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Advanced Studies in Biology, Vol. 7, 2015, no. 8, 377 - 388
HIKARI Ltd, www.m-hikari.com
http://dx.doi.org/10.12988/asb.2015.5634
Optimal Concentration of PEG-Coated Fe3O4
Nanoparticles for Generation of Reactive Oxygen
Species in Human-Derived Amniotic Membrane
Stem Cells
Maryam Naseroleslami 1, Kazem Parivar 1, Samideh Khoei 2
and Nahid Aboutaleb 3,4 *
1 Department of Biology, Science and Research Branch
Islamic Azad University, Tehran, Iran
2 Medical Physics Department, School of Medicine
Iran University of Medical Sciences, Tehran, Iran
3 Physiology Research Center, Tehran, Iran
4 Department of Physiology
Iran University of Medical Sciences, Tehran, Iran
*Corresponding author
Copyright © 2015 Maryam Naseroleslami et al. This article is distributed under the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Abstract
Introduction: Superparamagnetic iron oxide nanoparticles (SPIONs) are widely
used for various biomedical applications. Although the potential benefits of
SPIONs are considerable, it has been shown that SPION uptake can cause
intracellular stress via the generation of reactive oxygen species (ROS).
Therefore, it is necessary to find the proper dose of SPIONs for bio medical
applications and treatment and diagnosis. The main goal of this study was to find
a concentration of SPIONs that can induce low intracellular levels of ROS.
Methods: After synthesis SPIONs were incubated with human-derived amniotic
membrane stem cells (hAMCs) in 5-200 μg/mL concentrations. Level of ROS was
378 Maryam Naseroleslami et al.
then measured using fluorimetry with Rhodamine 123 oxidation-sensitive
fluorescent probe uploading. Results: Data analysis showed that there was no
significant increase in generation of ROS in the cells with the particle
concentration range of 5-100 μg/mL. However, at concentrations higher than
100 μg/mL, generation of ROS increased. Conclusion: SPIONs coated with
polyethylene glycol (PEG) in concentrations ≤100 μg/mL have excellent labeling
efficiency and biocompatibility for hAMCs. Higher SPION concentrations
induced the generation of ROS in labeled cells.
Keywords: hAMCs, cell labeling, iron oxide nanoparticles, ROS
Introduction
Superparamagnetic iron oxide nanoparticles have been approved for
medical applications, diagnosis and cell therapy [1]. Factors such as size, shape
and surface charge of nanoparticles can determine their cellular internalization
and distribution as well as their efficacy. The requirements for use of
nanoparticles in biomedical applications include high magnetization value, size
≤100 nm, narrow particle size distribution and specific surface coating [2].
Various materials have been evaluated for SPION surface coating such as
polymers, biomolecules, metals and silica to make them more biocompatible.
PEG coating improves labeling efficiency with subtle distribution of SPION
complexes in cells [1]. The biological toxicity of nanoparticles highly depends on
the organ or cell types exposed primarily. The toxicokinetics of uptake, transport,
metabolism and accumulation of nanoparticles in different organs and their final
excretion are equally important when discussing nanotoxicity [3, 4]. Iron overload
due to the degradation of SPIONs has also been presumed to cause cytotoxicity.
Several adverse effects such as generation of ROS, membrane and DNA damage
have been reported in SPION-labeled cells [1, 5]. ROS, as the name implies,
derive and present higher reactivity than molecular oxygen with redox activity [6,
7]. The recent interest in the role of free radical processes in cellular physiology
and pathology has resulted in a demand for methods of quantification of ROS
production in living cells. Thus, detection of ROS relies on detecting end products
formed when specific compounds react with ROS. These include measurements
of oxidation of dichlorofluorescein (H2DCF), Rhodamine 123 (RH123) or
hydroethidine (DHE). Rhodamine 123, an anionic dye, is capable of penetrating
into the mitochondrial matrix and causing photoluminescence quenching
dependent on mitochondrial transmembrane potential [8]. In fact, RH123 can be
oxidized by various ROS thereby serving as an indicator of the degree of general
oxidative stress [9]. This study aimed to evaluate the effect of nanoparticles on
hAMCs because these cells have anti-inflammatory properties, secrete growth
factors, have high differentiation rate and are easily accessible and affordable[10].
