Accepted Manuscript

In vitro genotoxicity and cytotoxicity of polydopamine-coated

magnetic nanostructures

Anna Woźniak, Magdalena Walawender, Dominika Tempka,

Emerson Coy, Karol Załęski, Bartosz F. Grześkowiak, Radosław
Mrówczyński

PII: S0887-2333(17)30208-4
DOI: doi: 10.1016/j.tiv.2017.07.022
Reference: TIV 4071
To appear in: Toxicology in Vitro
Received date: 9 January 2017
Revised date: 3 July 2017
Accepted date: 22 July 2017

Please cite this article as: Anna Woźniak, Magdalena Walawender, Dominika Tempka,
Emerson Coy, Karol Załęski, Bartosz F. Grześkowiak, Radosław Mrówczyński , In vitro
genotoxicity and cytotoxicity of polydopamine-coated magnetic nanostructures,
Toxicology in Vitro (2017), doi: 10.1016/j.tiv.2017.07.022

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 ACCEPTED MANUSCRIPT

In vitro genotoxicity and cytotoxicity of polydopamine-coated magnetic

nanostructures

Anna Woźniak*a, Magdalena Walawendera, Dominika Tempkab, Emerson Coya, Karol Załęskia,

Bartosz F. Grześkowiaka, Radosław Mrówczyński*a

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a
NanoBioMedical Centre, Adam Mickiewicz University in Poznan, Umultowska 85, 61-614 Poznan,

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Poland
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Department of Medical Biophysics, University of Lodz, Pomorska 141/143, 90-236 Lodz, Poland

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*Corresponding author: Anna Woźniak. NanoBioMedical Centre, Adam Mickiewicz University in
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Poznan, Umultowska 85, 61-614 Poznan, Poland, tel. +48 61 829 67 07, fax +48 61 829 52 91,
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[email protected]

Radosław Mrówczyński NanoBioMedical Centre, Adam Mickiewicz University in Poznan,

Umultowska 85, 61-614 Poznan, Poland e-mail: radosł[email protected]

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Co-authors’ emails:
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Magdalena Walawender [email protected]

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Dominika Tempka [email protected]

Emerson Coy [email protected]

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Karol Załęski [email protected]

Bartosz F. Grześkowiak [email protected]

*Radosław Mrówczyński [email protected]

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Abstract

Synthesis of magnetic nanoparticles and magnetic nanoclusters was performed by the co-

precipitation method or solvothermal synthesis, respectively, followed by oxidative polymerization of

dopamine, resulting in a polydopamine (PDA) shell. The nanomaterials obtained were described using

TEM, FTIR and magnetic measurements.

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For the first time, cyto- and genotoxicity studies of polydopamine-coated nanostructures were

performed on cancer and normal cell lines, providing in-depth insight into the toxicity of such

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materials. The tests conducted, e.g. ROS, apoptosis and DNA double-break of the nanomaterials

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obtained revealed the low toxicity of these structures. Thus, these results prove the biocompatibility

and low genotoxicity of these materials and provide new data on the toxicity of PDA-coated materials,
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which is of great importance for their biomedical application.
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Highlights

1. Novel polydopamine-coated magnetic nanoparticles and nanoclusters are proposed.

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2. The mechanism relies on their multimodal potential in biomedical applications.

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3. Cyto- and genotoxicity in vitro are determined as low and acceptable.

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Keywords: magnetic nanoparticle; nanotechnology; polydopamine; genotoxicity; cytotoxicity

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1. Introduction

Nanotechnology is a modern and intensive developing branch of science and has attracted the

attention of many laboratories. The main utility of this technology is that it uses the unique properties

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of nanoparticles (NPs) with respect to their macroscale counterparts. These changes significantly

improve their mechanical, optical, and electrical properties. Due to their size, which are similar to that

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of biomolecules, nanoparticles can pass through the cellular lipid bilayer and potentially be used in

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biological systems (Zhang et al., 2008). Thus, over the last decade there has been dynamic growth in

research in nanotechnology for biomedical applications. One of the most frequently investigated types
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of nanoparticle in this field of science are magnetic nanoparticles (MNPs). MNPs are characterized by

excellent superparamagnetic properties, a large surface area and the possibility of surface chemical
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modifications and biofunctionalization. The processes of synthesizing magnetic nanoparticles offers

the possibility of modification, which produces nanomaterials in of different size and purity. Applying
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magnetic nanoparticles to biomolecules or drug delivery increases the efficiency of such systems.
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Combining various properties of MNPs provides multimodal nanocarriers that can be used in targeted
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therapy, theranostics or hyperthermia.

One of the most important factors determining the utility of such types of nanoparticles in
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biomedicine is their toxicological profile. Nanotoxicology concerns the cytotoxicity and genotoxicity
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of nanoparticles on living cells or organisms. Exposure of in vitro systems to magnetic nanoparticles

may lead to them being damaged and/or to their death. The most commonly investigated

morphological and biochemical changes in cells include cell membrane permeability, enzyme activity,

proliferation studies, free radical production and changes in genetic material (Tang et al., 2015). The

majority of the changes described may lead to cell death. Reactive Oxygen Species (ROS) is well

known to play an important role in many cellular processes, such as cell signaling and regulation,

activation of signaling cascades and apoptosis, and alternate death pathways (Hancock et al., 2001;

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Burhans and Heintz, 2009; Li et al., 2003; Chen et al., 2009). A highly regulated and controlled

process of apoptosis induces changes in the content and location of proteins or other surface structures,

activates proteases (caspases) and destroys DNA and cellular body. Genotoxicity is also an emerging

subject in nanotoxicological research. Fragmentation of nucleic acids is the final product of genetic

material degradation, but it is a key issue in determining DNA damage via cell signaling pathways, as

well as epigenetic interactions (Thongkumkoon et al., 2014; Tanaka et al., 2007).

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In general, nanoparticle toxicity is highly complex and may depend on the purity, type of

synthesis, coating, shape, concentration and biological system being tested. Therefore, toxicity tests

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should be performed on every type of nanoparticle and after every step of synthesis with the use of

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different compositions and under various conditions. The lack of strict regulations concerning

physicochemical and biological evaluation of nanoparticles leads to a great need to provide extensive,
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careful analysis, in particular for biomedicine.

