This document describes a study that synthesized Fe3AlO6 nanoparticles using a soft chemical approach and characterized their structure and properties. It then assessed the cytotoxicity of the nanoparticles in human neural stem cells (hNSCs) through several assays. Specifically, Fe3AlO6 nanoparticles were synthesized via hydrolysis of a single-source molecular precursor under controlled conditions. X-ray diffraction and transmission electron microscopy confirmed the formation of monophasic nanocrystalline particles around 16.5 nm in size. Cytotoxicity assays including MTT, LDH and FDA were performed on hNSCs treated with varying concentrations of the nanoparticles. Scanning electron microscopy was also used to examine changes in cell morphology and nanoparticle distribution within

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Soft chemical approach for the synthesis,
characterization and safety assessment of novel
Fe3AlO6 nanopowder in human neural precursor cells
ARTICLE in JOURNAL OF BIONANOSCIENCE · MARCH 2015
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Taimur Athar
Indian Institute of Chemical Technology
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Deccan College of Medical Sciences
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Deccan College of Medical Sciences
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Retrieved on: 28 September 2015

2. RESEARCHARTICLE
Copyright © 2015 American Scientific Publishers
All rights reserved
Printed in the United States of America
Journal of
Bionanoscience
Vol. 9, 1–10, 2015
Soft Chemical Approach for the Synthesis and
Characterization of Novel Fe3AlO6
Nanopowder and Assessment of Its
Cytotoxicity in Human Neural Stem Cells
Taimur Athar1 ∗
, Alabass Razzaq1
S. S. Mahamad Shafi1
, Sandeep K. Vishwakarma2
,
Avinash Bardia2
, Md. Aejaz Habeeb2
, Syed AB Paspala2
, and Aleem Ahmed Khan2 3
1
OBC, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, Andhra Pradesh, India
2
Central Laboratory for Stem Cell Research and Translational Medicine, Centre for Liver Research and Diagnostics,
Deccan College of Medical Sciences, Hyderabad, AP, India
3
Salar-E-Millat Research Centre for Cellular and Molecular Medicine,
Deccan College of Medical Sciences, Hyderabad, AP, India
In present study we synthesized kinetic controlled Fe3AlO6 nanopowder by hydrolysis of single
source molecular precursor in the presence of sodium hydroxide. Structural and morphological
properties of the particle were characterized by XRD, TEM, FT-IR, UV-vis-spectroscopy and ther-
mal analysis. The nanocrystalline structure with monophasic was obtained after annealing. The
mean particle size (16.5 nm) was calculated by using X-ray diffraction pattern. The crystallite size
and phase purity depends with temperature along with molar ratio of the molecular precursor, pH,
temperature, reaction time along with their synthetic methodology. In this context in vitro culture
model system provides a better approach to understand the basic biology of stem cells transplan-
tation in controlled behavioral biological environment. Hence in present study in vitro cytotoxicity
was assessed in human neural stem cells (hNSCs) using most commonly used assays i.e., MTT,
LDH and FDA. SEM analysis was performed for the hNSCs incubated with Fe3AlO6 nanopowder to
assess the changes in cell morphology and distribution of nanoparticles within the cell. Internaliza-
tion of nanoparticles in hNSCs (after 2 h of incubation) was clearly observed under a conventional
inverted microscope in closed 0.4 m aperture. The results obtained from this study represents
the potential usefulness of Fe3AlO6 nanoparticles further help to track transplanted NSCs in animal
models further to assess its half life and with high contrast in long-term cell fate determination.
Keywords: Sol–Gel, Powder Synthesis, Characterization Techniques, in Vitro Cytotoxicity.
1. INTRODUCTION
Chemistry of nanoparticle has attracted the attention since
last one decade to understand a transformation from
molecular precursor into solid state materials with well
defined structural unit for better understanding the con-
version from micro to nanoscale. The synthesis of func-
tional heterometallic framework with desired properties
remains a challenge to the synthetic chemists. Metal oxide
nanoparticles act as a core material to provide a plat-
form for surface functionalization, due to their exceptional
physical, mechanical, electronic, thermal and chemical
properties.1–16
Soft molecular precursor with exceptional
physico-chemical properties help to synthesize a functional
∗
Author to whom correspondence should be addressed.
heterometallic oxide with unique physical and chemical
properties. Based on structural-surface reactivity plays a
critical role in fabrication of future nanodevices. Due to its
unique architectural framework has received more atten-
tion both in basic and applied research which help for
fabricating the devices for its futuristic applications in gas
sensors, solar cells, doped semiconductors, dye-sensitized
solar cells, transistors, electrodes for lithium ion batter-
ies, catalyst supports and as well as for super capacitors.
Synthetic route, the type of molecular precursors used
and along with synthetic parameters plays a key role to
control crystal engineering to design desired functional
properties.17–26
The synthesis of Fe3AlO6 nanopowder was
mainly controlled by chemical and thermodynamic param-
eters thereby help to improve the functional properties.
J. Bionanosci. 2015, Vol. 9, No. xx 1557-7910/2015/9/001/010 doi:10.1166/jbns.2015.1282 1

3. RESEARCHARTICLE
Soft Chemical Approach for the Synthesis and Characterization of Novel Fe3AlO6 Athar et al.
