Study the biological effect of nanosize ferric oxides prepared from low cost organic materials against human breast cancer cells

Article abstract of DOI:10.1016/j.inoche.2018.08.001

The aim of this paper is to fully exploit the medical potential of α?Fe₂O₃ as a promising material for anticancer agent. Herein, Fe₂O₃ nanoparticles (NPs) have been synthesized using thermal decomposition method

Full text of DOI:10.1016/j.inoche.2018.08.001

Accepted Manuscript
Study the biological effect of nanosize ferric oxides prepared from
low cost organic materials against human breast cancer cells
Mohamed S. Hegab, Samy M. El-Megharbel, M.M. AL-
Majthoub, Abdel Majid A. Adam, Moamen S. Refat
PII:
DOI:
Reference:
S1387-7003(18)30496-9
doi:10.1016/j.inoche.2018.08.001
INOCHE 7067
To appear in:
Inorganic Chemistry Communications
Received date:
Revised date:
Accepted date:
31 May 2018
11 July 2018
1 August 2018
Please cite this article as: Mohamed S. Hegab, Samy M. El-Megharbel, M.M. AL-
Majthoub, Abdel Majid A. Adam, Moamen S. Refat , Study the biological effect of
nanosize ferric oxides prepared from low cost organic materials against human breast
cancer cells. Inoche (2018), doi:10.1016/j.inoche.2018.08.001
This is a PDF file of an unedited manuscript that has been accepted for publication. As
a service to our customers we are providing this early version of the manuscript. The
manuscript will undergo copyediting, typesetting, and review of the resulting proof before
it is published in its final form. Please note that during the production process errors may
be discovered which could affect the content, and all legal disclaimers that apply to the
journal pertain.

ACCEPTED MANUSCRIPT
Study the biological effect of nanosize ferric oxides prepared from low cost organic
materials against human breast cancer cells
Mohamed S. Hegab1, Samy M. El-Megharbel 2,3*, M.M. AL-Majthoub2, Abdel
Majid A. Adam2 and Moamen S. Refat2,4
1Deanship of Supportive studies (D.S.S.), Taif University, Al-Hawiah, Taif, P.O. Box 888
Zip Code 21974, Saudi Arabia
2Department of Chemistry, Faculty of Science, Taif University, Taif, Saudi Arabia
3Department of Chemistry, Faculty of Science, Zagazig University, Zagazig, Egypt
4Department of Chemistry, Faculty of Science, Port Said University, Port Said, Egypt
*Corresponding author: S.M.El-Megharbel
E-mail address: samyelmegharbel@yahoo.com
1

ACCEPTED MANUSCRIPT
Abstract
The aim of this paper is to fully exploit the medical potential of α-Fe2O3 as a
promising material for anticancer agent. Herein, Fe2O3 nanoparticles (NPs) have been
synthesized using thermal decomposition method for the iron(III) Schiff base (N,N`-
disalicylidene-l,2-phenylenediamine (H2dsp), N,N`-disalicylidene-3,4-diaminotoluene
(H2dst), 4-nitro-N,N`-disalicylidene-1,2-phenylenediamine (H2ndsp) and N,N`-
disalicylidene ethylenediamine (H2salen)) complexes as precursors. The calcined of
Fe2O3 NPs were comprehensively characterized based on UV-Vis, FT-IR, SEM, TEM
and XRD. The band gap energy of the synthesized α-Fe2O3 NPs particles are 1.75-1.92
eV range. Evaluation of cytotoxicity against human breast cancer cell line (MCF-7) for
the synthesized α-Fe2O3 hematite nanoparticles was performed.
Keywords: α-Fe2O3 hematite, Schiff base, nanoparticles, XRD, FT-IR, UV.
2

ACCEPTED MANUSCRIPT
1- Introduction
Many experimental techniques were performed to make development in the
preparation of iron oxide NPs [1-5], the common techniques are thermal decomposition,
co-precipitation, electrochemical, hydrothermal, sonochemical, microemulsion [1,2],
laser pyrolysis [3], iron reducing bacteria [4,5]. These procedures dependent on essential
parameters e.g. stability, low cost, grain size and biocompatibility of Fe2O3 NPs. Simple
organic molecules are a good precursors, which resist the agglomeration of iron oxide
NPs through the synthesis process enhance the medical compatibility of iron oxide nano-
particles [6]. The search of the best way to prepare iron oxide NPs and their composites
is mainly related to the great input of these particles in biomedical applications [7-9]
being easily attached to bioactive molecules. The iron oxide NPs have been used as light
scattering labels and luminescent optical markers [7,8] because of their potential
applications as contrasting materials for magnetic resonance imaging [10,11], in vitro cell
separation [12], targeted drug delivery [13] and hyperthermia [14]. It can be noted that
particle size and surface morphology play an important role in the microbial applications
[15,16]. The side effect of residual iron oxide nanoparticles with their toxicity and high-
level accumulation on target tissue or organ can be overcame by functionalized bioactive
molecules [17]. The magnetic nanoparticles have been used in a cancer diagnosis and
therapy [18], drug delivery compounds [19] and in water remediation [20]. Magnetic
random-access memory devices [21].
Schiff bases are capable of forming coordinate bonds with many of metal ions
through both azomethine group and phenolic group or via its azomethine or phenolic
groups [22,23]. A large number of Schiff bases and their complexes are significant
interest and attention because of their biological activity including anti-tumor,
antibacterial, fungicidal and anti-carcinogenic properties [24,25]. Tetradentate Schiff
3