One of the most important issues that influences the toxicity of magnetic nanoparticles is
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surface chemistry. Bare nanoparticles often are hydrophobic. However, functionalization with

functional groups, ligands or cationic polymers increases their dispersion properties, biocompatible
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profile, and cellular uptake, due to electrokinetic interaction (Jin et al., 2011). Coating the surface of
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MNPs with a polymer not only improves the biocompatible profile of magnetic nanoparticles, but also
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provides functional groups for further modifications with biomolecules or drugs. However, differential

cellular responses were described due to various surface modifications (Yang et al., 2013).
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It was proved that poly(vinylalcohol)-coated SPIONs (Superparamagnetic iron oxide

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nanoparticles) decreased antigen processing and the CD4+ T cell stimulation capability of human

monocyte-derived dendritic cells. Yu et al. showed that dextran and polyethylene glycol coatings

reduced iron oxide NPs' cytotoxicity in aortic endothelial cells compared to bare ones. They

investigated MNPs of 5 and 30 nm in diameter using a Live/Dead assay, ROS and Cell Length and

Actin Cytoskeleton (Yu et al., 2012). Yang et al. also examined MNPs of different sizes (10, 100–150

nm) with different functional groups (–OH, –NH2) and coated with tetraethyl orthosilicate (TEOS), 3-

aminopropyltrimethoxysilane (APTMS) or TEOS/APTMS. They tested those nanoparticles with a

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CCK-8 assay, LDH assay and tail content of DNA and concluded differential cellular responses

(normal fibroblasts, fibrosarcoma cells) (Yang et al., 2013). A careful study of the literature also

revealed data describing the extremely low level of cytotoxicity and genotoxicity of thiolated (SH) and

S-nitrosated (S-NO) iron oxide superparamagnetic nanoparticles on healthy (3T3, human lymphocytes

cells, and Chinese hamster ovary cells) and cancer (MCF-7) cell lines (Seabra et al., 2014).

These research papers presented the toxicity of magnetic nanoparticles in contexts of biosafety

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that indicate good biocompatible properties, depending on the surface coating. However, there is a

need to develop a modern/novel coating which will possess a hydrophilic and stabilization function

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andwill also be able to serve as a donor of a functional group for further biomolecule (drugs, nucleic

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acid, antibody, peptides) attachment, as well as a nanostructure compound with physicochemical

properties enabling hyperthermia or phototherapy. Due to PDA's unique and specific properties, there
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is a possibility to bind biologically active biomolecules in a highly efficient and effective way

(Mrówczyński et al., 2016). It is also possible to achieve a local toxic effect by controlled NIR
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radiation and heating (Chen et al., 2016).

Polydopamine (PDA) is one of the most promising bioinspired coating polymers that allows to
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straightforward modification of coated objects, and exhibits adhesive properties towards various
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materials. PDA is formed by the oxidation of dopamine and is composed of dihydroxyindole,

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indoledione, and dopamine units (Liebscher et al., 2013). Other structures were also proposed in the

literature, where units of polymer linked covalently or via hydrogen bonding. The recently exploited
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features of polydopamine in biomedicine are photothermal properties, which allows the use of mere
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polydopamine or polydopamine nanomaterials in photothermal therapy, where cancer cells are

destroyed by an irradiating photothermal agent with a laser beam, resulting in temperature elevation.

What is of greater importance, polydopamine has been used to increase the photothermal properties of

magnetic nanoparticles which exhibit such features but are not big enough for application. Thus,

combining magnetic nanomaterials with a polydopamine coating provides a versatile nanoplatform

which can be applied in cancer thermal therapy and after the functionalization of such composites can

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be used for preparing multifunction nanomaterials for targeted drug and nucleic acid delivery, guided

photothermal therapy and MRI.

Even though magnetic nanomaterials coated with polydopamine have already been

successfully applied in vivo, proving their effectiveness, there still exists a significant need to provide

complex cytotoxicity and genotoxicity analysis of such nanostructures to determine their wide

biocompatibility profile for biomedical applications. Consequently, for the purpose of this study, the

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two most commonly used magnetic nanomaterials - magnetite nanoparticles (MNP 2) and magnetic

nanoclusters (MNP 3), both coated with polydopamine - were chosen due to their promising

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application as nanocarriers in biomedicine and the lack of information regarding their comprehensive

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cytotoxic data. To our best knowledge, so far no report has been produced which deals with intense in

vitro studies of polydopamine-coated nanomaterials and their influence and cytotoxicity on cancer
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cells and normal human fibroblasts.
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2. Materials and Methods

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2.1. Synthesis of nanoparticles

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2.1.1. MNP 2
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Firstly, magnetic nanoparticles were obtained according to the literature protocol with a slight
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modification as follows: FeCl2.4H2O(1.72 g, 8.65 mmol) and FeCl3.6H2O (4.7 g, 17.38 mmol) were

dissolved in water (80 mL) and degassed. The temperature was then elevated at 85 oC and an ammonia

solution (20 mL) was added. Heating was continued for 30 minutes, followed by the addition of citric

acid (2 g, 10.4 mmol) in 8 mL of water. Stirring continued for 1.5 h at 95 oC. The mixture was cooled

down to RT and the resulting nanomaterial was washed with distilled water (3x200 mL) and finally

redispersed in 100 mL of water.

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Next, the magnetic nanoparticles that had been obtained (1.4 g) were redispersed in 70 mL of H2O and

70 mL of Tris buffer (pH=8.5, 10 mmol) followed by the addition of dopamine hydrochloride (140

mg, 0.74 mmol, c=1 mg mL-1). Stirring under air access continued for 6 h and then the nanoparticles

were collected by an external magnetic field and washed with water (3x200 mL). Finally, they were

redispersed in water (250 mL).

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2.1.2. MNP 3

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Magnetic clusters were synthesized via the solvothermal method. FeCl3.6H2O (0.54 g) was dissolved

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in ethylene glycol with the addition of sodium acetate, sodium acrylate and then transferred into a

sealed Teflon autoclave and heated at 200 oC for 10 h. After cooling down to RT, the particles were
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washed with water and EtOH and dried in a vacuum at 40 oC. In the next step 25 mg of the result

material was redispered in 25 mL of water and 25 mL of Tris buffer using ultrasounds. Then dopamine
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hydrochloride (25 mg, 0.13 mmol) was added and continued to stirr for 2 h. Then nanoparticles were

collected by an external magnetic field and washed with water.

