Based on previous inves-
tigations, the drug cleaved from the nanoparticles inside
cells.24
2.3. Synthesis of 4-[3,5-bisIJ2-hydroxyphenyl)-1,2,4-triazol-1-yl]
benzoic acid (deferasirox)
The synthesis of deferasirox, (DFX or ICL670) was first
reported in 1999.25 In 2005, deferasirox became the first
FDA-approved oral alternative for the treatment of iron
overload and was subsequently approved in the EU in
2006.26 This iron chelator is used for the treatment of iron
overload in certain types of anemia, such as that caused by
β-thalassemia, and the treatment of other toxic metal
overload.27,28 Cancer cells typically require more iron than
normal cells to mediate their generally rapid DNA synthesis
and growth.29 The main aim of the present study was to in-
vestigate the potential antitumor activity of deferasirox
loaded on iron oxide nanoparticles via APTMS as a spacer
with no chelated iron ions.
The compound, 2-(2-hydroxyphenyl)-4H-3,1-benzoxazin-4-one
was prepared according to a previously reported procedure
with some modifications.30,31 The compounds, 4-hydrazino-
benzoic acid (11.5 mmol, 1.75 g) and Et3N (11.5 mmol, 1.16
g) were dissolved in boiling EtOH (80 mL). Then,
2-(2-hydroxyphenyl)-4H-3,1-benzoxazin-4-one (10.45 mmol,
2.50 g) was added to the clear solution, and the reaction mix-
ture was refluxed for an additional 2 h. After completion of
the reaction, the solution was cooled down to room tempera-
ture, and water was added until the first sign of precipitation
was observed. The mixture was then concentrated to a total
volume of 50% under reduced pressure, and aqueous 6 M
HCl (40 mL) was added. The resulting solid was filtered,
washed with water and dried for 24 h in vacuum. Yellow pow-
der; (3.11 g, yield = 80%); m.p. 264–266 °C; IR (KBr) ν: 3317,
2540, 1680 (CO), 1607, 1517, 1495, 1431, 1351, 1221, 988,
2. Experimental
2.1. Chemicals
1
752 cm−1; H NMR (C3H6O-d6400 MHz): δ 7.00 (s, 1H), 7.01–
All solvents were purchased from Merck Co. Triethylamine (NEt3),
4-hydrazino-benzoic acid, and 2-(2-hydroxyphenyl)-4H-3,1-
benzoxazin-4-one were purchased from Merck without further
purification; 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hy-
drochloride (EDC) and 7-hydroxybenzotriazole hydrate (HOBt)
were purchased from Fluka (Germany).
RPMI-1640 medium, Dulbecco's modified eagle's medium
(DMEM) and fetal bovine serum (FBS) were purchased from
GIBCO (Gaithersburg, USA). Penicillin and streptomycin were
purchased from Biochrom AG (Berlin, Germany). MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yel-
low tetrazole) was purchased from Sigma Co., Ltd. Cisplatin
was purchased from Sigma Aldrich.
7.04 (m, 3H), 7.39 (m, 2H), 7.48 (d, 1H), 7.53 (d, 2H), 8.15 (d,
2H), 8.19 (d, 1H) 10.00 (s, OH), 10.78 (s, OH) ppm; 13C NMR
(C3H6O-d6, 75 MHz): δ 113.7, 113.9, 116.6 (CH), 117.0 (CH),
119.5 (CH), 119.8 (CH), 124.0 (2 CH), 126.9 (CH), 130.4 (2
CH), 130.5, 130.7 (CH), 131.4 (CH), 132.6, 141.9 (CH), 152.1,
155.6, 156.4, 160.4, 165.7 (CO) ppm; M.S. (70 eV) m/z (%):
374 (M+); anal. calc. for C21H15N3O4: C, 67.56; H, 4.05; N,
11.25. Found: C, 67, 76; H, 3.85; N, 11.14.
2.4. Synthesis of Fe3O4 NPS
The magnetite nanoparticles (Fe3O4 MNPs) were synthesized
by a reported chemical co-precipitation technique with ferric
and ferrous ions in alkaline solution, with some modifica-
tions.32,33 FeCl2·4H2O (9.25 mmol) and FeCl3·6H2O (15.8
mmol) were dissolved in deionized water (150 mL) under Ar
atmosphere at room temperature. An NH4OH solution (25%,
50 mL) was then added dropwise (drop rate = 1 mL min−1) to
the stirring mixture at room temperature to reach a reaction
pH of 11. The resulting black dispersion was continuously
2.2. Apparatus
NMR spectra were recorded on Avance Bruker-400 MHz
spectrometers. All chemical shifts in NMR experiments are
reported as ppm and were referenced to residual solvent.