A simple, efficient and practical wet method was under-
taken to synthesize an ultra-pure materials with lim-
ited surface defects in short time and agglomerization
to enhance functional properties by better understanding
the kinetic–thermodynamic parameters during the reac-
tion conditions. The self-assembled framework of Fe3AlO6
nanopowder with a precise physical, chemical composi-
tion and morphological properties was synthesized; het-
erometallic oxide was characterized with the help of XRD,
TEM, FT-IR, UV and TG-DTA studies for its use in many
fields of science and technology.
Solid state
synthesis
Soft Synthesis
Particle Properties
Controlled
Controlled
Not
Under
Advantages and disadvantages of synthetic method.
In recent years stem cell research has attracted tremen-
dous attention due to its enormous potential in medical
applications. However, before their therapeutic appliances
there is a need to develop more advanced technologies
for better understanding of fate, engraftment, distribution
and functional perspectives following stem cells transplan-
tation. In vitro culture model system provides a better
approach to understand the basic biology of stem cells
transplantation in controlled behavioral biological environ-
ment. In addition to this more accurate and non-invasive
methods are needed for tracking their translocation after
transplantation. Various methods along with characteri-
zation techniques used to understand a cell tracking by
gene modification by using green fluorescent protein (GFP)
or luciferase, however these techniques limit their in tis-
sue penetration and are applicable for only small animal
models.27–30
Additional concerns have been behavioral and
functional changes in cells due to incorporation of foreign
genes.
The emerging technology by using nanomaterials that
can be internalized by cells, it has showed encouraging
results in many animal experiments as well as in some
clinical trials.31–36
Till date a number of nanoparticles have
been developed but derivatives of iron oxide nanoparti-
cles have shown better vehicle for drug delivery for the
synthetic molecules to target defective organ/tissue across
the blood-brain barrier.37–42
However, very little is known
about their internalization and effect in cellular activities
which further help to assess their cellular therapy based
on molecular imaging techniques play an important role to
investigate the behavior and the ultimate feasibility of stem
cell transplantation. In the present study Fe3AlO6 nanopar-
ticles cytotoxicity was assessed in human neural stem
cells (hNSCs) using most commonly assays i.e., mito-
chondrial dehydrogenase (MDH), lactate dehydrogenase
(LDH) and fluorescein di-acetate (FDA) cell membrane
integrity assays. Scanning electron microscopy (SEM) was
performed to identify the interaction between nanoparicles
and cell surface. Transmission electron microscopy (TEM)
analysis was done for the neural stem cells to assess the
intracellular distribution of nanoparticles within the cells.
2. EXPERIMENTAL DETAILS
2.1. Chemicals
Ferric chloride, potassium hydroxide and organic solvents
were purchased from Aldrich and used as such without
further purification except organic solvents. The organic
solvent was dried as reported in the literature.43 44
In the
experiment the de-ionized water (18.2 M ) was used
which was purchased from Millipore.
2.2. Experimental Procedure
Heterometallic oxide was prepared by a ligand exchange
reactions in which a molar ratio of precursor was
treated with an excess of water–isopropanol mixture.
5 gm (30.769 mmol) of FeCl3 was taken with 4.9 gm
(87.5 mmol) of KOH in 40 ml of de-ionized water. Then it
was stirred and refluxed for 4 hr at 85 C. The progress of
reaction was monitored with the help of pH. It was then fil-
tered and washed with a mixture of 95% Isopropanol+5%
water till the filtrate becomes neutral. The brown nano-
material was obtained and then dried under vacuum for
overnight at 80 C gives good yield (90%). It was calci-
nated at 800 C in dry air and good yield was obtained.
The reaction was carried out as shown in the flow chart:
2.3. Characterization
Structural and morphological properties of Fe3AlO6
nanoparticles were characterized by using analytical
techniques. FTIR spectroscopy was recorded at room
temperature in the range of 4000 to 400 cm−1
by using
KBr pellets with the help of Perkin-Elmer spectrometer
GX model with a wave number resolution of 4 cm−1
.
Optical properties were investigated by using UV-spectra,
recorded on a GBC UV-Visible Cintra spectrometer with
wave length of 200–800 nm. Thermal behavior, phase
purity and crystalline transformation were confirmed by
thermal studies by using TGA-DTA (Mettler Toledo star,
Columbus, OH). The powder was heated in dry air
from room temperature to 1000 C with the heating
2 J. Bionanosci. 9, 1–10, 2015

4. RESEARCHARTICLE
Athar et al. Soft Chemical Approach for the Synthesis and Characterization of Novel Fe3AlO6
FeCl3 2.5KOH
3ClFe(OH)2
–2KCl
– 3 HCl
– 3H2O
Al(OH)3
Fe3AlO6
A flow chart for the heterometallic oxide nanomaterials.
rate of 5 C/min. X-ray diffraction studies were car-
ried out with Bragg angle ranging from 0 to 80 on
a Siemens (Cheshire, UK) D 500 X-ray diffractometer.
EDAX was taken in an Oxford link ISIS-300 instru-
ment. Morphological features were investigated by using
a Philips Tecnai G2
FEI 12 Transmission electron micro-
scope with a operating system at 80–100 KV. The sam-
ples for TEM were prepared in ethanol-toluene mixture.