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Preparation of MNP 2 and MNP 3 is presented in Scheme 1.

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Scheme 1. Preparation of polydopamine-coated magnetite and magnetite nanoclusters covered with

polydopamine.

2.2. Physicochemical characterization

Transmission electron microscopy (TEM) images were recorded on a JEM-1400 microscope made by

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JEOL (Japan). The accelerating voltage was 120 kV. A small amount of the sample was placed on a

copper measuring grid (Formvar/Carbon 200 Mesh made by TedPella (USA)) after 5 minutes of

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sonication in deionized water. Next, the sample was dried in a vacuum desiccator for 24 hours. FTIR

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spectra were recorded on a Bruker Tensor 27 spectrometer in KBr pallets. Magnetization

measurements were performed using a MPMS-XL SQUID magnetometer. Temperature-dependent

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magnetization was studied in the temperature range 2-300 K. Magnetization, as a function of the

magnetic field ±30 kOe, was performed at 5 and 300 K. The zeta (ζ) potential of the MNPs suspension
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in water was determined by electrophoretic light scattering using a Malvern Zetasizer Nano ZS (UK).
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2.3. Cyto- and genotoxicity studies

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The KB cells (epidermal carcinoma derivative of HeLa cells) were cultured in EMEM -

Eagle's Minimum Essential Medium (Sigma-Aldrich), containing 10% fetal bovine serum (FBS,
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Sigma-Aldrich) and 1% antibiotics (penicillin 100 µg mL-1, streptomycin 100 µg mL-1, Sigma-
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Aldrich). The MSU-1.1 cells (normal human fibroblasts) were cultured in DMEM - Dulbecco's

Modified Eagle's medium, Sigma-Aldrich supplemented as described above. The cells were

maintained under sterile conditions (cabinet with laminar air flow) and incubated at 37 °C in a

humidified atmosphere supplemented with 5% CO2. Cell morphology and confluence were evaluated

using a Leica DMIL LED inverted microscope.

The KB cell line was chosen for this study as representative cancer cells, the MSU-1.1 cell

line served as a control cell line.

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For all the analysis, cells were prepared as follows: 70% confluence cells were trypsinized,

seeded at a concentration of 2x105/mL (MSU-1.1 cells) and 1.5x105/mL (KB cells) in microtiter plates

(tissue culture grade, 24 wells, flat bottom) and cultured for 24 h. Next, the medium was replaced with

a MNP 2 or MNP 3 suspension in a complete medium (concentrations of 16, 32, 64, 100, 300 µg mL-
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) and incubated in three time intervals (24, 48 and 72 h). All experiments were carried out in triplicate

in three independent biological evaluations. For each experiment, untreated cells were read as a

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negative control and cells incubated with 10% dimethyl sulfoxide – DMSO (Sigma-Aldrich) served as

a positive control.

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2.3.1. Oxidative stress assay (Muse® Oxidative Stress Kit, Merck Millipore)
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An oxidative stress assay allows for the quantitative measurement of superoxide radicals in

cells undergoing oxidative stress. The cells (70% of confluence) were prepared as described above
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(see section 2.3) and incubated with MNP 2 or MNP 3 suspensions in a complete medium. At the end

of incubation (24, 48 and 72 h), the cells were treated according to the manufacturer’s protocol. The
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cells were prepared in a 1X Assay Buffer and then incubated with 190 µl of Oxidative Stress working
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solution (37 oC, 30 min). The samples were then mixed thoroughly and run on a Muse® Cell
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Analyzer (Merck Millipore).

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2.3.2. Apoptosis assay (Muse™ Annexin V & Dead Cell Kit, Merck Millipore)
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Apoptosis tests based on Annexin V and PI staining allows the percentage of live, apoptotic

and dead cells to be estimated. The cells were prepared and incubated with MNP 2 or MNP 3

suspensions in a complete medium as described in section 2.3, and maintained according to the

manufacturer's instructions. The cells were resuspended in a medium containing at least 1% FBS. 100

μL of Muse™ Annexin V & Dead Cell Reagent and 100 μL of cells were added to each tube. The cell

suspension was incubated (RT, 20 min) and loaded onto a Muse® Cell Analyzer.

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2.3.3. DNA damage assay (Muse™ Multi-Color DNA Damage Kit, Merck Millipore)

A DNA damage assay enables the activation of ATM (Ataxia telangiectasia mutated kinase)

and H2AX (H2A histone family, member 2) signaling pathway in the cells exposed to nanoparticles to

be detected. The cells were prepared and incubated with a MNP 2 or MNP 3 suspension in a complete

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medium (procedure described in section 2.3). At the end of exposure (24, 48 and 72 h), the cells were

treated according to the manufacturer’s procedure. The cells were fixed in a Fixation Buffer (10 min

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on ice), then permeabilized with a Permeabilization Buffer (10 min on ice). After the washing stage,

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200 000 cells were incubated with 10 µL of the antibody cocktail (5 µL anti-phospho-ATM (Ser1981),

PE and 5 µL of anti-phospho-H2AX (Ser139), PECy5 conjugated) in 90 µL of 1X Assay Buffer per

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sample (RT, 30 min, in dark). The cells were then resuspended in a 200 µL 1X Assay Buffer and

acquired on a Muse® Cell Analyzer.

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2.4. Statistical analysis

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Each experiment was performed in at least three independent repeats. The responses measured
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were compared to the control and the corresponding samples with MNP 2 or MNP 3. The differences

between two unmatched groups were evaluated by the t-student parametric test or U Mann–Whitney
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nonparametric test. The difference was considered statistically significant at p value< 0.05. The data
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was statistically analyzed in Statistica (StatSoft).

3. Results

3.1. Physico-chemical characterization

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Figure 1. TEM picture of MNP 2 coated with PDA(a), magnetic nanoclusters (b) and FTIR spectra of
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nanomaterial MNP 2 and MNP 3 (c).