Chemical shifts are reported in parts per million and the sig-
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stirred for 1 h at room temperature and then collected by 3. Biological studies
magnet.
3.1. Cell culture methods
Human breast cancer cells MCF-7 (ATCC HTB-22), human
cervical epithelial carcinoma HeLa (ATCC CCL-2), human co-
lon cancer cell line HT-29 (ATCC HTB-38), human leukemia
cell line K-562 (ATCC CCL-243), mouse neuroblastoma cell
line Neuro-2a (ATCC CCL-131), and mouse fibroblast L-929
cell line (ATCC CCL-1) were obtained from the American Type
Culture Collection (ATCC; Manassas, VA, USA) and cultured
at 37 °C in a humidified atmosphere of 5% CO2 in air. HeLa
cells were cultured in Dulbecco's modified eagle's medium
(DMEM) with 0.1 mM nonessential amino acids, 2 mM gluta-
mine, 1.0 mM sodium pyruvate, and 5% fetal bovine serum,
at 37 °C in an atmosphere of 5% CO2. Cells were plated in
96-well sterile plates at a density of 1 × 104 cells per well in
100 μL of medium and incubated for 1 h. Also, MCF-7 and
HT-29 cells were cultured in Dulbecco's modified eagle's me-
dium (DMEM) containing 10% fetal bovine serum, 100 units
per mL of penicillin and 100 μg mL−1 of streptomycin. L-929
and K-562 cells were cultured in RPMI-1640 containing 10%
fetal bovine serum, 100 units per mL of penicillin and 100 μg
mL−1 of streptomycin. This study was approved by the Ethics
Committee of Ferdowsi University of Mashhad, Mashhad,
Iran and the Pharmaceutical Research Center, School of
Pharmacy, Mashhad University of Medical Sciences, Mash-
had, Iran. Also, basic pre-clinical research is needed before
these can be recommended for human administration.
2.5. Surface coating of MNPs by APTMS (MNP-APTMS)
The silanization of magnetic nanoparticles with APTMS
(MNP-APTMS) was done according to a previously reported
procedure by Lui et al.34
The MNPs were dispersed in 100 mL of methanol/toluene
(volume ratio, 1 : 1) as solvent and then sonicated for 30 min
and heated up to 95 °C until 50 mL of the solvent was evapo-
rated. Thereafter, methanol (50 mL) was added. The opera-
tion was repeated three times to ensure that the solution was
completely anhydrous. Subsequently, the volume of reaction
mixture was fixed at 100 mL by the addition of methanol
followed by APTMS addition and stirred for a certain time (30
min) at 30 or 70 °C under N2. The product was washed sev-
eral times with anhydrous ethanol and distilled water by
magnetic decantation and dried under vacuum. The product
was washed with toluene and Soxhlet extraction was
performed for 24 h and finally, it was dried at 40–50 °C for
6 h.
2.6. The reaction of APTMS surface-functionalized MNPs with
deferasirox
Deferasirox (4.2 mmol) was dissolved in 100 ml of toluene
and was then added to 1.1 g of the previous sample in the
presence of EDC (50 mg)/HOBt (40 mg) to activate the carbox-
ylate groups of deferasirox for amide bond formation.35 EDC/
HOBt (also NHS) crosslinking leads to amide bond formation
between activated carboxyl groups and amine groups. This
mixture was refluxed under stirring for 24 h and was then fil-
tered and washed with 100 ml of dry toluene (Soxhlet extrac-
tion) for 24 h. The solid product was dried at room tempera-
ture under vacuum and is abbreviated as MNP-APTMS-DFX.
The preparation procedure for deferasirox-loaded iron oxide
nanoparticles is shown in Scheme 1.