ACCEPTED MANUSCRIPT
bases are well known to coordinate with various metal ions forming stable compounds
and some of these complexes are recognized as oxygen carriers [26]. Literature survey
reveals that little work has been done on tetradentate Schiff base complexes of Fe(III), on the
other hand, the four N,N`-disalicylidene-l,2-phenylenediamine (H2dsp), N,N`-
disalicylidene-3,4-diaminotoluene (H2dst), 4-nitro-N,N`-disalicylidene-1,2-phenylene
diamine (H2ndsp) and N,N`-disalicylidene ethylenediamine (H2salen) Schiff base ligands
have been studied with divalent metal ions such as Ni(II) and Zn(II) [27,28]. Great attention
has been paid to the synthesis of α-Fe2O3 hematite nanoparticles, due to their application
in various areas of modern technology [1-3]. This study focuses on the synthesis of
iron(III) Schiff base complexes obtained from N,N`-disalicylidene-l,2-phenylenediamine
(H2dsp), N,N`-disalicylidene-3,4-diaminotoluene (H2dst), 4-nitro-N,N`-disalicylidene-
1,2-phenylenediamine (H2ndsp) and N,N`-disalicylidene ethylenediamine (H2salen)
simple organic compounds. The complexes were characterized by elemental analysis
CHN, FT-IR, UV–Vis spectroscopy, conductivity measurement and magnetic moment.
The spectroscopic studies suggested the octahedral structures for the synthesized four
iron(III) complexes. Then the complexes were used as precursors for preparation of
Fe2O3 NPs via solid-state thermal decomposition route. The chemical structure of the
iron(III) oxide is studied by FT-IR, XRD, SEM and TEM. The goal of this paper is to
synthesis of Fe2O3 nanoparticles using a simple low cost chemical route and studied the
effect of nanostructure form and optical absorption properties against clinically important
of cancer cell lines of MCF-7.
4

ACCEPTED MANUSCRIPT
2- Experimental
2-1- Chemicals
All chemicals in this work were fine and procured from Sigma-Aldrich Chemical
Company. All solvents were used of spectroscopic grade.
2-2- Synthesis of H2dsp, H2dst, H2ndsp and H2salen Schiff base chelates (Fig. 1)
The Schiff base ligands (H2dsp, H2ndsp and H2salen) have been synthesized by
the method of Garg and Kumar [27,28]. These three NNOO tetradentate Schiff base
ligands were prepared by condensations between diamines and hydroxyaldehydes in
ethanol/methanol with 1:2 stoichiometry and were purified by recrystallization from a
dichloromethane/hexane mixed solvent through the partial evaporation of the more
volatile dichloromethane.
H2dst Schiff base ligand was prepared by dissolving 25 mmol (3.050 g) of 3,4-
diaminotoluene in 100 mL of ethanol and stirred for 3 h. After that 50 mmol (6.10 mL) of
salicylaldehyde was mixed in 150 mL of ethanol. The 3,4-diaminotoluene solution was
added to the salicylaldehyde solution using an overhead stirrer for complete mixing.
Some yellow precipitate resulted. The crude product was re-crystallized from
dichloromethane/hexane (1:2) mixed solvent.
2-3- Synthesis of iron(III) complexes
1 mmol of FeCl3.6H2O and 1 mmol of each (H2dsp, H2dst, H2ndsp and H2salen)
Schiff base compounds were dissolved in 50 mL of ethanol to form homogeneous
solutions. Few drops of ammonia solution were added until pH 6–8. The mixtures were
allowed to reflux under stirring for 3 h. After that, the resulting mixtures were cooled at
room temperature and filtrated then washed three times by ethanol to removed un-reacted
5