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Magnetic nanoparticles covered with a layer of PDA (MNP 2) with a diameter of 12±2 nm, as shown

by transmission electron microscopy (Figure 1a), were prepared by the routine co-precipitation

method from iron salts in a basic environment followed by oxidative polymerization of dopamine. A

thin layer of polydopamine was visible on the surface of the magnetic nanoparticles. The particles

were almost spherical in shape, although slightly agglomerated. The PDA-coated magnetic

nanoclusters (MNP 3) were papered via well-established protocol using the solvothermal method

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followed by coating with a PDA shell also using the dip method. The magnetic nanoclusters obtained

were 80±10 nm (Figure 1b). These spheres were continuously coated with polydopamine with a very

good distribution of spheres. Moreover, the successful coating of nanomaterials was proven not only

by TEM but also by FTIR. In the spectrum of MNP 2 and MNP 3 characteristic peaks between 1410

and 1620 cm-1 for polydopamine were observed, proving that a polydopamine shell was deposited on

the surface of the nanomaterials (Figure 1c). Additionally, the zeta potential of the nanomaterials

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prepared was also investigated. The zeta potential of the MNP 2 and MNP 3 suspension in water was -

30.9 mV and -32.5 mV, respectively.

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The magnetic properties of synthesized nanocomposites determined by SQUID measurements are

shown in Fig. 4. The saturation of magnetization determined from M(H) curves at room temperature is
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67 emu/g and 58 emu/g for MNP 2 and MNP 3, respectively. These values are high enough to control

magnetic particles easily by an external magnetic field. Additionally, the magnetic behavior of MNP 2
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was investigated at 5 K. It was found that at 5 K MNP 2 exhibits a hysteretic character with a

coercivity of Hc = 200 Oe. However, at 300 K there is only a small hysteretic contribution with a H c ~
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5 Oe – left inset, which is probably caused by clusterization of some part of the nanoparticles.
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Nevertheless, at 300 K they behave more like superparamagnetic materials. The maximum of the ZFC
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curve at TB = 233 K is defined as the blocking temperature of the superparamagnetic nanoparticles.

The ZFC curve's maximum width is probably also connected with the clusterization process between
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some part of the nanoparticles. This is in agreement with TEM data, which also showed a small
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clusterization of MNP 2.

Surprisingly, MNP 3 does not exhibit superparamagnetic behavior. The room temperature the M(H)

curve of MNP 3 shows hysteretic character – with Hc = 37 Oe, which point to the ferri/ferromagnetic

state of the sample at room temperature. We also observed that there is no maximum in the ZFC curve.

Thus, MNP 3 did not show superparamagnetic features.

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Figure 2. Magnetization versus applied magnetic field for MNP 2 at 5 and 300 K. The left inset shows

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the small hysteretic contribution in the M(H) curve at 300 K. The right inset shows the zero-field
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cooled (ZFC) and field cooled (FC) temperature dependence of magnetization for MNP 2 under an

applied field of 100 Oe (a). Magnetization versus applied magnetic field for MNP 3 at 300 K. The
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inset shows the ZFC and FC curves for MNP 3 at 100 Oe (b).

3.2. Cyto- and genotoxicity studies

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3.2.1. Oxidative stress assay

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The first test performed (oxidative stress assay) was devoted to fluorescent evaluation of

Reactive Oxygen Species (ROS) in the cells examined. This assay is based on cell permeable
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dihydroethidium (DHE). The DHE during the reaction with superoxide anions undergoes oxidation to

form a DNA-binding fluorophore ethidium bromide or a structurally similar product which intercalates

with DNA, resulting in red fluorescence. In this assay, two types of cell populations can be detected:

ROS(-) live cells and ROS(+) presenting ROS cells.

The exposure time intervals (24, 48 and 72 h) of MNP 2 toward KB cells resulted in the

highest level of ROS(+) cells at the concentration of 300 µg mL-1 and were estimated at approximately

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15.24%, 12.33% and 13.545%, respectively (Figure 3 a,b,c). The KB cells' response after incubation

with MNP 3 was also low, even at the highest concentration and the longest incubation time (Figure 3

d,e,f).

Statistically significant differences from the negative control at p value< 0.05 for: MNP 2:

after 48 and 72 h for ROS (-) and ROS (+) cells for concentrations of 100 and 300 µg mL-1; for MNP

3: after 24 h for ROS (-) cells for concentrations of 32, 64 and 100 µg mL-1, for ROS (+) cells for

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concentration of 64 µg mL-1, after 48 h for ROS (-) cells for concentrations of 32, 64, 100 and 300 µg

mL-1, for ROS (+) cells for concentrations of 64, 100 and 300 µg mL-1, after 72 h for ROS (-) cells for

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concentrations of 64, 100 and 300 µg mL-1.

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Figure 3. Oxidative stress analyses in KB cells after 24 (a), 48 (b), 72 h (c) exposure to MNP 2 and

after 24 (d), 48 (e) and 72 h (f) exposure to MNP 3.

Incubation of MNP 2 with MSU-1.1 for 24 h and 48 h cells did not result in significant

induction of ROS (Figure 4 a,b). However, an increased percentage of ROS(+) cells was observed

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after 72 h. The level of ROS(+) cells was nearly the same for all MNP 2 concentrations examined and

was approximately 30.49% (Figure 4 c). After 24 h and 48 h the MNP 3 examined caused a very low

percentage of ROS(+) cells (Figure 4 d,e). Longer exposition time (72 h) induced oxidative stress in

cells at the level of 19.66% (Figure 4 f).

Statistically significant differences from the negative control at p value< 0.05 for: MNP 2:

after 24 h for ROS (-) cells for concentrations of 32, 64, 100 and 300 µg mL-1, for ROS (+) cells for all

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concentrations, after 48 h for ROS (-) and ROS (+) cells for concentration of 64 µg mL-1, after 72 h for

ROS (-) cells for concentrations of 64 and 100 µg mL -1; for MNP 3: after 72 h for ROS (-) and ROS

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(+) cells for concentrations of 16, 32, 64 and 100 µg mL-1.

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Figure 4. Oxidative stress analyses in MSU-1.1 cells after 24 (a), 48 (b), 72 h (c) exposure to MNP 2

and after 24 (d), 48 (e) and 72 h (f) exposure to MNP 3.

3.2.2. Apoptosis assay

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The early stage of apoptosis can result in permeabilization of the cell membrane, leading to a

move outside phosphatidylserine, an important interior phospholipid. An apoptosis microcapillary

flow cytometer test combines two reagents, where each specifies a different type of cell population.

The first reagent is fluorescently labelled annexin V, which can specific binds to phosphatidylserine.