Suspension of Fe3AlO6 nanoparticles was put in carbon-
coated copper grid. Dynamic light scattering (DLS) char-
acterization was carried out by a zetasizer 3000 HS∧.
DRS UV/Vis studies were recorded with the help of
UV/Vis spectrometer Carry-5000. At room temperature
photo luminiscence spectral studies were done by using a
Jobin-Yvion Fluorolog-3 spectrofluorimeter using the 360
nm excitation line with Xenon lamp (450 W) .The sample
was prepared in methanol by ultrasonication.
2.4. Cell Preparation and in Vitro Culture
Human fetal sub ventricular zone derived neural stem cells
(NSCs) were enriched using prominin-1 by magnetic acti-
vated cell sorting (MACS) and resuspended in Neurocult
basal medium (Stem Cell Technologies, Canada). The via-
bility of prominin-1 enriched hNSCs was determined by
trypan blue exclusion method. The cells were counted
using hemocytometer. 1×104
viable cells/ml were seeded
in each well with 96 well culture plate at 37 C with
5% CO2 for 24 h. Medium was replaced with serum free
NeuroCult Proliferation (Stem Cell Technologies, Canada)
supplemented with 20 g/ml epidermal growth factor
(EGF), 10 g/ml basic-fibroblast growth factor (b-FGF),
100 U/ml penicillin and 100 g/ml streptomycin.
2.5. Assessment of Fe3AlO6 Cytotoxicity
2.5.1. MTT Assay
Cytotoxicity of Fe3AlO6 on hNSCs was assessed using
the mitochondrial oxidation assay to quantify the mito-
chondrial activity. Briefly, hNSCs (1×104
cells/well) were
seeded in a 96 well culture plate with 100 l of NeuroCult
Proliferation Medium for 24 h, 48 h and 72 h along with
Fe3AlO6 nanoparticles at concentration of 10 g/ml to
1 mg/ml. Cells cultured without nanoparticles were used as
control. The exhausted medium was aspirated and the cells
were washed with 100 l/well of 1X Phosphate Buffer
Saline (PBS; Gibco) and incubated with 100 l of 1 mg/ml
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) for 4 h. The purple formazan crystals were
solubilized by incubating the cells with acidified Iso-
propanol for additional 2 h. The absorbance was measured
at 570 nm using microplate reader (BIORAD). All mea-
surements were performed in triplicate to avoid any tech-
nical error and four independent experiments were carried
out to verify the data.
2.5.2. LDH Assay
Fe3AlO6 cytotoxicity was assayed by measuring the
amount of released lactate dehydrogenase (LDH) from
dead cells with using many concentrations LDH assay kit
(Promega WI). 1×104
viable hNSCs were incubated with
200 l of Fe3AlO6 (10 g/ml to 1 mg/ml) for 24 h and
then mixed with 50 l of reagent. The mixture was incu-
bated for 30 min at room temperature. The reaction was
stopped by adding 50 l of stop solution. Absorbance was
measured at 490 nm using a microplate reader (BIORAD).
2.5.3. FDA Membrane Integrity and
Cell Viability Assay
Quantification of cell viability and membrane integrity
was performed by Fluorescein diacetate (FDA). Briefly,
1 × 104
viable cells/well were seeded in each well with
24 well culture plate in NeuroCult Proliferation Medium.
After 24 h, cells were treated with a suspension of 200 l
Fe3AlO6 nanoparticles (1 mg/ml). The effect on cellular
viability was evaluated after 48 h by adding 100 l of FDA
(0.1 mg/ml). Cells with FDA were incubated for 5 min
at 22 C–25 C and fluorescence was measured by Carl
Zeiss microscope at 10X and 40X magnifications. All the
images were documented using Axiovert Version 4.2 soft-
ware (Carl Zeiss, Germany). The cell viability was estab-
lished by the ratio between viable (green) and dead (no
fluorescence) cells counted on several microscopic fields.
2.5.4. Scanning Electron Microscopy (SEM) Studies
SEM was used to characterize the cellular morphology and
adhesion of Fe3AlO6 on cell surface. For SEM analyses
hNSCs were fixed overnight with 2.5% gluteraldehyde in
0.1 M phosphate buffer (pH 7.2) solution at 4 C and
then post fixed with the help of aqueous Osmium tetraox-
ide for 4 h. After dehydration the sample with alcohols
(10%, 30%, 50%, 70% and 100%) and dried to critical
point drying with CPD unit. The samples were processed
and then mounted over the stubs with double-sided carbon
conductivity tape, and thin gold layer coating by using an
automated sputter coater (JEOL JFC-1600) for 3 min and
J. Bionanosci. 9, 1–10, 2015 3

5. RESEARCHARTICLE
Soft Chemical Approach for the Synthesis and Characterization of Novel Fe3AlO6 Athar et al.
then scanned with SEM (JOEL-JSM 5600) as per the stan-
dard procedures at RUSKA Lab’s College of Veterinary
Science, Hyderabad.45
2.6. Identifying Fe3AlO6 Nanoparticles in hNSCs
Fe3AlO6 nanoparticles (1 mg/ml) were mixed with 2 ×
104
hNSCs and incubated at 4 C for 2 h. Cells were
washed twice with 1X PBS and placed between high qual-
ity glass slide cover (22 ×22 mm, D263M, Schott) which
were suspended above the objective lens. The use of regu-
lar microscopic slides was avoided because they generate
diffractive noises due to large number of surface imper-
fections, particularly at the nanoscale. Nanoparticles were
visualized by using an inverted conventional microscope
(Carl Zeiss, Germany) in transmission mode. A 40X objec-
tive lens with a numeric aperture of 0.4 m was used with
a high resolution (1324×1024) 16-bit monochrome cooled
CCD camera.