3.2. MTT assay in human cancer cell lines
Fe3O4-APTMS-DFX was tested for cytotoxic activity against hu-
man breast cancer cells (MCF-7), human cervical epithelial
carcinoma (HeLa), human colon cancer cell line (HT-29), hu-
man leukemia cell line (K-562), and mouse neuroblastoma
cell line (Neuro-2a); mouse fibroblast, L-929 cell line, and cis-
platin was used as a comparative standard by MTT assay. Cell
viability was evaluated by using a colorimetric method based
Scheme 1 Preparation procedure of deferasirox-loaded iron oxide nanoparticles.
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on the tetrazolium salt, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide], which is reduced by living
cells to yield purple formazan crystals. Cells were seeded in
96-well plates at a density of 2–5 × 104 cells of MCF-7, HeLa,
HT-29, K-562, Neuro-2a and L-929 per well in 200 μL of cul-
ture medium and left to incubate overnight for optimal ad-
herence. After careful removal of the medium, 200 μL of a di-
lution series of Fe3O4-APTMS-DFX in fresh medium were
added and incubation was performed at 37 °C/5% CO2 for 72
h. Fe3O4-APTMS-DFX was first solubilized in DMSO, diluted
in medium and added to the cells in final concentrations be-
tween 20 nM and 200 μM. The percentage of DMSO in the
cell culture medium did not exceed 0.3%.36 Cisplatin was
first solubilized in saline and was then added at the same
concentrations used for the other compounds. At the end of
the incubation period, the conjugate was removed and the
cells were incubated with 200 μL of MTT solution (500 μg
ml−1). After 3–4 h at 37 °C, 5% CO2, the medium was re-
moved and the purple formazan crystals were dissolved in
200 μL of DMSO by shaking. The cell viability was evaluated
by measurement of the absorbance at 570 nm using a STAT
FAX-2100 microplate reader (Awareness Technology, Palm
City, FL, USA). The cell viability was calculated by dividing
the absorbance of each well by that of the control wells (cells
treated with medium containing 0.3% DMSO). Each experi-
ment was repeated at least three times and each point was
determined from at least three replicates.
analysis by flow cytometry. Flow cytometry was performed
with a Partec PAS flow cytometer (Partec GmbH, Germany)
with Fe3O4-APTMS-DFX and cisplatin as a reference.
Also, apoptosis was detected using an in situ cell death de-
tection kit (Boehringer Mannheim Corp., Indianapolis, IN),
as described by Narla et al. and Zhu et al. Cells were incu-
bated with all compounds in 0.3% DMSO or 1 : 16 diluted
plasma samples from DFX-treated mice for 48 h at 37 °C,
and were fixed, permeabilized, incubated with a reaction mix-
ture containing TdT- and FITC-conjugated dUTP, and
counterstained with propidium iodide. Cells were transferred
to slides and viewed with a confocal laser scanning micro-
scope (Bio-Rad MRC 1024) mounted on a Nikon Eclipse E800
series upright microscope, as reported previously.37,38
3.4. Statistical analysis
IC50 values were expressed as mean standard deviation (SD)
from at least three independent experiments. Statistical tests,
including one-way ANOVA, Tukey multiple comparison, or
unpaired Student's t-tests were performed using SPSS, version
17 software. A p value of less than 0.05 was considered
significant.
4. Results
The present study has reported the synthesis of iron oxide-
based deferasirox (DFX) loaded nanoparticles (Fe3O4@DFX).
The surface of the magnetic nanoparticles (MNPS) was first
coated with (3-aminopropyl)trimethoxysilane (APTMS) and
was then linked with deferasirox via the amidation reaction
between –NH2 and –COOH to form well-dispersed surface-
functionalized biocompatible MNPs. The obtained nano-
particles were thoroughly characterized by various spectro-
scopic and microscopic methods, such as SEM, FT-IR, TGA,
and VSM.
3.3. Apoptosis assay for compound MNP-APTMS-DFX by flow
cytometry
Evaluation of apoptosis by flow cytometry is generally accom-
plished by methods that use annexin V-FITC as vital dye,
which binds phosphatidyl serine exposed on the cell surface
at the beginning of this process. The differentiation between
apoptotic and necrotic cells can be performed by simulta-
neous staining with propidium iodide (PI). Therefore,
annexin V-FITC was used as a marker of phosphatidyl serine
and PI as a marker for dead cells. This combination allows
differentiation among early apoptotic cells (annexin V-posi-
tive, PI-negative), late apoptotic/necrotic cells (annexin V-pos-
itive, PI-positive), and viable cells (annexin V-negative, PI-
negative).