ACCEPTED MANUSCRIPT
o
materials and dried at 60 C. The purity of four iron(III) complexes were checked using
TLC plates. Several attempts to obtain a single crystal suitable for X-ray crystallography
failed. However, the spectroscopic and magnetic data are able to predict the possible
structures of the synthesized iron(III) Schiff base complexes (see Fig. 2). The
experimental results are in a good agreement with those of calculated values for the
o
suggested formula with high melting points 250 C, indicating the thermal stability of
the synthesized complexes. The analytical and physical properties of complexes I-IV are
summarized as follows.
[Fe(H2dsp)(Cl)(H2O)] complex I, F.Wt (C20H16ClFeN2O3), M.Wt (423.65 g/mol),
Mp  250 oC. Anal.: (Calc.) Found, %: C, (56.70) 56.39; H, (3.81) 3.87; N, (6.61) 6.59;
Fe, (13.18) 13.09. ΛM = 19 Ω−1cm2mol−1. [Fe(H2dst)(Cl)(H2O)] complex II, F.Wt
(C21H18ClFeN2O3), M.Wt (437.68 g/mol), Mp  250 oC. Anal.: (Calc.) Found, %: C,
(56.63) 56.43; H, (4.15) 4.06; N, (6.40) 6.32; Fe, (12.76) 12.54. ΛM = 13 Ω−1cm2mol−1.
[Fe(H2ndsp)(Cl)(H2O)] complex III, F.Wt (C20H15ClFeN3O5), M.Wt (468.65 g/mol), Mp
 250 oC. Anal.: (Calc.) Found, %: C, (51.26) 51.08; H, (3.23) 3.11; N, (8.97) 8.90; Fe,
(11.92) 11.87. ΛM = 17 Ω−1cm2mol−1. [Fe(H2salen)(Cl)(H2O)] complex IV, F.Wt
(C16H16ClFeN2O3), M.Wt (375.61 g/mol), Mp  250 oC. Anal.: (Calc.) Found, %: C,
(51.16) 51.08; H, (4.29) 4.17; N, (7.46) 7.39; Fe, (14.87) 14.65. ΛM = 10 Ω−1cm2mol−1.
2-4- Synthesis of Fe2O3 oxide
Iron(III) oxide, Fe2O3 was synthesized by the thermal decomposition technique
[1,2]. The mentioned iron(III) Schiff base complexes were well grinding to confirm the
homogeneous powder and then heated at 800 oC for 3 h in a classic furnace. The residual
solid powders were washed several times with hot methanol to remove the organic
impurities.
6

ACCEPTED MANUSCRIPT
2-5- Instrumentations
The microanalytical analyses of %C, %H and %N percentages were calculated
using a Perkin Elmer CHN 2400 (USA). The iron metal ion content was determined
using the atomic absorption technique, an Atomic Absorption Spectrometer model PYE-
UNICAM SP 1900. The molar conductivities of iron(III) complexes with 10-3 mol/cm3
concentration in DMSO solvent were measured using Jenway 4010 conductivity meter.
The UVVis absorption spectra were recorded in DMSO solvent within 800-200 nm
range using a UV2Unicam UV/Vis Spectrophotometer fitted with a quartz cell of 1.0
cm path length. The infrared spectra with KBr discs were recorded on Bruker FTIR
Spectrophotometer (4000–400 cm-1). SEM images were obtained using a Jeol Jem1200
EX II Electron microscope at an acceleration voltage of 25 kV. X-ray diffraction patterns
of the samples were recorded on X Pert Philips X-ray diffractometer. All the diffraction
patterns were obtained by using CuK1 radiation, with a graphite monochromator at
0.02/min scanning rate. The transmission electron microscopy images were performed
using JEOL 100s microscopy. Magnetic measurements were carried out on a Sherwood
Scientific magnetic balance in the micro analytical laboratory using Gouy method.
Calibration: Two very good solid calibrants are Hg[Co(CNS)4] and [Ni(en)3](S2O3). They
are easily prepared pure, do not decompose or absorb moisture and pack well. Their
susceptibilities at 20˚C are 16.44×10-6 and 11.03×10-6 c.g.s. Units, decreasing by
0.05×10-6 and 0.04×10-6 per degree temperature raise respectively, near room
temperature. The cobalt compound, besides having the higher susceptibility, also packs
rather densely and is suitable for calibrating low fields, while the nickel compound with
lower susceptibility and density is suitable for higher field [29]. Here we are used
Hg[Co(CNS)4] only as calibrant.
7