3. STATISTICAL ANALYSIS
All the data were expressed as the mean±standard devia-
tion. Statistical analyses of means were calculated by using
one way and two way analysis of variance using Graph
Pad Prism software (version V). The differences were sta-
tistically significant at p value of < 0 05.
4. RESULTS AND DISCUSSION
The reaction temperature plays a vital role in studying the
growth mechanism for the synthesis of well defined nano-
structure framework with precise desired surface morphol-
ogy and crystalline nature. In soft chemical approach the
SN2 mechanism takes place in which the nucleophile OH−
group is added to metal cation to increase the coordination
number based on the electronegativity of the metal ion.
The properties of functional Fe3AlO6 nanopowder strongly
depend on its structural morphology, size and orientation
of the crystal shape. Based on the type of molecular pre-
cursor used and as well as in controlling reactions param-
eters help to attain satisfactory nucleation for the growth
of particle size with uniform size and its distribution.
Fig. 1. FT IR spectra of Fe3AlO6 nanopowder after calcinations.
FT-IR shows the presence of peaks which are char-
acteristic to the material. A presence of weak peak at
1062 cm−1
, 1450 cm−1
and a strong absorption peak at
554.55 cm−1
and 483.43 cm−1
occurs due to the asym-
metric and symmetric stretching vibrations of Fe–O and
Al–O band, thereby supporting the formation of Fe3AlO6
nanopowder (Fig. 1). These bands become stronger after
annealing thereby supporting the formation of a strong and
well-defined Fe–O–Al–O frame-work after the removal
of surface impurities. The peak at 3422.2 cm−1
and
1610 cm−1
shows the presence of stretching vibration and
deformation frequency due to OH bond. The absorption of
water takes place during the sample preparation.46–48
Optical properties were studied with the help of UV-
Visible spectrum (Fig. 2). A broad prominent excitonic
absorption band gap was observed in the region at 270 nm
and 320 nm shows the presence of Fe–O and Al–O bond
which support the presence of small size with homogeneity
with different size regime. It is not possible to observe a
clean shift with respect to the functional properties related
to particle size due to ageing. The UV studies are same
both in freshly prepared sample as well as the sample
prepared after ageing thereby supports that ageing phe-
nomena has no impact in controlling the functional prop-
erties. The synthesized nanomaterial is a porous material
with open voids, which support that the absorption band
gaps changes with chemical composition of the sample and
grain size due to quantum size effects.49–51
The formation of Fe3AlO6 nanoparticle take places by
controlling the nucleation rate by better understanding the
molecular seeding effect along with its growth mecha-
nistic approach in the quantum confinement regime. The
morphological activities of nanoparticles were influenced
with their reaction conditions along with type of single
source precursor used. The crystallinity, chemical compo-
sition and phase purity were examined at room temper-
ature after calcination. The diffraction peaks support the
presence of face-centered tetragonal phase with good crys-
tallinity as supported by the presence of sharp diffraction
peaks (Fig. 3). It is difficult to assign a JCPDS file num-
ber because the nanomaterial was synthesized for the first
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6. RESEARCHARTICLE
Athar et al. Soft Chemical Approach for the Synthesis and Characterization of Novel Fe3AlO6
Fig. 2. UV-visible absorption spectra of Fe3AlO6 nanopowder after
calcinations.
time. The particle size was calculated by using Scherrer
formula and it was calculated to be 16.25 nm, thereby
supporting the formation of Fe3AlO6 nanopowder in a
controlled way with respect to temperature. No charac-
teristic peaks related to impurities of Fe/Al, Fe(OH)3 and
Al(OH)3 were observed there by supporting the formation
of nanoparticle with an ultra purity.52–54
The thermal analysis was carried out to understand the
changes taking place in nanomaterial with phase purity
to build up crystalline structure with the help of self–
assembly after the removal of surface impurities as illus-
trated in (Fig. 4). The first step involves the removal of
unbound water at 80–90 C as appeared with the presence
of exotherm peak, followed by removal of volatile materials
from the particle surface at 150–250 C as observed with
Fig. 3. XRD spectra of Fe3AlO6 nanopowder after calcinations.
