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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Figures:

Figure 1. Spherical shape of a synthesized iron oxide nanoparticle under an

electron microscope

Figure 2. Hydrodynamic size of magnetic nanoparticles

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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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RN1

Gate: R1

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40

60

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FL2 CD34-PE

coun

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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

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SSC

-

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FL1

CD4

5-FI

TC

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FL1 CD45-FITC

coun

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SSC

-

R1

Gate: R1

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FL2 CD14-PE

FL1

CD4

5-FI

TC

Q1: 18.54% Q2: 0.41%

Q3: 80.82% Q4: 0.22%

Gate: R1

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FL2 CD14-PE

coun

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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

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FL1

CD1

05-F

ITC

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FL1 CD105-FITC

coun

ts

1 10 100 10000

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FL2 CD166-PE

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100

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-

R1

Gate: R1

1 10 100 10001

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FL2 CD166-PE

FL1

CD1

05-F

ITC

Q1: 0.00% Q2: 95.26%

Q3: 0.22% Q4: 4.51%

Gate: R1

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40

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FL1 CD105-FITC

coun

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Gate: R1

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FL2 CD166-PE

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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