Thus, further studies are required to assess the effect of iron oxide nanoparticles
on these cells to find a non-toxic dose of these nanoparticles for use to label and track cells with MRI after injection into the target tissue. This study aimed to evaluate
Optimal concentration of PEG-coated Fe3O4 nanoparticles 379
the effect of different concentrations of PEG-coated SPIONs on hAMCs Because
we want Monitoring of in vivo migration of labeled hAMCs with MRI after
intra myocardial injection in Next study.
Materials and Methods
Particle preparation:
Nanoparticles were synthesized by classic co-precipitation of iron oxide in
an alkaline solution in presence of polyethylene glycol. Next, magnetic
nanoparticles were characterized by various physicochemical means such as
transmission electron microscopy (TEM), dynamic light scattering (DLS) and
infrared spectroscopy (FT-IR).
Isolation and cultivation of human mesenchymal cells from the amniotic sac:
Term placentas from pregnancies with normal evolution were taken via
normal delivery without using any invasive method (Milad Hospital) after
obtaining written informed consent of the pregnant women. The harvested
placentas were transported in cold phosphate buffered saline (PBS) solution in a
thermally insulated container on ice. To obtain msenchymal cells, the amniotic
membrane was mechanically separated from the subjacent chorion by detachment.
The membrane fragments were transferred to Petri dishes and blood vessels were
removed. The tissue was washed with PBS solution and mechanically crushed by
a blade to obtain quite small pieces of tissue. The pieces of tissue dete wideerew
by collagenase and incubated for four hours. Then, they were centrifuged at
1500×g for five minutes and the pellets were suspended in low glucose
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum (FBS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL
streptomycin (Gibco Co., Germany). The cells were plated in 25 cm2 flasks
supplemented with DMEM and maintained in 5% CO2 atmosphere at 37°C. The
medium was refreshed 48-72 hours after the initial culture to remove non-
adherent cells and the adherent cells were grown to confluence before each
passage. The medium was refreshed twice a week.
Cell characterization by flow cytometry:
In order to ensure that the isolated cell was a mesenchymal cell, we
performed flow cytometric assay. Cells were stained with specific antibodies for
flow cytometry. In brief, cultured hAMCs were washed twice in PBS and
harvested using 0.25% trypsin/EDTA. The cells were then washed with PBS and
divided into groups for antibody staining. Each aliquot contained approximately 5
× 103 cells. The antibodies were used to detect cell surface antigens namely
CD90, CD105, CD166, CD45 and CD34. All antibodies were conjugated with
fluorescein-isothiocyanate and phycoerythrin. The cells were stained at 4℃ for 30
minutes. After incubation, the cells were washed with PBS and re-suspended in
500 µL of PBS. Analysis was performed with a FACSCalibur flow cytometer
(BectinDekenson, USA).
380 Maryam Naseroleslami et al.
Osteogenic Differentiation:
Osteogenic differentiation was performed with medium DMEM-HG
supplemented with 10 μM dexamethasone, 10 nM vitamin D3, and 50 mg
ascorbic phosphate for 21 days. Calcium deposits were evidenced by 2% Alizarin
Red staining for 10 minutes PBS solution. Then viewed with invert microscop.
Adipogenic differentiation:
Cells were cultivated for 21 days in DMEM-HG supplemented with 1 μM
dexamethasone, 10 μg/mL insulin, and 200 μM indomethacin. The cells were
stained with 0.5% Oil Red O for 10 minutes to reveal the intracellular
accumulation of lipid-rich vacuoles. Then viewed with invert microscop.
All reagents used in this experiment were from Sigma Co.
Cell culture:
Human derived amniotic membrane stem cells were cultured in DMEM
supplemented with 10% FBS at 37°C and 5% CO2 in a humidified atmosphere.
Cells were subcultured upon reaching 80% to 90% confluence. All experiments
were performed on third-fifth passage cells.
Magnetic cell labeling:
SPIONs were added directly to the cell culture at concentrations of 0,5, 10,
20, 30, 40, 50, 75, 100, 125, 150, 175 and 200 µg /mL. After 48 hours of
incubation with cells, culture media were removed and cells were washed three
times with PBS buffer to wash out non-internalized particles and use them for
subsequent assays.