Fig. 4. TGA-DTA of Fe3AlO6.
the appearance of exothermic peak, then finally leads to
the formation of crystalline nanoparticle with phase purity
at 585 C as illustrated by a presence of an exothermic
peaks. DTA curve shows an endothermic peak at 80 C;
correspond to the evaporation of the absorbed water. An
exothermic peak at approximately 500 C occurs in DTA,
can be assumed to be associated with the conversion of
nanomaterial after the decomposition of inorganic residues
which lead to the surface purity. The formation of crys-
talline nanoparticle with phase purity takes place at 585 C
as illustrated by a presence of an endothermic peak.55
The SEM image shows the presence of particle with var-
ious shapes and size with agglomeration leads to a limited
homogeneity thereby supporting the formation of flakes
(Fig. 5). The selective growth in the particle takes place
due to presence of high surface energy and inter/intra par-
ticle interactions. Shape and size of the particle depends
with pH conditions along with synthetic methodology. It
is clear from the SEM figure that densification takes place
J. Bionanosci. 9, 1–10, 2015 5

7. RESEARCHARTICLE
Soft Chemical Approach for the Synthesis and Characterization of Novel Fe3AlO6 Athar et al.
Fig. 5. SEM of Fe3AlO6.
with a significant occurrence of microstructural material
which helps to build a hierarchical nanostructure with the
help of molecular forces. The purity was further confirmed
by EDX showing the presence of Fe:Al:O in the stoichio-
metric ratio with C6 space group.56
The TEM image shows the distribution of size with
tetragonal shape with particle size of 16.25 nm which
agrees well with the particle size calculated with the help
of Scherrer equation (Fig. 6). The inset shows the SAED
pattern which confirms the presence of dark diffraction
rings, thereby supporting that the formation of crystalline
nature with irregular orientation having multifaceted shape
and its wide distribution. The method of preparation plays
a key role in controlling the surface energy to restrict the
agglomerisation.57
To understand the mechanistic approach for the forma-
tion of particle via self-assembly size and a zeta poten-
tial measurement was carried out with the help of DLS
(Fig. 7). The sample formed was forced to undergo
hydrolysis followed by thermal treatment. The sample
was dried in vacuum and then redispersed in water and
methanol. DLS measurement shows the particle diameter
with 77.30 nm having a zeta potential of −3.03 mV in
water supporting that dispersant solvents, which plays an
important role in determining the particle diameter as well
Fig. 6. TEM of Fe3AlO6.
as synthetic methodology and along with a type of molec-
ular precursor used. Zeta potential shows clear, consistent
information with source of stabilization for controlling the
surface energy.
The room-temperature photo luminiscence spectra with
an excitation show the presence of emission at 434.99 nm
with required morphology and dimension due to quan-
tum confinement effect. Emission in the visible region
take place with combinations of photoexcited hole with
an excited state between the metal-ligand charge transfer
having a specific structural defect and as well as oxy-
gen vacancies within metal ion. In Fe3AlO6 nanoparti-
cles, a reduction in the band gap can be measured by
using optical absorption techniques (Fig. 8). Fe and Al
is tri-coordinated, due to the presence of oxygen vacan-
cies, nanomaterials shows effective electronic and chem-
ical properties by controlling the stoichiometric ratio for
its use in catalytic reactions and other applications.58
Based on characterization technique the following plausi-
ble structure has been suggested (Fig. 9).
4.1. Cytotoxicity Assessment
The purpose of this investigation was to evaluate safety
and its general mechanisms involved in induced neuro-
toxicity with Fe3AlO6 in vitro culture model system. The
nanoparticles toxicity was assessed with the help of cells
selection by using appropriate cytotoxicity assay is of vital
important. Hence in present study all potential interfer-
ences were considered to avoid false-positive results.
4.1.1. MTT Assay
Fe3AlO6 cytotoxicity to hNSCs was assessed by measur-
ing the activity of cellular mitochondrial dehydrogenase
(MDH) which is based on the reduction with MTT to for-
mazan by MDH. The variable concentrations of Fe3AlO6
nanoparticles ranging from 10 g/ml to 1 mg/ml did not
show any influence in the cellular mitochondrial dehydro-
genase activity after 24 hours, 48 hours and 72 hours incu-
bation with survival rate below 80% as shown in (Fig. 10).
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8. RESEARCHARTICLE
Athar et al. Soft Chemical Approach for the Synthesis and Characterization of Novel Fe3AlO6
Fig. 7. Formation of particle via self-assembly size and a zeta potential measurement with the help of DLS.
The graphs represent the mean standard deviation with
four independent MTT assays experiments show biocom-
patiblity in >80% cell viabilities are often biocompatible
with hNSCs upto 1 mg/ml concentration. Therefore it is
concluded that the absence of toxicity may be due to (a)
Surface interaction of the particle with cell and (b) Due to
hydrophilic surface interaction which prohibit the release
of metal ion in the cell. The exact mechanism of nanoparti-
cles action within the cell is still unknown; however in vivo
and in vitro experiments shows that metal oxide nanoma-
terials can produce reactive oxygen species (ROS) which
ultimately can damage DNA, alter the gene expression and
also impede several cell signaling pathways.59 60
The MTT
results give insight information about non-cytotoxicity due
to nonproductive oxygen free radicals after incubation for
72 h.
4.1.2. LDH Assay
In vitro cytotoxicity assay was performed by using LDH
with incubation time of 24 h by using Fe3AlO6 nanopar-
ticles. Total cell lysate was used as positive control to
assess the kit sensitivity for 100% with the release of lac-
tate dehydrogenase from the dead cells after incubation.