4.1. Characterization of deferasirox-loaded magnetic nano-
particles (MNP-APTMS-DFX)
The conjugates were thoroughly characterized using various
spectroscopic and microscopic methods, such as FT-IR, TGA,
and SEM-EDS.
Cells lines (5 × 105) were seeded, treated with the
conjugate (Fe3O4-APTMS-DFX) and incubated for 24 h at a
concentration close to the IC50 at 37 °C. Following treatment,
the cells were harvested by trypsinization and centrifugation
at 1000 rpm for 5 min. The supernatant was removed, and
the cell pellet was washed in PBS, followed by two washes in
binding buffer (10 mM HEPES, 150 mM NaCl, 5 mM KCl, 1.8
mM CaCl2, 1 mM MgCl2). The cells were incubated with an
annexin V/FITC antibody (5 ml in 100 ml binding buffer) at 4
°C for 15 min in the dark. Samples were washed in binding
buffer, and the supernatant was discarded. The pellet was re-
suspended in 490 ml binding buffer, and 10 ml propidium
iodide (10 mg ml−1 in PBS) was added to the samples before
Fig. 1 (a) Infrared spectra of magnetic nanoparticles (MNPs), (b) MNPs
modified with 3-(aminopropyl)trimethoxysilane (MNP-APTMS) and (c)
deferasirox anchored on MNP-APTMS (MNP-APTMS-DFX).
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Fig. 1 shows the FT-IR spectrum of as-synthesized MNP-
APTMS-DFX. Uncoated Fe3O4 MNPs Fig. 1(a) show a broad
peak around 580 cm−1 due to the stretching of Fe–O bonds.39
Fig. 1(b) shows the FT-IR spectrum of Fe3O4-APTMS; the char-
acteristic peaks of Fe3O4 are shifted to 597 cm−1. The bands
at 1030 and 935 cm−1 could be attributed to the stretching vi-
bration of the Si–O bond. C–N stretching vibration and N–H
bending bands appeared at 1150 and 1550 cm−1.40 The peaks
around 2910 cm−1 and a broad absorption peak around 3430
cm−1 could be attributed to the stretching vibrations of CH2
and NH2 groups, respectively. The FT-IR spectrum of Fe3O4-
APTMS-DFX (Fig. 1(c)) indicates new peaks at 1462, 1605, and
1683 cm−1 due to the aromatic CC stretch and CN stretch
of deferasirox. Furthermore, an amide carbonyl group ap-
pears at 1710 cm−1. C–H stretching of aromatic systems and
propyl groups is observed at 2921–3100 cm−1. The broad ab-
sorption peak at 3435 cm−1 is attributed to the O–H groups
of deferasirox and the N–H stretching band of the amide. All
the observed bands revealed that the surface of the Fe3O4
NPs was successfully modified with organic moieties, in
agreement with the results reported in the literature.41
To examine the thermal stability and the organic moiety
loaded on the surface of the MNPs, thermogravimetric analy-
sis was used. Fig. 2(a) shows the typical TGA curve of Fe3O4-
APTMS. The initial weight loss up to 250 °C was probably due
to the removal of surface hydroxyls and surface adsorbed wa-
ter. The weight loss at 250–500 °C could be attributed mainly
to the evaporation and subsequent decomposition of surface-
bonded APTMS. The Fe3O4 MNPs can be transformed to
Fe2O3 and the weight would increase when the temperature
is above 500 °C. As shown in Fig. 2(b), the weight loss of
Fe3O4-APTMS-DFX is much higher than that of Fe3O4-APTMS
in the temperature range 250–500 °C, which confirms the
loading of DFX on the Fe3O4-APTMS surface. The morphology
of Fe3O4 nanoparticles before and after deferasirox loading
was studied with scanning electron microscopy (SEM). As
shown in Fig. 3(a) and (b), the nanoparticles had a small size
and a uniform spherical shape. The energy dispersive spec-
trum (EDS) indicated the presence of Fe, O, C, Si, and N ele-
ments (Fig. 4). This analysis confirms that the magnetic
nanoparticles were modified by organic moieties.
Fig.
3
SEM of: (a) Fe3O4 modified with 3-(aminopropyl)-
trimethoxysilane (MNP-APTMS) and (b) deferasirox anchored on MNP-
APTMS (MNP-APTMS-DFX).