The second component is fluorescent propidium iodide (PI), which has the ability to pass a

permeabilized cell membrane and reach the DNA of late apoptotic or dead cells. Combining these

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reagents allows for fluorescence detection of the four types of cells analyzed: living (annexin V

negative, PI negative), early apoptotic (annexin V positive, PI negative), late apoptotic (annexin V

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positive, PI positive), and dead (annexin V negative, PI positive).

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The KB cells exhibited all four types of the aforementioned subgroups. After 24, 48 and 72 h

incubation with MNP 2, the average percentages of live cells in all the concentrations examined were
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stable: 83.723%, 82.71% and 83.60%, respectively. During the duration of the analysis we observed

an increasing contribution of late apoptotic cells and a simultaneous decrease in the percentage of
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early apoptotic cells. This growth corresponded to the time of incubation and nanoparticle

concentrations (Figure 5 a,b,c). Incubation with MNP 3 also resulted in a high profile of live cells
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expressed as follows: 77.52% (24 h), 83.40% (48 h) and 88.45% (72 h). In this case, we observed a
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decreasing subgroup of early and late apoptotic cells. In both types of magnetic nanoparticles tested,
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cells undergo apoptosis but dead cells were reported as being at a very low level (Figure 5 d,e,f).

Statistically significant differences from the negative control at p value< 0.05 for MNP 2: after
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24 h for live cells for concentration of 300 µg mL-1, for late apoptotic cells for concentration of 300
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µg mL-1, after 72 h for live cells for concentration of 300 µg mL-1; for MNP 3: after 24 h for live

cells for concentrations of 100 and 300 µg mL-1, for late apoptotic cells for concentrations of 32, 64,

100 and 300 µg mL-1, after 48 h for live cells for concentrations of 32 and 64 µg mL-1, for late

apoptotic cells for all concentrations.

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Figure 5. Apoptosis analyses in KB cells after 24 (a), 48 (b), 72 h (c) exposure to MNP 2 and after 24
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(d), 48 (e) and 72 h (f) exposure to MNP 3.

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After exposure of the MSU-1.1 cells to MNP 2, we observed an increased percentage of late

apoptotic cells with the highest value being reached after 72 h. At the same time, a subpopulation of
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dead cells grew but did not exceed 3% (Figure 6 a,b,c). A similar cellular response was detected

during MSU-1.1 cell incubation of MNP 3 (Figure 6 d,e,f).

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Statistically significant differences from the negative control at p value< 0.05 for MNP 2: after

24 h for live cells for concentrations of 64, 100 and 300 µg mL -1, for early apoptotic cells for

concentrations of 32, 64, 100 and 300 µg mL-1, for late apoptotic cells for concentration of 64 µg mL-1,

after 48 h for early apoptotic cells for concentrations of 16, 32 and 64 µg mL-1, for 72 h for early

apoptotic cells for all concentrations; for MNP 3: after 24 h for early apoptotic cells for concentrations

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of 32 and 64 µg mL-1, after 48 h for early apoptotic cells for concentration of 64 µg mL -1, for dead

cells for concentration of 300 µg mL-1.

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Figure 6. Apoptosis analyses in MSU-1.1 cells after 24 (a), 48 (b), 72 h (c) exposure to MNP 2 and

after 24 (d), 48 (e) and 72 h (f) exposure to MNP 3.

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3.2.3. DNA damage assay

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DNA damage (double-strand breaks) can result in activation of ATM protein, which is a

member of the phosphoinositide 3-kinase (PI3K)-related Ser/Thr protein kinase family (Bakkenist and

Kastan, 2003). Activated ATM initiates critical cellular signaling pathways and phosphorylates of

various downstream factors, including P53, CHK2, SMC1, NBS1, and histone H2AX (Burma et al.,

2001; Powers et al., 2004; Wu et al., 2000). The level of phospho-H2AX increases and accumulates at

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the sites of DNA damage, and is thus an indicator of DNA damage (Mahmoudi et al., 2011). This Kit

includes two directly conjugated antibodies, a phospho-specific ATM (Ser1981)-PE and a phospho-

specific H2AX-PECy5 and allows detection of the phosphorylation state of ATM and histone H2AX

simultaneously in a population of cells. This approach makes it possible to find three subpopulations

of the cells being analyzed: negative (e.g. no DNA damage), ATM phosphorylated only, phospho-

H2AX only, and DNA double-strand breaks (ATM + H2AX co-activated).

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The data showed that MNP 2 caused no DNA damage after 24 h of incubation with cancer cell

line KB. The highest percentage of ATM phosphorylated cells (4.61%) was observed at a

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concentration of 64 µg mL-1 (Figure 7 a). After 48 h and 72 h of incubation, those levels had

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decreased slightly and reached their highest value after 48 h 0.55% for a concentration of 16 µg mL -1

and after 72 h 1.64% for a concentration of 100 µg mL -1 (Figure 7 b,c). The population of phospho-
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H2AX cells was very low and after 24 h of incubation the highest level was established at the point

0.8% (concentration of 300 µg mL-1), after 48 h it was 4%, and after 72 h increased to 0.58% (both at a
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concentration of 16 µg mL-1) (Figure 7 a,b,c). The analysis also provided the percentage of DNA

double-strand break (ATM + H2AX co-activated) cells. Those values were the most visible after 24 h
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of incubation at point 4.6% (concentration of 32 µg mL-1), after 48 h it was 0.45% (concentration of 16

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µg mL-1), and after 72 h 0.45% (concentration of 100 µg mL-1) (Figure 7 a,b,c).

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DNA damage assay of MNP 3 also caused no significant changes. After 24 h of incubation

with MNP 3 the KB cell line resulted in a very low percentage of ATM phosphorylated cells, with the
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highest value being 4.92% for a concentration of 300 µg mL -1 (Figure 7 d). Decreased values were
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observed after 48 h (0.55% - 64 µg mL-1) and 72 h (0.62% - 32 µg mL-1) (Figure 7 e,f). The highest

percentage of the phospho-H2AX cells after exposure of 24 h was 0.87% (concentrations of 32 µg mL-
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) (Figure 7 d). A longer time of incubation caused a slightly increased level of phospho-H2AX

activated cells and was detected at the highest level of 2.88% (48 h, concentration of 32 µg mL -1) and

3.1% (72 h, concentration of 16 µg mL-1) (Figure 7 e,f). DNA double-strand breaks after the

introduction of MNP 3 to the cells being analyzed did not reach a high level and presented as follows:

after 24 h of incubation the highest value of this type of cell population was 2.04% (a concentration of

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16 µg mL-1), after 48 h it was 0.44% (a concentration of 64 µg mL-1) and after 72 h it was 0.6% (a

concentration of 16 µg mL-1) (Figure 7 d,e,f).