Fig. 8. Fluorescence image.
Fig. 9. Plausible structure of Fe3AlO6 nanoparticle.
Figure 11 clearly shows that there was steady release of
LDH from cells treated with nanoparticles and were com-
pared with controlled concentrations. In general nanopar-
ticles didn’t cause LDH leakage from hNSCs presenting
high cell viability retaining cell membrane integrity.
Fig. 10. Mitochondrial dehydrogenase activity (MDH) showed that cell
viability was almost similar to control (untreated) in hNSCs with differ-
ent concentrations of Fe3AlO6 nanoparticles with increasing time period
of incubation. MTT assay did not show any statistically significant dif-
ference in MDH activity at different concentrations of nanoparticles used
at different time periods (n = 3, p > 0 05).
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9. RESEARCHARTICLE
Soft Chemical Approach for the Synthesis and Characterization of Novel Fe3AlO6 Athar et al.
Fig. 11. Toxicity of Fe3AlO6 nanoparticles was measured by the release
of cytosolic enzyme LDH in vitro. hNSCs were exposed to nanoparti-
cles for 24 h at concentration of 10 g/ml to 1 mg/ml. The released
LDH quantified in the treated culture supernatant which was found non-
significant as compared to control whereas LDH activity in lysed cells
yielded the total activity (100%).
4.1.3. FDA Membrane Integrity and Cell Viability
FDA assay showed even green fluorescence towards the
cell membrane after 24 hr both in controls as well as
Fig. 12. FDA assay showing green fluorescence in all the cells representing utmost membrane integrity in both the normal and nanoparticles treated
cells (scale bar: 10 m).
Fig. 13. SEM analyses revealed Fe3AlO6 nanoparticles crystalline structures and their interaction with hNSCs on their surface. Nanoparticles were
adhered and grouped at the cellular membrane without any damage.
cells treated with 1 mg/ml of Fe3AlO6 nanoparticles. Cell
membrane was found intact in the samples incubated with
nanoparticles (Fig. 12). The green fluorescence occurs with
a enzymatic dissociation of FDA into fluorescein. Treated
cells show similar green fluorescence in cytoplasmic region
with high esterase activity and cell viability which suppor-
tan integrity of cell membrane as measured by LDH assay.
These result support a fully matched with MTT assay data
with as high enzymatic activity within the cell system.
4.1.4. SEM Analysis
SEM analysis was used to confirm the cell morphology
with the presence of nanoparticles at the cell surface.
Both cylindrical and spherical clusters were attached to the
hNSCs due to inter/intra cellular diffusion forces as well as
hydrostatic attraction. The particle gets attached with the
cell due to hydrostatic forces, thereby reducing the surface
energy.
4.2. Internalization of Fe3AlO6 Nanoparticles
Figure 14(A) shows Fe3AlO6 nanoparticles (16.5 nm)
generate characteristic optical rings (approximately
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10. RESEARCHARTICLE
Athar et al. Soft Chemical Approach for the Synthesis and Characterization of Novel Fe3AlO6
Fig. 14. Identification of Fe3AlO6 nanoparticles by using a conventional microscope (A) Single nanoparticle produces an optical ring around it and
increases the resolution 1000 times larger than its nominal size (white arrow). Self-assembly of nanoparticles increases the resolution and appears in
rod-shape with increases resolution (red arrow, Scale bar: 10 m) (B) Internalization of nanoparticles in hNSCs (after 2 h of incubation) was clearly
observed under 40X objective of a conventional inverted microscope in closed 0.4 m aperture (Scale bar: 20 m). White arrows indicate interlized
nanoparticles within the cell. (C) Cells without nanoparticles did not show any signature in conventional microscope and were found difficult to track
due to low resolution in closed aperture (Scale bar: 20 m).
5 m–10 m) when viewed in a conventional inverted
microscope with its minimum condensed closed aperture.
Optical rings around a single nanoparticle increase the res-
olution almost 1000 times for locating and tracking parti-
cles in situ without any requirement of labeling. However
some of the nanoparticles tend to aggregate into clusters,
resulting substantially larger diameter than an individual
nanoparticle. These clusters were easy to identify form-
ing larger signatures of higher contrast with a rod-shape,
rather than a circle, at their centre. This property offers the
prospect of tracking the interaction of nanoparticles with
functionalized surfaces or their self-assembly. Their poten-
tial application is illustrated in Figure 14(B) for 16.5 nm–
diameter Fe3AlO6 nanoparticles interacting with hNSCs.
Nanoparticles at periphery within the cell were easily iden-
tified in closed condensed aperture of conventional micro-
scope at 40X objective. Different size of optical signatures
for different nanoparticles within the cell clearly demon-
strates that the particles are at different depths in the cell.
These results represent a significant advancement in earlier
work on location and tracking of particles.61
5. CONCLUSIONS
The state of the art of heterometallic nanopowder were
successfully synthesized in good yield via soft chemi-
cal approach by controlling experimental conditions which
help to open a new opportunities by understanding both
fundamental and as well as the technological research. Wet
synthetic approach helps to control characteristic physi-
cal parameters such as specific surface, particle size and
purity with well defined shape at low temperature with
good yield. The particle size can be adjusted by controlling
the reaction temperature. Our approach provides a general
and flexible method to prepare other type of metal oxide
nanoparticles in large scale by retaining the functional
properties intact for its various environmental and bio-
logical applications with well defined incubation period.