ACCEPTED MANUSCRIPT
2-6- Anti-cancer activity
Mammalian cell lines: MCF-7 cells (human breast cancer cell line) was obtained
from VACSERA Tissue Culture Unit.
Chemicals Used: Dimethyl sulfoxide (DMSO), crystal violet and trypan blue dye
were purchased from Sigma (St. Louis, Mo., USA). Fetal Bovine serum, DMEM, RPMI-
1640, HEPES buffer solution, L-glutamine, gentamycin and 0.25% Trypsin-EDTA were
purchased from Lonza. Crystal violet stain (1%): It composed of 0.5% (w/v) crystal
violet and 50% methanol then made up to volume with ddH2O and filtered through a
Whatmann No.1 filter paper.
Cell line Propagation [30,31]:
The cells were propagated in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, HEPES
buffer and 50µg/ml gentamycin. All cells were maintained at 37ºC in a humidified
atmosphere with 5% CO2 and were subcultured two times a week. Cytotoxicity
evaluation using viability assay: For cytotoxicity assay, the cells were seeded in 96-well
plate at a cell concentration of 1×104 cells per well in 100µl of growth medium. Fresh
medium containing different concentrations of the test sample was added after 24 h of
seeding. Serial two-fold dilutions of the tested chemical compound were added to
confluent cell monolayers dispensed into 96-well, flat-bottomed microtiter plates
(Falcon, NJ, USA) using a multichannel pipette. The microtiter plates were incubated at
37ºC in a humidified incubator with 5% CO2 for a period of 48 h. Three wells were used
for each concentration of the test sample. Control cells were incubated without test
sample and with or without DMSO. The little percentage of DMSO present in the wells
(maximal 0.1%) was found not to affect the experiment. After incubation of the cells for
at 37°C, various concentrations of sample were added, and the incubation was continued
8

ACCEPTED MANUSCRIPT
for 24 h and viable cells yield was determined by a colorimetric method. In brief, after
the end of the incubation period, media were aspirated and the crystal violet solution
(1%) was added to each well for at least 30 minutes. The stain was removed and the
plates were rinsed using tap water until all excess stain is removed. Glacial acetic acid
(30%) was then added to all wells and mixed thoroughly, and then the absorbance of the
plates were measured after gently shaken on Microplate reader (TECAN, Inc.), using a
test wavelength of 490 nm. All results were corrected for background absorbance
detected in wells without added stain. Treated samples were compared with the cell
control in the absence of the tested compounds. All experiments were carried out in
triplicate. The cell cytotoxic effect of each tested compound was calculated. The optical
density was measured with the microplate reader (SunRise, TECAN, Inc, USA) to
determine the number of viable cells and the percentage of viability was calculated as [1-
(ODt/ODc)]x100% where ODt is the mean optical density of wells treated with the tested
sample and ODc is the mean optical density of untreated cells. The relation between
surviving cells and drug concentration is plotted to get the survival curve of each tumor
cell line after treatment with the specified compound. The 50% inhibitory concentration
(IC50), the concentration required to cause toxic effects in 50% of intact cells, was
estimated from graphic plots of the dose response curve for each conc. using Graphpad
Prism software (San Diego, CA. USA).
3-Results and discussion
3-1-Identification of iron(III)Schiff base complexes
The results of microanalytical and physical analyses of the synthesized iron(III)
N,N`-disalicylidene-l,2-phenylenediamine (H2dsp), N,N`-disalicylidene-3,4-diamino
9

ACCEPTED MANUSCRIPT
toluene (H2dst), 4-nitro-N,N`-disalicylidene-1,2-phenylenediamine (H2ndsp) and N,N`-
disalicylidene ethylenediamine (H2salen) Schiff base complexes are summarized in
experimental section. These complexes are stable and hygroscopic in static air, higher
melting points, insoluble in water and most of organic solvents except for DMSO and
DMF. The elemental analyses results confirm that these solid complexes were isolated
with 1:1 M:L stoichiometry.
3-1-1-Conductance measurements
Molar conductance is one of an important analyzed tools that help to recognize to
the arrangement of chelates and geometrical structures around central metal ions of the
new synthesized complexes within the limits of their solubility. Also the molar
conductance values indicate that the anions may be exhibits outside the coordination
sphere or inside or absent. Firstly, the conductance values of the free H2dsp, H2dst,
H2ndsp and H2salen Schiff base ligands in DMSO solvent (1.0×10-3 mol) were in the
range (4.50-7.00) Ω-1cm-1mol-1. Molar conductance (ΛM) of 10-3 M DMSO solutions of
the four iron(III) Schiff base complexes was determined. The values for iron(III)
complex are within the range of 3441 Ω-1 cm2 mol-1 consistent with 1:1 electrolyte [32].
Thus these complexes behaves as 1:1 nonelectrolyte; consequently, one chloride ion is
present inside the coordination sphere [33]. This supported a method of testing the degree
of ionization of the complexes, the molecular ions that a complex liberates in solution (in
case of presence anions outside the coordination sphere), the higher will be its molar
conductivity and vice versa [33]. These results were strongly supported with the micro
analysis data, where Cl ions are not detected by addition of AgNO3 solution, this tested
well matched with CHN data, the chloride ion can be detected inside the coordination
sphere of the complex by the degradation of the complex using concentrated nitric acid.
10