ROS assay:
Treated cells were collected and resuspended in PBS, which contained 10 μM
Sigma RH123 and incubated at 37 ºC for 30 minutes. When the mitochondrial
membrane potential depletes, the RH123 is released from the mitochondria and
produces fluorescence. Samples were analyzed by fluorimetry; which was
performed with an excitation wavelength of 422 nm and an emission wavelength
of 520 nm.
Statistical analyses:
All values were expressed as mean± standard deviation from three
experiments. Statistical analyses were performed using SPSS version 21.0. Data
were analyzed for statistical significance by one-way ANOVA with post hoc
Bonferroni correction for multiple comparisons. A P-value of less than 0.05 was
considered statistically significant.
Optimal concentration of PEG-coated Fe3O4 nanoparticles 381
Results
Particle preparation:
PEG-coated SPION samples were successfully obtained dnw had spherical
morphology and average particle size of 20 nm. Morphological examinations of
the nanoparticles were performed using a TEM (Figure 1) and the diameter of the
nanoparticles was analyzed by DLS (Figure 2). Also, FT-IR analysis of SPIONs
and surface-modified SPIONs was performed to characterize the surface nature of
these particles and study the coating properties of the magnetite surface (Figure
3).
Cell harvesting from the amniotic membrane:
We succeeded in isolating and culturing mesenchymal cells from the
placental amniotic membrane. At first, the cells had a round shape and as this
process progressed they became elongated, finally acquiring a typical fibroblastic
morphology (Figure 4).
Cell characterization by flow cytometry:
We conducted flow cytometric analysis of the third passage cells and
monitored the expression of five CD markers (CD34, CD45, CD90, CD105 and
CD166) to determine whether the cells displayed a mesenchymal stem cell
phenotype. The cells that expressed CD90, CD166 and CD105 showed a negative
reaction to hematopoietic markers such as CD34 and CD45 (Figure 5).
Osteogenic Differentiation and Adipogenic differentiation:
Adipogenic differentiation of hAMCs was apparent after 21 days of
incubation with adipogenic medium. By the end of the third week, half of the cells
contained Oil red O-positive lipid droplets. The colonies of hAMCs were
subjected to Alizarin red staining 21 days after the initiation of osteogenic
differentiation. Intense Alizarin red staining of the colonies confirmed that
calcium deposition had occurred (Figure 6).
ROS assay:
Different concentrations of SPIONs had cytotoxic effects on hAMCs
compared to the control group after 30 minutes of incubation. The generated ROS
was 0.58%±0.53%, 0.73%±0.24%, 0.88%±0.23%, 1.48%±1.26% , 2.15±0.77,
3.32 ± 0.72, 6.96± 1.19, 13.17± 0.83 (P<0.05), 25.63± 0.91 (p<0.005), 34.58±
5.44 (p<0.005), 49.96± 1.49 (p<0.005) and 56.05± 1.54 (p<0.005) at iron
concentrations of 5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175 and 200 µg /mL,
respectively.The suitable dose of SPION for use on hAMCs was calculated to be
100 µg /mL (Figure 7).