4.1.1. The magnetization properties of magnetic nano-
particles loaded with deferasirox. The magnetic properties of
the prepared MNPs were studied by vibrating sample magne-
tometry (VSM). The magnetic hysteresis loops of Fe3O4 and
MNP-APTMS-DFX are shown in Fig. 5. The saturation magne-
tization (Ms) values at room temperature were 64.9 emu g−1
and 35.1 emu g−1, respectively. The Ms value of MNP-APTMS-
DFX was much lower than that of the Fe3O4 MNPs due to the
silica coating and DFX loading, which provide less magnetic
moment per unit mass than that of ferromagnetic core re-
gions, which would lead to a decrease in the Ms. As shown in
Fig. 5c, a dark homogeneous dispersion was observed in the
absence of an external magnetic field, while in the presence
of an external magnetic field, the black nanoparticles were
attracted to the wall of the capped tube and the solution be-
came transparent.
4.1.2. Dynamic light scattering and zeta-potential mea-
surements. The hydrodynamic diameter and zeta potential of
the compounds in all the steps were determined using dy-
namic light scattering (DLS) on a Malvern Nano ZS instru-
ment (Malvern Instruments, UK). The results are presented
as mean SD for three independent measurements (Table 1).
The particle size of MNPs-APTMS was 44 nm, which was re-
duced after loading of DFX. Due to the presence of the amino
group, MNP-APTMS conjugates had a positive zeta potential
Fig.
2 TGA of (a) iron oxide nanoparticles modified with
3-(aminopropyl)trimethoxysilane (MNP-APTMS) and (b) deferasirox
anchored on MNP-APTMS (MNP-APTMS-DFX).
Fig. 4 The EDS spectrum of deferasirox anchored on MNP-APTMS
(MNP-APTMS-DFX).
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5. Discussion
Magnetite nanoparticles (MNPs) have been widely used in
biomedical fields due to low toxicity and good compatibil-
ity.3,9,42,43 These are one of the FDA-approved materials used
in vivo and have been utilized in the magnetic separation of
biological entities, hyperthermia treatment, magnetic reso-
nance imaging (MRI), and drug delivery.9 MNPs with high
magnetization values and ultrafine particle sizes can be ma-
nipulated by an external magnetic field (MF), by which hu-
man tissues can be penetrated, indicating that employing
these nanoparticles for targetable drug delivery is possi-
ble.4,44 To develop a more suitable drug delivery system com-
posed of magnetite nanoparticles, some work needs to be
done to diminish particle agglomeration, reduce cytotoxicity,
and improve compatibility.45–48 Many candidates have been
considered for functionalization of MNPs to achieve these
goals, including polymers, biomolecules, surfactants, and or-
ganic and inorganic materials. In this study, the potential of
iron oxide nanoparticles loaded with deferasirox as effective
anticancer compounds in vitro has been investigated. Be-
cause of interesting pharmacological activities of
deferasirox, especially as anti-tumor agent,29 treatment of
toxic metals overload,27,28 and treatment of iron overload in
certain types of anaemia (β-thalassemia), ability of this drug
were considered in this study.
The present study has reported the synthesis of iron
oxide-based deferasirox (DFX)-loaded nanoparticles (Fe3-
O4@DFX). After characterization of magnetic nanoparticles
(MNPs) by various methods, Fe3O4@DFX was screened for
antitumor activity against MCF-7, HeLa, HT-29, K-562, Neuro-
2a, and L-929 cell lines, and cisplatin was used as a compara-
tive standard in an MTT assay. The results of the MTT assay
were shown as IC50 values. In comparison with the IC50 of
cisplatin, compounds with IC50 values less than 30 μM were
considered to be strongly cytotoxic; compounds with IC50
values in the range 30–50 μM were considered to be moder-
ately cytotoxic; compounds with IC50 values in the range of
50–100 μM were considered to be weakly cytotoxic; and com-
pounds with IC50 values over 100 μM were considered to be
inactive.49
Fig. 5 Magnetization curve of the (a) bare Fe3O4, (b) MNP-APTMS-
DFX, (c) photograph of the separation of magnetic nanoparticles under
an external magnetic field.
in the range 15.1–18 mV, which decreased as the degree of
loading increased. However, sufficient positive charge existed
on all MNP-APTMS-DFX, which is necessary for the interac-
tion of MNP-APTMS-DFX with negatively charged cell mem-
branes. The particle size of all the MNP-APTMS-DFX was
found to be in the range 27–38 nm by DLS analysis (Table 1),
indicating a narrow size distribution.