ACCEPTED MANUSCRIPT
3-1-2-Magnetic moments and electronic spectral studies of complexes
The experimental magnetic susceptibility measurements of four iron(III) Schiff
base complexes gave values within 5.11-5.42 B.M. range that supported the high spin
state of iron(III) ion in the octahedral geometry complexes [34]. These values are very
close to the spin only value of 5.9 B.M. iron(III) complexes with a low spin-high spin
equilibrium are found to be having a subnormal magnetic moment [35]. The UV–vis
spectra of the free H2dsp, H2dst, H2ndsp and H2salen Schiff base ligands and their four
iron(III) complexes were scanned in DMSO solvent. The electronic spectra of free Schiff
base ligands display two electronic transitions, one of them exhibited at range 200-280 nm due to
ππ* transition for the molecular orbitals of the phenolic chromophore. These peaks were
shifted to higher wavelength after complexation, this may be due to the donation of lone pair of
electrons on the oxygen atom of the phenoxy group toward iron(III) central metal atom [36]. The
second electronic transition presence within range of 300-400 nm can be assigned to nπ*
transition due to molecular orbitals of azomethine group C=N chromophore and the benzene ring.
These transition bands have been shifted under complexation confirmed that, the nitrogen atom of
imine group coordinated to the iron(III) metal ion [37]. The iron(III) Schiff base complexes
exhibit three transition bands around 580-730, 300-380 and 200-260 cm−1 due to
6
6A1g→4T2g, A1g→4T1g and L→MCT or M→LCT transitions suggesting high spin
octahedral geometry [38].
3-1-3- FT-IR spectra of iron(III) Schiff base complexes
FTIR spectra of the iron(III) Schiff base complexes are display in Fig. 3. The
distinguish infrared spectral bands of free H2dsp, H2dst, H2ndsp and H2salen Schiff base
ligands and its iron(III) complexes are assigned and listed in Table 1. The spectra of the
free Schiff base ligands exhibit a medium broad intensity bands within 25003353 cm-1
11

ACCEPTED MANUSCRIPT
range which were assigned to the intramolecular Hbonding vibration (OHN). These
spectral bands are disappear under complexation (see Table 1). The stretching vibration
motions of the C=N azomethine groups for the free H2dsp, H2dst, H2ndsp and H2salen
Schiff base ligands are observed at range 16101640 cm-1. These bands were shifted to
higher frequencies after complexation toward iron(III) metal ion, that confirmed the
participation of nitrogen atom of azomethine group in coordinated to the iron(III) metal
ion [39]. The bands which were exhibited at 13101350 cm-1 range in case of free Schiff
base ligands can be assigned to the phenolic (CO) group vibrations [39]. Upon
complexation, these bands displace towards higher or lower frequencies attributed to the
chelation of oxygen to the iron(III) metal ions. In case of H2ndsp Schiff base iron(III)
complex, the bands at 1510 and 1340 cm-1 are assigned to the nitro group attached to
H2ndsp Schiff base ligand undergo, this meaning that nitro group is uncoordinated to the
iron(III) metal ions. The new bands presence at 450-460 and 420425 cm-1 range are
assigned to the ν(FeO) and ν(RuN) modes, respectively [39]. In case of iron(III)
complexes, the vibration bands exhibited within 3400 cm-1 are assigned to stretching
vibration mode of ν(OH) coordinated water molecule [40].
Table 1: FTIR frequencies (cm-1) of free Schiff base ligands and its iron(III) complexes
Compounds
Assignments
ν(C=N)
ν(OH) H2O
ν(OH)
2500-2680
--
ν(C-N)
1400
1455
1400
1460
1380
1400
1370
ν(ph-O) ν(Fe-O)
ν(Fe-N)
--
H2dsp
I
--
3415
--
1610
1604
1615
1608
1615
1605
1640
1325
1359
1350
1377
1330
1340
1310
--
460
--
425
--
H2dst
II
2550-2750
--
3420
--
460
--
425
--
H2ndsp
III
3338-3353
--
3422
--
460
--
420
--
H2salen
2650-2750
12

ACCEPTED MANUSCRIPT
IV
3420
--
1603
1395
1343
450
425
3-2-Identification of iron(III) oxide
The [Fe(Sb)(Cl)(H2O)] (Sb = H2dsp, H2dst, H2ndsp and H2salen) Schiff base
complexes were used as simple organic precursors for preparation of hematite Fe2O3
nanoparticles (NPs) via solid-state thermal decomposition route at 800 oC.
3-2-1-Infrared spectrum of synthesized Fe2O3
The FTIR spectrum of Fe2O3 in Fig. 4, the strong bands at 526 and 443 cm-1 are
assigned to the stretching vibration mode of Fe–O [41].
3-2-2-UV-vis spectrum of synthesized Fe2O3
The α-Fe2O3 in nano-crystalline state is one of the most successful materials
utility for photocatalytic and photoelectrochemical applications owing to its narrow band
gap ~ 2.0 eV, which could collect up to 40% of the solar spectrum energy. The α-Fe2O3
NPs exhibits great stability in aqueous solutions and good magnetic properties [42].
The UV–Vis absorption spectra of α-Fe2O3 hematite synthesized with thermal
decomposition of [Fe(Sb)(Cl)(H2O)] (Sb = H2dsp, H2dst, H2ndsp and H2salen) Schiff
base complexes as an organometallic precursors show that the absorption spectra curves
display a broad absorption in the range of 250–750 nm wavelength (Fig. 5). These data
are agreement with data presented in literature [43]. The optical band gap (Eg) for α-
Fe2O3 hematite NPs was calculated using Tauc plot [44].
αhν = A(hν  Eg)n
(Tauc plot)
13