382 Maryam Naseroleslami et al.
Discussion
Superparamagnetic iron oxide nanoparticles are important for medical
applications. Despite extensive applications of these nanoparticles, exposure of
cells to these nanoparticles may induce the release of ROS in cells [11, 12]. The
toxicity of SPIONs is strongly dose-dependent. Moreover, it depends on the way
it is expressed i.e. mass versus particles versus surface area. The choice of more
biocompatible iron oxide-based magnetic particles would resolve the problem of
cytotoxicity. Coating of these nanoparticles with an additional biocompatible
layer may improve the colloidal stability and biocompatibility [13]. We used PEG
for coating of SPIONs. At low levels of ROS production, cells initiate a protective
response to guarantee their survival, but excessive production of ROS can initiate
cellular tissue damage by modifying and denaturing lipids, proteins and DNA,
which seriously compromises cell viability and may induce a variety of cellular
responses via the generation of secondary reactive species, leading to cell death by
necrosis or apoptosis [11, 12]. We demonstrated that intracellular oxidative stress
indicated by ROS production in SPION labeled cells occurred in a concentration
range from 5 to 200 μg /mL, which was detected using Rhodamine 123. Our data
indicated that SPION labeling dose-dependently affected hAMCs in vitro and we
showed that the PEG coating of SPION was not toxic to cells at concentrations
less than 125 μg /mL with respect to controls. Furthermore, nanoparticles at
concentrations between 5-100 µg /mL were biocompatible and suitable for use in
vivo and for therapeutic purposes. This result was in accord with the findings of a
previous experiment on generation of ROS in cells loaded with SPION in
vitro.Naqvi et al, in 2010 evaluated the cytotoxicity of iron oxide nanoparticles
based on their concentration via assessment of increase in oxidative stress
markers. They cultured human macrophages and added nanoparticles to this
medium. Using an ascending concentration of iron oxide nanoparticles increased
ROS, which subsequently caused cell injury and cell death [14]. Hoskins et al.
showed that iron nanoparticles increased the generation of ROS.
Production of oxidative stress factors caused cell death [15]. Studies have
shown that iron metabolism damages the SPION surface, increases the
concentration of iron ions and induces oxidative stress [16]. Thus, it seems that
high dose injection of these nanoparticles causes DNA damage, cell necrosis and
apoptosis via oxidative stress [4, 5]. The results of the previous studies as well as
the current study showed that cell damage due to iron oxide nanoparticles was
dose-dependent. Thus, use of proper dose of iron oxide nanoparticles is very
important to prevent cell damage or cell death due to oxidative stress. Our results
revealed that generation of ROS in cells labeled with a specific dose of SPION
was not significantly different from that in controls. Thus, SPIONs at a specific
concentration can be used for detection of cells.
Optimal concentration of PEG-coated Fe3O4 nanoparticles 383
Conclusion
Considering the extensive applications of nanoparticles in medicine, the
effects of SPIONs on cells must be evaluated. In this study, we assessed the
effects of SPIONs on generation of ROS by measuring the level of ROS in cells
treated with various doses of SPIONs. We observed that the intracellular level of
ROS gradually increased with an increase in concentration of nanoparticles. Our
data may be useful for efficiently calibrating the best SPION dose to minimize
their harmful effects on cells.
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Optimal concentration of PEG-coated Fe3O4 nanoparticles 385
Figures:
Figure 1. Spherical shape of a synthesized iron oxide nanoparticle under an