The tuning of functional properties in the precursor along
with concentration of solvent and thermodynamic-kinetic
parameters help to better understand the synthesis along
nanotechnology parameters for its effective use in nanode-
vices. In this perspective, accurate prediction of cell phe-
notype changes due to cell to cell and cell-nanoparticles
interaction is an essential step further to designate proper
boundaries for their applications. Interestingly in present
study, Fe3AlO6 did not show any sort of cytotoxicity to
the hNSCs with variable concentrations representing high
grade of biocompatibility with the human neural cells.
These studies help to fully evaluate the cytotoxicity and
to provide more reliable in-depth safety data with vari-
ous parameters to predict sub-lethal effect of nanoparti-
cles on cellular changes, which help to better understand
the interaction of nanomaterials with human neural cells.
The results obtained from this study represents the poten-
tial usefulness of Fe3AlO6 nanoparticles further help to
track transplanted NSCs in animal models further to assess
its half life and with high contrast in long-term cell fate
determination.
Conflict of Interest
None.
Acknowledgment: None.
References and Notes
1. Y. S. Chaudhary, D. Chinthalapelly, U. M. Bhat, P. K. Naik, and
D. Khushalani, J. Mater. Chem. 31, 18 (2008).
2. D. L. Fedlheim, C. A. Foss, Metal Nanoparticles, Synthesis, Char-
acterization and Applications, Marcel Dekker, New York (2002).
3. G. Cao, Nanostructures and Nanomaterials, Imperial College Press,
Covent Garden, London (2004).
4. D. Vollath, Nanomaterials, WILEY-VCH Verlag, GmbH & Co.
KGaA, Weinheim (2008).
5. A. Muller, A. K. Cheetham, and C. N. R. Rao, Nanomaterials
Chemistry: Recent Developments and New Directions, Wiley-VCH,
WILEY-VCH Verlag, GmbH & Co. KGaA, Weinheim (2007).
6. R. Backov, Soft Mater. 2, 452 (2006).
J. Bionanosci. 9, 1–10, 2015 9

11. RESEARCHARTICLE
Soft Chemical Approach for the Synthesis and Characterization of Novel Fe3AlO6 Athar et al.
7. V. M. Rotello, Nanoparticles: Building Blocks for Nanotechnology,
Springer, Spring Street: New York (2004).
8. L. M. Marzan and P. V. Kamat, Nanoscale Materials, Kluwer Aca-
demic Publishers Springer, Berlin (2003).
9. J. L. G. Fierro, Metal Oxide: Chemistry and Applications, Taylor &
Francis, Broken (2005), p. 6000.
10. A. K. Bandyopadhyay, New Age International (2007).
11. P. Yang, Edit. The Chemistry of Nanostructured Materials, World
Scientific Publishing Co., Washington D.C., U. S. A (2003).
12. J. A. Rodriguez and M. F. Garcia, Synthesis, Properties and Applica-
tion of Oxide Nanomaterials, John Wiley and Sons, Hoboken, New
Jersey (2007).
13. K. J. Klabunde and R. M. Richards, Nanoscale Materials in Chem-
istry, 2nd edn., Wiley, New York (2012).
14. Y. M. Jose and R. Mehl, Award Metallurgical and Material Trans-
actions A 29, 713 (1997).
15. P. N. Kapoor, D. Heroux, R. S. Mulukutla, V. Zaikovskii, and K. J.
Klabunde, J. Mater. Chem. 13, 410 (2003).
16. C. L. Carnes, P. N. Kapoor, and K. J. Klabunde, J. Chem. Mater.
7, 2922 (2002).
17. P. N. Kapoor, A. K. Bhagi, R. S. Mulukutla, and K. J. Klabunde,
Nanosci. and Nanotech. (2007).
18. M. S. Hegde, K. Nagaveni, and S. Roy, Pramana 65, 641 (2005).
19. S. Szafert, T. John, and P. Sobota, Dalton Tran. 46, 6509 (2008).
20. B. Li, M. Li, C. Yao, Y. Shi, D. Ye, J. Wu, and D. Zhao, J. Mater.
Chem. 23, 6742 (2003).
21. J. Y. Ahn, W. D. Kim, J. H. Kim, J. K. Lee, J. M. Kim, and S. H.
Kim, Nanomater. (2011).
22. S. O. Brien, L. Brus, and C. B. Murray, J. Am. Chem. Soc.
123, 12085 (2001).
23. M. Niederberger and G. Garnweitner, Chem. A European J. 12, 7282
(2006).
24. J. J. Urban, W. S. Yun, and H. Park, J. Am. Chem Soc. 124, 1186
(2002).
25. I. Bilecka, I. Djerdj, and M. Neiderberger, Chem. Commun. 7, 886
(2008).
26. G. A. Seisenbaeva, V. G. Kessler, R. Pazik, and W. Strek, Dalton
Trans. 26, 3412 (2008).