ACCEPTED MANUSCRIPT
where α is the absorption coefficient, A is constant, hν is photon energy, and Eg is the
allowed energy gap, n = ½ for allowed direct transition and n = 2 for allowed indirect
½
transition [45]. Fig. 6 shows the plot of (αhν) versus hν. The energy gap can be
determined from the intercept of the linear absorption edge part with the energy axis. The
decrease in the particle size of synthesized α-Fe2O3 with different H2dsp, H2dst, H2ndsp
and H2salen Schiff base precursors are consequently due to increasing the optical band
gap of 1.75–1.92 eV where, the relation between them is inverse relationship.
3-2-3-XRD spectrum of synthesized Fe2O3
Fig. 7 refers to the X-ray powder diffraction spectrum of the synthesized αFe2O3
NPs based on thermal decompositions technique for the [Fe(Sb)(Cl)(H2O)] (Sb = H2dsp,
H2dst, H2ndsp and H2salen) Schiff base complexes as precursors. The crystallinity and
phase structure were discussed dependent on Fig. 7. The diffraction patterns of this
spectrum are detected at 2θ = 24.14, 33.18, 35.51, 40.75, 49.35, 54.03, 57.51, 62.32,
63.78 and 71.81o which may be assigned to 012, 104, 110, 113, 024, 116, 018, 214, 300
and 119 crystalline structures attributed to pure single rhombohedral phase of αFe2O3
NPs [46]. The narrow sharp peaks indicate that the hematite products are highly
crystalline, implying that the high purity of synthesized hematite nanoparticles is
obtained by using this precursors. These XRD data are agreement with the JCPDS card
no: 33-0664. The crystallite size is evaluated by Scherer’s equation [47].
D = K/βcosθ
K is the shape factor (0.89),  is the wavelength (0.15418 nm, CuKα), β is the full width
at half maximum (FWHM), and θ is the Bragg diffraction angle. The (104) Bragg
reflection was selected to calculate the crystallite size α-Fe2O3, the average of crystalline
sizes associated with XRD data ranged between 4050 nm. The sizes of nanoparticles
14

ACCEPTED MANUSCRIPT
range were proved that the particle size increased with the raise of the molecular weights
of precursor because the higher atomic weights enhanced the merge of crystal nucleus,
agglomeration of particles and enhancement in the density of nucleation centers in the
synthesized samples [48,49].
3-2-4- Scanning electron micrographs (SEM) of Fe2O3
Fig. 8A-D shows a scanning electron microscopy photos of αFe2O3 NPs which
were synthesized from thermal decomposition technique of iron(III) complexes of H2dsp,
o
H2dst, H2ndsp and H2salen Schiff base precursors at 800 C at 10,000X magnification.
The crystallinity of the αFe2O3 nanoparticles were associated and the grain sizes are
nearly uniform morphology with different features dependent on the chemical structures
of H2dsp, H2dst, H2ndsp and H2salen Schiff base precursor chelates. The surface
morphologies of synthesized αFe2O3 solid powders were investigated using SEM
analytical tool as shown in Fig. 8A-D. These showed that the synthesized samples possess
solid powder clusters, geometrical-like, flowers-like and ice crushed-like shapes with
uniform size distributions for αFe2O3 NPs synthesized from H2dsp, H2dst, H2ndsp and
H2salen Schiff base precursor, respectively. The chemical compositions of αFe2O3 NPs
was synthesized from thermal decomposition of iron(III) H2ndsp Schiff base complex at
o
800 C was examined using EDX as refer in the Fig. 9. The EDX spectrum refers that
this spectrum is consisted of Fe and O, without any other elements detected in the
spectrum, this meaning the association of αFe2O3 NPs in pure form.
3-2-5- Transmittance electron micrographs (TEM) of Fe2O3
Particle size and uniform morphological of the αFe2O3 are determine using the
analyze of TEM micrographs. The TEM images of αFe2O3 NPs resulted from the
15