A strong rationale for continued research into the develop-
ment of new compounds with this structure improved its
therapeutic application. Therefore, the newly synthesized
compound was screened for its in vitro growth inhibitory ac-
tivities against five human and animal cultured cell lines,
namely human breast cancer cells (MCF-7), human cervical
epithelial carcinoma (HeLa), human colon cancer cell line
(HT-29), human leukemia cell line (K-562), mouse neuroblas-
toma cell line (Neuro-2a), and mouse fibroblast L-929 cell
line, as well as normal cells, by the MTT assay method. In
this study, cisplatin and MNPs were used as positive and neg-
ative controls, respectively. In vitro cytotoxicity of the com-
pounds was assessed by MTT bioassay in different cancer
cells after 72 h of drug exposure. We also investigated the cy-
totoxicity of the compounds at different incubation times, 24
and 48 h. Our results showed that our compounds do not
have any cytotoxic effects on cell lines at these incubation
times (24 and 48 h). The results are shown as IC50 values af-
ter 72 h continuous exposure (Table 2). The effect of the syn-
thesized compound on the mouse fibroblast cell line (L-929)
was evaluated as control, simultaneously. Our results con-
firmed that the synthesized compound has no cytotoxic ef-
fects on L-929.
The IC50 values of MNP-APTMS-DFX and cisplatin as a
comparative standard were measured in all cell lines. Such
measurements were done after 72 h of incubation using con-
centrations of several compounds in the range 20 nM to 200
μM. The selection of this concentration range was based on
our investigations in previous studies. According to our
knowledge, up to now only one study has investigated the
anti-proliferative effects of deferasirox.34 Higher concentra-
tions over 200 μM are in a flat concentration range in the
IC50 curve and can be placed in a drug-resistance range. As
shown in our results, 200 μM is a non-toxic concentration to
normal cells.
Table 1 Hydrodynamic diameter and zeta potential of MNP, MNP-
APTMS and MNP-APTMS-DFX
Compound
MNP
70
MNP-APTMS MNP-APTMS-DFX
44 35
Dynamic light
scattering DLS (nm)
Zeta potential (mV) (−) 6.9 1.35 (+) 16.8 2.65 (+) 1.9 0.43
The IC50 values obtained for MNP-APTMS-DFX were be-
tween 8.50 and 89.1 μM (the cases up to 100 μM have been
ignored), while those found for the cisplatin as positive
The experiments were done in triplicate. Data were expressed as the
mean of the triplicate.
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Table 2 Cytotoxic activity of MNP, MNP-APTMS and MNP-APTMS-DFX tested against MCF-7, HeLa, HT-29, K-562, Neuro-2a cancer cell lines and
L-929 after 72 h continuous exposure
IC50 SDa (μM)
Compound
MCF-7
HeLa
HT-29
K-562
Neuro-2a
L-929
MNP
94.3 7.9
87.6 8.8
16.9 3.53
53.9 6.1
5.94 1.47
155.9 10.9
100.4 11.5
36.1 4.75
35.5 3.9
133.9 11.8
67.0 7.8
16.9 2.93
18.5 3.62
19.3 3.46
105.1 9.8
81.5 7.3
8.50 2.24
87.9 7.5
24.7 4.71
183.2 11.7
106.7 9.5
89.1 9.53
100.7 9.4
103 9.8
241.3 17.5
126.5 10.7
141.3 12.5
102.3 9.5
0.7 0.2
MNP-APTMS
MNP-APTMS-DFX
DFX
Cisplatin
0.45 0.13
a
The concentration of the complex required to inhibit cell growth by 50%. The experiments were done in triplicate. Data were expressed as the
mean of the triplicate. IC50 > 100 μM is considered to be inactive.
control ranged between 0.45 and 100 μM (Table 2). However,
the cytotoxic activity was approximately similar for all com-
pounds tested and was threefold more than cisplatin against
the two examined cancer cell lines (HT-29 and K-562).
The IC50 values were obtained using deferasirox concentra-
tions in the nanoparticles, as described by Buchman et al.50
In this method, the total nitrogen in each stage was obtained
by elemental analysis. We used the ninhydrin quantitative
test for determination of primary amines before and after
drug (deferasirox)-loading on silica nanoparticles.