Statistically significant differences from the negative control at p value< 0.05 for MNP 3: after

48 h for pATM cells for concentration of 64 µg mL -1, after 72 h for negative cells for concentration of

300 µg mL-1, for phospho-H2AX for concentration of 300 µg mL-1.

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Figure 7. DNA damage analyses in KB cells after 24 (a), 48 (b), 72 h (c) exposure to MNP 2 and after

24 (d), 48 (e) and 72 h (f) exposure to MNP 3.

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The MSU-1.1 cells showed a very low percentage of pATM cells after exposition of MNP 2,

with the highest value after 72 h (4.36% at a concentration of 64 µg mL-1) (Figure 8 a,b,c). The

percentages of double-strand DNA breaks cells after 24 h and 48 h were low and an increase in these

was observed after 72 h (9.95% at a concentration of 100 µg mL -1) (Figure 8 a,b,c). The population of

phospho-H2AX cells increased most significantly after 24 h of exposition time (i.e. 6% at a

concentration of 16 µg mL-1). After 48 h and 72 h this population of cells decreased (Figure 8 a,b,c).
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MNP 3 toward MSU-1.1 cells showed a very low percentage of ATM phosphorylated cells after 24 h

and 48 h of incubation. However, this subgroup of cells increased after 72 h and reached 5% at a

concentration 100 µg mL-1 (Figure 8 d,e,f). Analysis also revealed an increased percentage of DNA

double-strand break cells with the highest value after 72 h (Figure 8 d,e,f). The percentage of phospho-

H2AX cells was rather low with a significant increase to the value 4.94% after 48 h at a concentration

of 100 µg mL-1 (Figure 8 d,e,f).

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Statistically significant differences from the negative control at p value< 0.05 for MNP 2: after

24 h for phospho-H2AX for concentration of 100 µg mL -1; for MNP 3: after 24 h for pATM cells for

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concentration of 100 µg mL-1, after 72 h for phospho-H2AX cells for concentrations of 100 and 300

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µg mL-1.
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Figure 8. DNA damage analyses in MSU-1.1 cells after 24 (a), 48 (b), 72 h (c) exposure to MNP 2 and

after 24 (d), 48 (e) and 72 h (f) exposure to MNP 3.

4. Discussion
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The aim of this paper was to investigate the cyto- and genotoxicity profile of polydopamine-

coated magnetic nanostructures. The synthesis of these included PDA-coated magnetic nanoparticles

(MNP 2) and nanoclusters (MNP 3). TEM characterization determined their shape as spherical with a

diameter 10 nm and 80-90 nm, respectively. PDA coating was confirmed using TEM and FTIR

analyses. Superparamagnetic properties were determined for MNP 2. Further work regarding the

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behavior of this material is ongoing in our laboratory. The zeta potential of prepared nanomaterials

revealed negatively charged samples. Those values indicate good stability of nanoparticle suspensions

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and are in close agreement with the values reported in the literature for PDA and PDA-coated

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materials. The charge of particles can influence their internalization, as well as their toxicity towards

cells. It was reported that negatively charged nanoparticles induce low cytotoxicity, although at the
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same time their internalization is diminished compared to the positively charged one (Gullotti et al.,

2013). On the other hand, nanoparticles found to have a high positive zeta potential show increased
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cytotoxicity and enhanced cellular internalization due to electrokinetic attraction (Jin et al., 2011).

Additionally, those nanoparticles are less stable in aquatic suspensions, as well as tending towards an
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aggregation and sedimentation process (Sze et al., 2003). However, in this research we decided to use
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nanoparticles in their native form and charged after coating with polydopamine, since this is the most
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common way they are used in various applications, including biomedicine.

In order to determine the profound profile of the toxicity of our materials we performed a 3
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test: oxidative stress assay, DNA damage and apoptosis, which are used for determining the
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cytotoxicity profile of nanomaterials. However, they are much more sophisticated than the routinely

used viability test. The two cell lines were chosen to determine the cellular response after NP exposure

in normal (fibroblasts) and cancer (KB) cells. It is important to show the biocompatible profile of the

NPs tested due to their future biomedical application, e.g. in vivo administration of anticancer drug

loaded MNP’s.

In the present study, ROS were dose-dependent and can elicit various further consequences

including apoptosis and DNA damage. The main cause of such a wide spectrum of negative

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implications lies in the ability of O2-, OH• and H2O2 lipids, proteins and nucleic acids peroxidation.

Externalization of phosphatidylserine was detected in the cell lines analyzed and thus it was possible

to estimate three populations of apoptosis-induced cells. The highest percentage was assigned to early

or late apoptotic ones. Recognition of ATM protein activation and phosphorylation of H2AX provided

in the further analyses allowed the precise determination of the two most frequent DNA damage

indicators, namely ATM phosphorylated only, phospho-H2AX only, with the occurrence of DNA

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double-strand breaks (ATM + H2AX co-activated) at a very low level. In the case of genotoxicity, we

observed time, dose and cell type-dependent mechanisms. Neither MNP 2 nor MNP 3 induced the

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most severe cellular injury as a response to nanostructure exposure, namely cell death.

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Comparing the unique structures of MNP 2 and MNP 3 which were tested, two different cell

types (KB – cancer, MSU-1.1 - normal) and the results observed, we tried to distinguish the most
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effective one in terms of inducing ROS, apoptosis or DNA damage. In the case of the KB cells'

oxidative stress test, the MNP 3 nanoparticles caused the highest percentage of ROS (+) cells
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(18.80%) at an MNP 3 concentration of 300 µg mL-1 after 48 h of incubation (Figure 3e). On the other

hand, the MSU-1.1 cells reached the highest level of response (39.24% ROS (+) cells) after 72 h of
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incubation with MNP 2 concentration of 32 µg mL-1 (Figure 4c). These values are statistically
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significant at p value< 0.05. An apoptosis assay revealed that MNP 3 induced the strongest response in
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KB cells (the lowest level of live cells – 73.89% after 24 h of incubation with an MNP 3 concentration

of 300 µg mL-1) (Figure 5d). This value is statistically significant at p value< 0.05. The opposite
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reaction of the MSU-1.1 cells was observed in the apoptosis test, because the lowest percentage of live
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cells – 67.39% - was a result of incubation with MNP 2 at a concentration of 16 µg mL-1 for 72 h

(Figure 6c). The percentage of negative KB cells after DNA damage investigation showed that MNP 2

significantly reduces the percentage of negative cells (90.81%, at a concentration of 64 µg mL -1 after

24 h of incubation) (Figure 7a). In the case of MSU-1.1 cells, 72 h of incubation with MNP 3 at a

concentration of 64 µg mL-1 resulted in a decreased percentage of DNA damage negative cells

(85.01%) (Figure 8f).