ACCEPTED MANUSCRIPT
o
complexity route and calcined at 800 C for 3 h are display in Fig. 10. After the thermal
o
decomposition in static air at 800 C, the αFe2O3 NPs were found within the range of
40–50 nm with medium spherical granules like-shape. In this study, the association of
nanoparticles size depends on the type of precursor used in the synthesis of αFe2O3. The
nanoparticle size of αFe2O3 in case of the thermal decomposition of iron(III) 4-nitro-
N,N`-disalicylidene-1,2-phenylenediamine (H2ndsp) Schiff base complex III at 800 oC is
better than other N,N`-disalicylidene-l,2-phenylenediamine (H2dsp), N,N`-disalicylidene-
3,4-diaminotoluene (H2dst), and N,N`-disalicylidene ethylenediamine (H2salen)
precursors. This can be assigned due to H2ndsp Schiff base chelate has been included the
nitro group that is acts as a foaming agent which reduce the particle size of nanoparticles
synthesized in the thermal decomposition process.
3-2-6- Anticancer assessments of synthesized Fe2O3
Evaluation of cytotoxic effects of synthesized αFe2O3 using H2dsp, H2dst,
H2ndsp and H2salen Schiff base precursor chelates were tested against breast carcinoma
cells (MCF-7 cell line) according to the method of Mosmann [30]. Cytotoxicity
evaluation using viability assay against MCF-7 cell line was investigated at different
µg/mL concentrations. Inhibitory concentration fifty (IC50) were found to be 4.10, 2.50,
0.90 and 3.50 µg for αFe2O3 obtained using H2dsp, H2dst, H2ndsp and H2salen
precursor complexes, respectively. These data were compared with inhibitory activity of
cis-platin (IC50 = 5.71 0.5 µg/mL) as standard drug against breast carcinoma cells.
Results revealed that αFe2O3 synthesized using H2ndsp precursor has showed significant
growth inhibition activity on tumor MCF-7 cell lines (IC50 = 0.90 µg/mL) compared to
their corresponding organic Schiff base ligands, so, this can be used as a potential anti-
cancer agent. Given the presence of nitro group on the aromatic ring (H2ndsp), it is
16

ACCEPTED MANUSCRIPT
realistic to expect that it is quite lipophilic, whilst the methyl or ethylene substituted
ligands (H2dst) and (H2salen) should be quite hydrophilic. Also, nitro group for the
H2ndsp Schiff base ligand was effective in breaking the agglomeration size and
generating Fe2O3 NPs. Findings from the majority of nanotoxicity studies conducted with
Fe2O3 NPs have found the production of reactive oxygen species as a major reason
behind cell death [50]. Fig. 11 is the diagrams of inhibitory activity of synthesized
αFe2O3 against breast carcinoma cells MCF-7 with different chelate precursors.
Conclusions
Fe2O3 NPs were successfully synthesized using thermal decomposition method of
the iron(III) Schiff bases complexity via a simple route. The formation of Fe2O3 NPs was
confirmed by spectroscopic (UV-Vis and FT-IR) and morphological (SEM, TEM and
XRD) tool of analyses. The synthesized Fe2O3 NPs were spherical and crystalline, with
an average size of 40-50 nm. The synthesized Fe2O3 NPs exhibited an efficient
anticancer effect against human breast cancer (MCF-7) cells. This study demonstrates
that fast, inexpensive and simple synthesis of Fe2O3 NPs using (H2dsp, H2dst, H2ndsp
and H2salen) Schiff bases could be a competitive alternative to conventional chemical
methods. It is also suggested that the synthesized Fe2O3 NPs with efficient anticancer
activity hold huge potential for medical and industrial applications.
17

ACCEPTED MANUSCRIPT
N OH
N OH
N
N
OH
OH
H3C
H2dsp
H2dst
N
N
OH
OH
N
OH
OH
O2N
N
H2ndsp
H2salen
Fig. 1. Chemical structures of N,N`-disalicylidene-l,2-phenylenediamine (H2dsp), N,N`-
disalicylidene-3,4-diaminotoluene (H2dst), 4-nitro-N,N`-disalicylidene-1,2-
phenylenediamine (H2ndsp) and N,N`-disalicylidene ethylenediamine (H2salen) Schiff
base chelates.
18

ACCEPTED MANUSCRIPT
Cl
Cl
N
N
O
O
N
N
O
O
Fe
Fe
H3C
OH2
OH2
Complex I
Complex II
Cl
Cl
N
N
O
O
N
O
O
Fe
Fe
N
O2N
OH2
OH2
Complex III
Complex IV
Fig. 2. Speculated structures of iron(III) complexes I: (H2dsp), II: (H2dst), III: (H2ndsp)
and IV: (H2salen) Schiff base chelates
19

ACCEPTED MANUSCRIPT
Fig. 3. FTIR spectra of iron(III) complexes I: (H2dsp), II: (H2dst), III: (H2ndsp) and IV:
(H2salen) Schiff base chelates
20