Furthermore, the cytotoxic properties of Fe3O4-APTMS-DFX
were compared to those of free deferasirox and the non-
functionalized nanoparticles, and the IC50 values of MNP and
MNP-APTMS were screened against these cancer cell lines.
Our results confirmed that MNP and MNP-APTMS were either
inactive against MCF-7, HeLa, HT-29, K-562, Neuro-2a, and
L-929 cell lines, or exhibited very poor activity against these
cancer cell lines.
In addition to cisplatin, the IC50 values of DFX were deter-
mined for a better comparison with the MNP-APTMS-DFX
conjugate. The results presented in Table 1 confirmed that in
all cell lines, magnetic nanoparticles functionalized with DFX
have higher cytotoxicity compared to deferasirox. It is notable
that the cytotoxicity of DFX is the same as both cisplatin and
MNP-APTMS-DFX against HT-29 as a normal cell line. Based
on the results of in vitro cytotoxicity studies of MNP-APTMS-
DFX, the mechanism of its cytotoxicity was evaluated using
apoptosis assay by a flow cytometry technique. The results
are shown in Table 3 and Fig. 6. The four areas in the dia-
grams stand for necrotic cells (Q1, left square on the top),
late apoptotic or necrotic cells (Q2, right square on the top),
live cells (Q3, left square at the bottom), and apoptotic cells
(Q4, right square at the bottom). As can be seen in Table 3
and Fig. 6, MNP-APTMS-DFX showed a high population of ap-
optotic cells (69.3%), nearly 1.2 fold higher than for cisplatin
(58.1%) at the same concentration. Also, flow cytometry re-
sults showed that the population of apoptotic cells in DFX is
more than in cisplatin but is less than in MNP-APTMS-DFX.
Nanoparticles loaded with anticancer drug were found to
show a higher apoptosis-inducing effect in cancer cell lines
than free drugs in vitro. These results are in agreement with
previous reports.51 The results of the current study demon-
strated that MNP-APTMS-DFX could induce apoptosis in
K-562 cancer cells. But to confirm the pro-apoptotic activity
of the conjugate, further investigation is needed to better un-
derstand the precise mechanism of action of this compound,
and basic animal studies are needed before it can be
recommended for human administration.
The population of apoptotic cells in DFX is more than in
cisplatin but is less than in MNP-APTMS-DFX.
Nanoparticles loaded with anticancer drug were found to
show a higher apoptosis-inducing effect in cancer cell lines
than free drugs in vitro. These results are in agreement with
previous reports.51 The results of the current study demon-
strated that MNP-APTMS-DFX could induce apoptosis in
K-562 cancer cells. But to confirm the pro-apoptotic activity
of the conjugate, further investigation is needed to better un-
derstand the precise mechanism of action of this compound
and basic animal studies are needed before it can be
recommended for human administration.
The induction of cell death by the MNP-APTMS-DFX con-
jugate was confirmed to be apoptosis using the TUNEL of ex-
posed 3′-OH termini of DNA with dUTP-FITC. As shown in
the confocal laser scanning microscopy images in Fig. 7,
(MNP-APTMS-DFX) treated K-562 leukemia cancer cells, ex-
amined for dUTP-FITC incorporation (green fluorescence)
Table 3 Percentages of the cell death pathways observed by the flow cytometry assay
Treatment
% vital cells
% apoptotic cells
% late apoptotic/necrotic cells
% necrotic cells
Control
DFX
MNP-APTMS-DFX
Cisplatin
77.5
35.4
24.2
34.1
16.8
32.3
37.6
30.8
4.6
28.0
31.7
27.3
1.1
4.5
6.5
7.8
These compounds were incubated for 24 h at a concentration of 5 μM, and the experiments were done in triplicate.
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Acknowledgements
The authors gratefully acknowledge the partial support of this
study (Grant No: 41256) by Ferdowsi University of Mashhad
Research Council. We are grateful for help provided by Buali
Research Institute, School of Pharmacy, Mashhad University
of Medical Sciences, Mashhad, Iran.
Fig. 6 Flow cytometric results after the exposure of K-562 cancer
cells. Four areas in the diagrams represent four different cell states:
necrotic cells (Q1), late apoptotic or necrotic cells (Q2), living cells
(Q3) and apoptotic cells (Q4).
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