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In summary, KB cells were more sensitive to MNP 3 nanoparticles and MSU-1.1 to MNP 2. We

observed that a particular type of cell (MSU-1.1 vs KB) presented a similar reaction to MNP 2 and

MNP 3 exposure. It is worth noting that those differences concern the percentage of subpopulations of

damaged cells, but the level of live, healthy cells was similar. In general, MSU-1.1 cells were slightly

more susceptible to MNP 2 and MNP 3 action. We should also take into consideration the fact that

cellular response may depend on the type of cell line. Cells in the logarithmic growth phase usually are

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more sensitive than cells in the stationary phase (Yang et al., 2013; Mahmoudi et al., 2010; Mahmoudi

et al., 2011).

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Taking into account the results described above, we suggest that oxidative stress might be the

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primary mechanism for toxicity of MNP 2 and MNP 3 in cancer KB and normal MSU-1.1 cells. Our

results are in agreement with observations reported by other authors on the effect of MNPs on
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cytotoxicity and gene expression. Mahmoudi et al. showed different levels of cytotoxicity and gene

expression patterns according to human cell types (HCM, BE-2-C, HEK293T) after exposure to
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SPIONs with various surface chemistries (bare, COOH, and NH2). For this purpose, both an MTT

assay and a DNA microarray analysis were used. They found that SPIONs-COOH altered genes
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associated with cell proliferative responses due to their reactive oxygen species (ROS) properties as
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well as inducing H2AFX gene, which was also investigated in our studies (Mahmoudi et al., 2011).
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The literature on this subject includes many scientific reports concerning the cytotoxicity of

magnetic nanoparticles and shows their impact on biological in vitro systems as being rather low
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(Yang et al., 2013; Seabra et al., 2014; Häfeli et al., 2009; Blank et al., 2011; Yu et al., 2012). Surface
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coatings, such as organic and inorganic polymers, peptides, fibronectin, and dextran result in improved

hydrophilicity and protection of biological objects from adverse toxic reactions (Mahmoudi et al.,

2010; Lin et al., 2008). In our previous study, which concerned MNPs for drug delivery, we presented

the results of a WST-1 assay and Live/Dead assay of PDA-coated magnetic nanoparticles on HeLa

cells and concluded their low cytotoxicity profile (Mrówczyński et al., 2016). Häfeli et al. showed that

magnetite nanoparticles (8-10 nm) coated with triblock copolymers containing a PEO tail of a length

of above 2 kDa are biocompatible and appropriate for in vivo application. That study was performed

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using a MTT test and prostate cancer cells (PC3 and C4-2), human umbilical vein endothelial cells

(HUVECs) and human retinal pigment epithelial (HRPE) (Häfeli et al., 2009).

The toxicity of magnetic nanoparticles and magnetic nanoclusters both coated with

polydopamine was evaluated on a cellular and genetic signaling level. According to the toxicity

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assays, both types of MNPs which were tested display a low, time- and dose-dependent effect on cells'

response in terms of oxidative stress, apoptosis and DNA damage. The normal MSU-1.1 cells were

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more sensitive to MNP exposure than cancer KB cells. We summarize that polydopamine as a coating

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layer of magnetic nanoparticles and nanoclusters is highly biocompatible. Additionally, DNA damage

tests performed for the first time for this type of coating did not show any significant alternations in
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the genetic material of the KB cells and slightly increased the response after long-term exposure to

MSU-1.1 cells. Our results relate to the possibility of a multifunctional biomedical application of
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these nanomaterials.
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Conflict of interest

The authors declare no competing financial interest.

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Acknowledgements

This work was supported by the National Science Centre under project number UMO-

2014/13/D/ST5/02793. We would like to thank Jacek Wojnarowicz (MSc) from the Laboratory of

Nanostructures, Institute of High Pressure Physics of the Polish Academy of Sciences, Warsaw,

Poland, for zeta potential analyses.

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References

Bakkenist, C. J., Kastan, M. B., 2003. DNA damage activates ATM through intermolecular

autophosphorylation and dimer dissociation. Nature 421, 499-506.

Blank, F., Gerber, P., Rothen-Rutishauser, B., Sakulkhu, U., Salaklang, J., de Peyer, K., Gehr P.,

Nicod, L. P., Hofmann, H., Geiser, T., Petri-Fink, A., von Garnier C., 2011. Biomedical nanoparticles

PT
modulate specific CD4+ T cell stimulation by inhibition of antigen processing in dendritic cells.

Nanotoxicology 5, 606–621.

RI
SC
Burhans, W., Heintz, N., 2009. The cell cycle is a redox cycle: Linking phase-specific targets to cell

fate. Free Radic Biol Med. 47, 1282-1294.

NU
Burma, S., Chen, B. P., Murphy, M., Kurimasa, A., Chen, D. J., 2001. ATM phosphorylates histone
MA

H2AX in response to DNA double-strand breaks. J. Biol. Chem. 276, 42462-42467.

D

Häfeli, U. O., Riffle, J. S., Harris-Shekhawat, L., Carmichael-Baranauskas, A., Mark, F., Dailey, J. P.,
E

Bardenstein, D., 2009. ATM Phosphorylates Histone H2AX in Response to DNA Double-strand
PT

Breaks. Mol Pharm. 6, 1417-1428.

CE

Chen, Y., Azad, M. B., Gibson S. B., 2009. Superoxide is the major reactive oxygen species regulating
AC

autophagy. Cell Death Differ. 16, 1040-1052.

Chen, Y., Ai, K., Liu, J., Ren, X., Jiang, C., Lu, L., 2016. Polydopamine-based coordination

nanocomplex for T1/T2 dual mode magnetic resonance imaging-guided chemo-photothermal

synergistic therapy. Biomaterials 77, 198-206.