ACCEPTED MANUSCRIPT
100
80
60
40
20
0
4000
3500
3000
2500
2000
1500
1000
500
Wavenumbers, cm-1
Fig. 4. FTIR spectrum of Fe2O3 synthesized from thermal decomposition of iron(III)
H2dsp Schiff base complex I at 800 oC
21

ACCEPTED MANUSCRIPT
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
(1)
0.2
(2)
(3)
(4)
0.1
0.0
250
300
350
400
450
500
550
600
650
700
750
Wavelength, nm
Fig. 5. UV-Vis spectra of Fe2O3 synthesized from thermal decomposition of iron(III)
o
complexes at 800 C (1): (H2dsp), (2): (H2dst), (3): (H2ndsp) and (4): (H2salen) Schiff
base chelates
22

ACCEPTED MANUSCRIPT
1.4
Eg = 1.75-1.92 eV
1.3
1.2
1.1
1.0
0.9
(h)1/2
0.8
0.7
0.6
0.5
0.4
0.3
0.2
(1)
(2)
(3)
(4)
1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 4.2
h (eV)
Fig. 6. Optical energy band gap plots of Fe2O3 synthesized from thermal decomposition
o
of iron(III) complexes at 800 C (1): (H2dsp), (2): (H2dst), (3): (H2ndsp) and (4):
(H2salen) Schiff base chelates
23

ACCEPTED MANUSCRIPT
600
500
400
300
200
100
0
10
20
30
40
50
60
70
80
2
Fig. 7. XRD spectrum of αFe2O3 synthesized from thermal decomposition of iron(III)
H2ndsp Schiff base complex at 800 oC
24

ACCEPTED MANUSCRIPT
Fig. 8. Scanning electron microscopy (SEM) photos of αFe2O3 synthesized from
o
thermal decomposition of iron(III) complexes at 800 C (A): (H2dsp), (B): (H2dst), (C):
(H2ndsp) and (D): (H2salen) Schiff base precursors
25

ACCEPTED MANUSCRIPT
Fig. 9. EDX spectrum of αFe2O3 synthesized from thermal decomposition of iron(III)
H2ndsp Schiff base complex at 800 oC
26

ACCEPTED MANUSCRIPT
Fig. 10. Transmission electron microscopy (TEM) photos of αFe2O3 synthesized from
o
thermal decomposition of iron(III) complexes at 800 C (A): (H2dsp), (B): (H2dst), (C):
(H2ndsp) and (D): (H2salen) Schiff base precursors
27

ACCEPTED MANUSCRIPT
100
80
60
40
20
0
A
B
C
D
Cisplatin
0
100
200
300
400
500
Concentration (g/mL)
Fig. 11. Inhibitory activity against breast carcinoma cells for the αFe2O3 synthesized
o
from thermal decomposition of iron(III) complexes at 800 C (A): (H2dsp), (B): (H2dst),
(C): (H2ndsp) and (D): (H2salen) Schiff base precursors
28

ACCEPTED MANUSCRIPT
Acknowledgement
This work was supported by grants from Vice President for Graduate Study and
Research, Taif University, Saudi Arabia under project Grants No. 5544-438-1.
References
1- L. LaConte, N. Nitin, G. Bao, Mater. Today 8, 32 (2005).
2- D. Patel, J.Y. Moon, Y. Chang, T.J. Kim, G.H. Lee, Colloid Surf. A 313–314, 91
(2008).
3- M. Zhao, L. Josephson, Y. Tang, R. Weissleder, Angew. Chem. Int. Ed. 42, 1375
(2003).
4- S. Mornet, S. Vasseur, F. Grasset, P. Veverka, G. Goglio, A. Demourgues et al.,
Prog. Solid State Chem. 34, 237 (2006).
5- P.D. Stevens, J. Fan, H.M.R. Gardimalla, M. Yen, Y. Gao, Org. Lett. 7, 2085
(2005).
6- W. Wu, Q. He, C. Jiang, Nanoscale Res. Lett., 3, 397 (2008).
7- Pardoe H, Chua-Anusorn W, St. Pierre TG, Dobson J, J. Magn. Magn. Mater.,
2001, 225:41-46.
8- Granick S, J Biol Chem 1942, 146:451-461.
9- Mornet S, Vasseur S, Garraset F, Duguet E, J Mater Chem 2004, 14:2161-2175.
10- Huh YM, Jun YW, Song HT, J Am Chem Soc 2005, 127:12387-12391.
11- Johnson WK, Stoupis C, Torres GM, Rosenberg EB, Ros PR, Magn Reson
Imaging 1996, 14(1):43-49.
12- Jordan A, Schotz R, Wust P, Schirra H, Schiestel T, Schmidt H, Felix R, J Magn
Magn Mater 1999, 194:185-196.
13- Jain TK, Morales MA, Sahoo SK, Leslie-Pelecky DL, Labhasetwar V, Mol
Pharm 2005, 2:194-205.
29