electron microscope
Figure 2. Hydrodynamic size of magnetic nanoparticles
-20,00
0,00
20,00
40,00
60,00
80,00
100,00
120,00
0,00 50,00 100,00 150,00 200,00 250,00 300,00
386 Maryam Naseroleslami et al.
Figure 3. FTIR spectroscopy of spion nanoparticles
Figure 4. Third passage stem cells isolated from the amniotic membrane at ×200
magnification
Optimal concentration of PEG-coated Fe3O4 nanoparticles 387
Figure 5. Flow cytometric analysis of hAMCs showing positive surface markers
for mesenchymal stem cells and negative surface markers for hematopoietic cells
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Q1: 1.23% Q2: 0.04%
Q3: 98.69% Q4: 0.04%
Gate: R1
1 10 100 10000
20
40
60
80
100
FL1 -
coun
ts
RN1
Gate: R1
1 10 100 10000
20
40
60
80
100
FL2 CD34-PE
coun
ts
RN2
partec PAS
Region Gate Ungated Count Count/ml %Gated Mean-x CV-x% Mean-y CV-y%
R1 <None> 2754 2754 - 91.80 92.80 29.21 80.20 45.47Q1 R1 98 34 - 1.23 1.80 28.35 4.06 30.74
Q2 R1 13 1 - 0.04 7.63 0.00 21.25 0.00
Q3 R1 2887 2718 - 98.69 1.01 10.95 1.17 31.20Q4 R1 2 1 - 0.04 7.16 0.00 1.00 0.00
RN1 R1 111 35 - 1.27 4.55 69.30 - -
RN2 R1 19 2 - 0.07 7.39 4.50 - -
File: cd45-f-cd14-pe_ Date: 10-03-2013 Time: 14:30:58 Particles: 2932 Acq.-Time: 40 s
0 50 100 150 200 2500
50
100
150
200
250
FSC -
SSC
-
1 10 100 10001
10
100
1000
FL2 CD14-PE
FL1
CD4
5-FI
TC
1 10 100 10000
20
40
60
80
100
FL1 CD45-FITC
coun
ts
1 10 100 10000
20
40
60
80
100
FL2 CD14-PE
coun
ts
0 50 100 150 200 2500
50
100
150
200
250
FSC -
SSC
-
R1
Gate: R1
1 10 100 10001
10
100
1000
FL2 CD14-PE
FL1
CD4
5-FI
TC
Q1: 18.54% Q2: 0.41%
Q3: 80.82% Q4: 0.22%
Gate: R1
1 10 100 10000
20
40
60
80
100
FL1 CD45-FITC
coun
ts
RN1
Gate: R1
1 10 100 10000
20
40
60
80
100
FL2 CD14-PE
coun
ts
RN2
partec PAS
Region Gate Ungated Count Count/ml %Gated Mean-x CV-x% Mean-y CV-y%
R1 <None> 2691 2691 - 91.78 90.49 28.22 81.11 43.24Q1 R1 598 499 - 18.54 1.63 40.58 6.07 160.73
Q2 R1 48 11 - 0.41 13.45 95.19 30.04 113.55
Q3 R1 2278 2175 - 80.82 1.04 20.97 1.19 39.50Q4 R1 8 6 - 0.22 7.97 30.63 1.30 45.20
RN1 R1 646 510 - 18.95 6.58 171.87 - -
RN2 R1 67 21 - 0.78 10.20 96.63 - -
File: cd105-f-cd166-pe Date: 10-03-2013 Time: 14:26:29 Particles: 3000 Acq.-Time: 31 s
0 50 100 150 200 2500
50
100
150
200
250
FSC -
SSC
-
1 10 100 10001
10
100
1000
FL2 CD166-PE
FL1
CD1
05-F
ITC
1 10 100 10000
20
40
60
80
100
FL1 CD105-FITC
coun
ts
1 10 100 10000
20
40
60
80
100
FL2 CD166-PE
coun
ts
0 50 100 150 200 2500
50
100
150
200
250
FSC -
SSC
-
R1
Gate: R1
1 10 100 10001
10
100
1000
FL2 CD166-PE
FL1
CD1
05-F
ITC
Q1: 0.00% Q2: 95.26%
Q3: 0.22% Q4: 4.51%
Gate: R1
1 10 100 10000
20
40
60
80
100
FL1 CD105-FITC
coun
ts
RN1
Gate: R1
1 10 100 10000
20
40
60
80
100
FL2 CD166-PE
coun
ts
RN2
partec PAS
Region Gate Ungated Count Count/ml %Gated Mean-x CV-x% Mean-y CV-y%
R1 <None> 2703 2703 - 90.10 91.86 28.18 79.50 45.39Q1 R1 0 0 - 0.00 - - - -
Q2 R1 2842 2575 - 95.26 47.09 60.34 13.29 143.28
Q3 R1 9 6 - 0.22 1.85 72.78 1.49 53.47Q4 R1 149 122 - 4.51 37.07 54.91 1.99 37.50
RN1 R1 2842 2575 - 95.26 13.29 143.28 - -
RN2 R1 2991 2697 - 99.78 46.64 60.41 - -
388 Maryam Naseroleslami et al.
Figure 6. Differentiation capacity of mesenchymal stem cell.Osteogenic
differentiation of hAMCs (left) and Adipogenic differentiation of hAMCs (right)
was determined after culturing for 21 days under osteogenic and adipogenic
conditions.
Figure 7. Cytotoxic analysis of SPIONs. Oxidative stress in cells after exposure
to various concentrations of SPIONs (5, 10, 20, 30, 40, 50, 75, 100, 125, 150, 175
and 200 µg /mL) for 30 minutes. Data are expressed as mean ± standard deviation
from three experiments as % of control cells. Statistical significance compared to
unlabeled cells as controls. *P <0.05 and **P <0.005.
Received: June 27, 2015; Published: August 7, 2015
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