26
 ACCEPTED MANUSCRIPT

Gullotti, E., Park, J., Yeo, Y., 2013. Polydopamine-based surface modification for the development of

peritumorally activatable nanoparticles. Pharm Res. 30, 1956–1967.

Hancock, J. T. , Desikan, R., Neill, S. J., 2001. Role of reactive oxygen species in cell signalling

pathways. Biochem Soc Trans. 29, 345-350.

PT
Jin, J., Gu, Y.-J., Man, C. W.-Y., Cheng, J., Xu, Z., Zhang, Y., Wang, H., Lee, V. H., Cheng, S. H.,

Wong, W. T., 2011. Polymer-Coated NaYF4:Yb3+, Er3+ Upconversion Nanoparticles for Charge-

RI
Dependent Cellular Imaging. ACS Nano 5, 7838-7847.

SC
Li, N., Ragheb, K., Lawler, G., Sturgis, J., Rajwa, B., Melendez, J. A., Robinson, J. P., 2003.
NU
Mitochondrial Complex I Inhibitor Rotenone Induces Apoptosis through Enhancing Mitochondrial

Reactive Oxygen Species Production. J Biol Chem. 278, 8516-8525.

MA

Liebscher, J., Mrówczyński, R., Scheidt, H. A., Filip, C., Hădade, N. D., Turcu, R., Bende, A., Beck,
D

S., 2013. Structure of Polydopamine: A Never-Ending Story? Langmuir 29, 10539-10548.

E
PT

Lin, M. M., Kim, D. K., El Haj, A. J., Dobson, J., 2008. Development of Superparamagnetic Iron

Oxide Nanoparticles (SPIONS) for Translation to Clinical Applications. IEEE Transactions on

CE

Nanobioscience 7, 298–305.
AC

Mahmoudi, M., Simchi, A., Imani, M., Shokrgozar, M. A., Milani, A. S., Häfeli, U. O., Stroeve, P.,

2010. A new approach for the in vitro identification of the cytotoxicity of superparamagnetic iron

oxide nanoparticles.Colloids and Surfaces B, 75, 300–309.

27
 ACCEPTED MANUSCRIPT

Mahmoudi, M., Laurent, S., Shokrgozar, M. A., Hosseinkhani, M., 2011. Toxicity Evaluations of

Superparamagnetic Iron Oxide Nanoparticles: Cell “Vision” versus Physicochemical Properties of

Nanoparticles. ACS Nano 5, 7263–7276.

Mrówczyński, R., Jurga-Stopa, J., Markiewicz, R., Coy, E. L., Jurga, S., Woźniak, A., 2016.

Assessment of polydopamine coated magnetic nanoparticles in doxorubicin delivery. RSC Adv. 6,

PT
5936-5943.

RI
Powers, J. T., Hong, S., Mayhew, C. N., Rogers, P. M., Knudsen, E. S., Johnson, D. G., 2004. E2F1

SC
Uses the ATM Signaling Pathway to Induce p53 and Chk2 Phosphorylation and Apoptosis. Mol.

Cancer Res. 2, 203-214.

NU
Seabra, A. B., Pasquôto, T., Ferrarini, A. C. F., Santos Mda, C., Haddad, P. S., Lima, de R., 2014.
MA

Characterization, Cytotoxicity, and Genotoxicity Evaluations of Thiolated- and S-Nitrosated

Superparamagnetic Iron Oxide Nanoparticles: Implications for Cancer Treatment. Chem. Res.
D

Toxicol. 27, 1207–1218.

E
PT

Sze, A., Erickson, D., Ren, L., Li, D., 2003. Zeta-potential measurement using the Smoluchowski

equation and the slope of the current-time relationship in electroosmotic flow. J. Colloid Interface Sci.
CE

261, 402–410.
AC

Tanaka, T., Huang, X., Halicka, H. D., Zhao, H., Traganos, F., Albino, A. P., Dai, W.,

Darzynkiewicz, Z., 2007. Cytometry of ATM Activation and Histone H2AX Phosphorylation to

Estimate Extent of DNA Damage Induced by Exogenous Agents. Cytometry A 71, 648-61.

28
 ACCEPTED MANUSCRIPT

Tang, Y., Shen, Y., Huang, L., Lv, G., Lei, C., Fan, X., Lin, F., Zhang, Y., Wu, L., Yang, Y., 2015. In

vitro cytotoxicity of gold nanorods in A549 cells. Environmental Toxicology and Pharmacology 39,

871-878.

Thongkumkoon, P., Sangwijit, K., Chaiwong, C., Thongtem, S., Singjai, P., Yu, L. D., 2014. Direct

nanomaterial-DNA contact effects on DNA and mutation induction. Toxicology Letters. 226, 90-97.

PT
Wu, X., Ranganathan, V., Weisman, D. S., Heine, W. F., Ciccone, D. N., O'Neill, T. B., Crick, K.

RI
E., Pierce, K. A., Lane, W. S., Rathbun, G., Livingston, D. M., Weaver, D. T., 2000. ATM

SC
phosphorylation of Nijmegen breakage syndrome protein is required in a DNA damage response.

Nature 405, 477-482.

NU
Yang, W., Lee, J., Hong, S., Lee, J., Lee, J., Han, D. W., 2013. Difference between Toxicities of
MA

Iron Oxide Magnetic Nanoparticles with Various Surface-Functional Groups against Human Normal

Fibroblasts and Fibrosarcoma Cells. Materials 6, 4689-4706.

E D

Yu, M., Huang, S., Yu, K. J., Clyne, A. M., 2012. Dextran and Polymer Polyethylene Glycol (PEG)
PT

Coating Reduce Both 5 and 30 nm Iron Oxide Nanoparticle Cytotoxicity in 2D and 3D Cell Culture.

Int. J. Mol. Sci. 13, 5554–5570.

CE
AC

Zhang, L., Gu, F. X., Chan, J. M., Wang, A. Z., Langer, R. S., Farokhzad, O. C., 2008. Nanoparticles

in Medicine: Therapeutic Applications and Developments. Clinical Pharmacology & Therapeutics 83,

761−769.

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Scheme 1
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Highlights

 Novel polydopamine-coated magnetic nanoparticles and nanoclusters are proposed.

 The mechanism relies on their multimodal potential in biomedical applications.

 Cyto- and genotoxicity in vitro are determined as low and acceptable.

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