27. Z. Li, A. Lee, M. Huang, H. Chun, J. Chung, P. Chu, et al., J. Am.
Coll. Cardiol. 53, 1229 (2009).
28. Z. Li, J. C. Wu, A. Y. Sheikh, D. Kraft, F. Cao, X. Xie, et al., Circul.
116, 46 (2007).
29. X. Xie, F. Cao, A. Y. Sheikh, Z. Li, A. J. Connolly, X. Pei, et al.
Clon. Stem Cells 9, 549 (2007).
30. M. G. Pomper, H. Hammond, X. Yu, Z. Ye, C. A. Foss, D. D. Lin,
et al., Cell Res. 19, 370 (2009).
31. W. Cai and X. Chen, Small 3, 1840 (2007).
32. J. M. Hare, J. H. Traverse, T. D. Henry, N. Dib, R. K. Strumpf, S. P.
Schulman, et al., J. Am. Coll. Cardiol. 54, 2277 (2009).
33. H. Guenou, X. Nissan, F. Larcher, J. Feteira, G. Lemaitre,
M. Saidani, et al., Lancet. 374, 1745 (2009).
34. Y. Fu, N. Azene, Y. Xu, and D. L. Kraitchman, Imaging Med. 3, 473
(2011).
35. R. M. Clelland, E. Wauthier, T. Tallheden, L. M. Reid, and E. Hsu,
Mol. Imaging Biol. 13, 911 (2011).
36. L. Wang, W. Su, Z. Liu, M. Zhou, S. Chen, Y. Chen, et al., Biomater.
33, 5107 (2012).
37. D. H. Kim and D. C. Martin, Biomater. 27, 3031 (2006).
38. M. Das, S. Patil, N. Bhargava, J. F. Kang, L. M. Riedel, S. Seal,
et al., Biomater. 28, 1918 (2007).
39. X. Li, W. Liu, L. Sun, Y. Fan, and Q. Feng, J. Biomater. Tissue Eng.
4, 994 (2014).
40. R. Tang, L. Zhang, J. Li, C. Wang, Z. Liu, Y. Han, and Q. Lin, J.
Biomater. Tissue Eng. 4, 1004 (2014).
41. J. Zhang, M. Liu, J. Zhang, B. Li, X. Y. Wang, and P. Han, J.
Biomater. Tissue Eng. 5, 135 (2015).
42. K. Parwez and S. V. Budihal, J. Bionanosci. 8, 61 (2014).
43. A. I. Vogel, Textbook of Quantitative Chemical Analysis, 5th edn.,
Longman, UK (1989).
44. W. L. F. Armoring and D. D. Perrin, Purification of Laboratory
Chemicals, 4th edn., Oxford, U.K (1996).
45. J. J. Bozzola, Russelle. Electron Microscopy Principles and Tech-
niques for Biologists 2nd edn., Jones and Bartlett Publishers, Sud-
bury, Massachusetts (1998), Vol. 19, p. 54.
46. B. Smith, et al., Infrared Spectral Interpretation: A Systematic
Approach, CrC Press, Boca Raton (1999).
47. K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coor-
dination Compounds, 5th edn., Wiley-Interscience (1997).
48. L. H. Little, Infrared Spectra of Absorbed Species, Academic Press,
New York (1966).
49. M. Niasaria, N. Mirb, and F. Davara, Polyhedron 28, 1111
(2009).
50. I. M. Watt, The Principles and Practice of Electron Microscopy, 2nd
edn., Cambridge University Press (1997).
51. A. Davydov, Molecular Spectroscopy of Oxide for Catalyst Surface,
John-Wiley and Sons, West Sussex, PO19 8SQ: England (2003).
52. B. D. Cullity, Elements of X-Ray DFiffraction. Addison-Wesley
Publishing Company, Inc., London (1967).
53. A. Guinier, X-Ray Diffraction in Crystals, Imperfect Crystals and
Amorphous Bodies, Dover Publications, New York (1994).
54. M. P. Klug, L. E. Alexander, X-Ray Diffraction Procedure for Poly-
crystalline and Amorphous materials, Wiley, New York (1974).
55. P. Gabbott, Principles and Applications of Thermal Analysis, Wiley-
Blackwell Publishing, U. S. A (2007).
56. D. L. G. L. Pavia, G. S. Kriz, and J. R. Vyvyan, Introduction to
Spectroscopy, 4th edn., rooks/Cole Cengage Learning, United State
(2009).
57. D. B. Williams and C. B. Carter, Sci. (2010).
58. G. Blasse and B. C. Grabmeier, Luminescent Materials, Springer-
Verlag, Berlin (1994).
59. L. Gao, J. Zhuang, L. Nie, J. Zhang, Y. Zhang, N. Gu, et al., Nature
Nanotech. 2, 577 (2007).
60. D. Parke and A. Sapota, Internatl J. Occup. Med. Env. Health. 9, 331
(1996).
61. N. Patterson, D. P. Adams, V. C. Hodges, M. J. Vasile, J. R. Michael,
and P. G. Kotula, Nanotech. (2008).
Received: xx Xxxx xxxx. Accepted: xx Xxxx xxxx.
10 J. Bionanosci. 9, 1–10, 2015