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 Diamond & Related Materials 127 (2022) 109149

Contents lists available at ScienceDirect

Diamond & Related Materials

journal homepage: www.elsevier.com/locate/diamond

Functionalized SPION immobilized on graphene-oxide: Anticancer and

antiviral study
Shaghayegh Kohzadi a, Najmeh Najmoddin a, *, Hadi Baharifar a, Mahdi Shabani b
a
Department of Biomedical Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran
b
Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

A R T I C L E I N F O A B S T R A C T

Keywords: The progressive and fatal outbreak of some diseases such as cancer and coronavirus necessitates using advanced
Graphene oxide materials to bring such devastating illnesses under control. In this study, graphene oxide (GO) is decorated by
Iron oxide superparamagnetic iron oxide nanoparticles (SPION) (GO/SPION) as well as polyethylene glycol functionalized
Chitosan
SPION (GO/SPION@PEG), and chitosan functionalized SPION (GO/SPION@CS). Field emission scanning elec­
Polyethylene glycol
Hyperthermia
tron microscopic (FESEM) images show the formation of high density uniformly distributed SPION nanoparticles
SARS-CoV-2 on the surface of GO sheets. The structural and chemical composition of nanostructures is confirmed by X-ray
diffraction and Fourier transform infrared spectroscopy. The saturation magnetization of GO/SPION, GO/
SPION@PEG and GO- SPION@CS are found to be 20, 19 and 8 emu/g using vibrating sample magnetometer.
Specific absorption rate (SAR) values of 305, 283, and 199 W/g and corresponding intrinsic loss power (ILP)
values of 9.4, 8.7, and 6.2 nHm2kg− 1 are achieved for GO/SPION, GO/SPION@PEG and GO/SPION@CS,
respectively. The In vitro cytotoxicity assay indicates higher than 70% cell viability for all nanostructures at 100,
300, and 500 ppm after 24 and 72 h. Additionally, cancerous cell (EJ138 human bladder carcinoma) ablation is
observed using functionalized GO/SPION under applied magnetic field. More than 50% cancerous cell death has
been achieved for GO/SPION@PEG at 300 ppm concentration. Furthermore, Surrogate virus neutralization test is
applied to investigate neutralizing property of the synthesized nanostructures through analysis of SARS-CoV-2
receptor-binding domain and human angiotensin-converting enzyme 2 binding. The highest level of SARS-
CoV-2 virus inhibition is related to GO/SPION@CS (86%) due to the synergistic exploitation of GO and chito­
san. Thus, GO/SPION and GO/SPION@PEG with higher SAR and ILP values could be beneficial for cancer
treatment, while GO/SPION@CS with higher virus suppression has potential to use against coronaviruses. Thus,
the developed nanocomposites have a potential in the efficient treatment of cancer and coronavirus.

1. Introduction
Cancer as one of the life-threatening diseases on human health,
causes almost 10.0 million deaths annually (2020), reported by world
health organization (WHO) in February 2020 factsheets [1]. Beyond
different reported treatments for cancer, including chemotherapy,
radiotherapy, and surgery, magnetic hyperthermia is a non-invasive
cancer therapy with minimal detrimental side effects [2]. In such
treatment, magnetic materials act as a heating source, generating heat
under the applied external magnetic field [3]. Cancer cells are typically
more susceptible than the healthy cells to mild heat (~ 42 ◦ C) due to
their less blood flow which consequently limits the heat dissipation to
the surrounding area [2,3]. Since tumor cells are more heat sensitive,
exploiting effective and specific targeting therapeutic agent (i.e., mag­
netic nanomaterials) would be a promising tool to combat cancer cells
[4].
The incidence (251 million confirmed cases) and mortality (5 million
deaths) of the newly emerged coronavirus SARS-CoV-2 require urgent
development of materials that can neutralize the virus function and
prevent the infection [5]. Several recent evidences have highlighted that
nanomaterials (e. g. graphene oxide, GO) have a high potential to
neutralize cell-protein interactions and prevent viral endocytosis due to
their large surface area or having various functional groups [6–8].
Moreover, it has been reported that chitosan shows inhibitory activity
against covid-19 [9,10]. Positively charged chitosan can interact with
the receptor-binding domain (RBD) of the virus and assemble protein-

* Corresponding author at: Department of Biomedical Engineering, Science and Research Branch, Islamic Azad University, Daneshgah Blvd, Tehran, Iran.
E-mail addresses: najmoddin@srbiau.ac.ir, najmoddin63@yahoo.com (N. Najmoddin).

https://doi.org/10.1016/j.diamond.2022.109149
Received 2 March 2022; Received in revised form 23 May 2022; Accepted 29 May 2022
Available online 3 June 2022
0925-9635/© 2022 Elsevier B.V. All rights reserved.
S. Kohzadi et al.
polymer complexes, which could inactivate the virus and obstruct its
connection with human cells [11].
Recently, superparamagnetic iron oxide nanoparticle (SPION) has
received immense attention in biomedical applications such as targeted
delivery of drug [12], magnetic resonance imaging (MRI) [13], tissue
engineering [14] and hyperthermia for cancer therapy [15] owing to its
magnetic property, low toxicity, resilient mechanical structure and no
remnant magnetization [16,17]. However, using this outstanding ma­
terial in vivo faces longstanding obstacles. Among them, particle
agglomeration, less colloidal stability in physiological media and short
circulation time in the blood are major challenges. To overcome particle
agglomeration and preserve narrow size distribution of SPION,
exploiting favorable support such as 2D materials (e. g. GO) could be a
good choice [18]. The high specific surface area of GO makes it a suit­
able candidate to be decorated with nanoparticles with rather good
distribution [19]. Moreover, GO has numerous functional groups such as
hydroxyl, carboxyl, and epoxy existed on its surface that makes possi­
bility for connection between GO sheets and many other molecules.
Many researchers have focused on using GO/iron oxide nanoparticles
composite in medical applications. For instance, Wang et al. [20]
controlled drug release of temozolomide from GO/iron oxide nano­
particles. Kumar et al. [21] synthesized GO/iron oxide nanoparticles for
radio-frequency hyperthermia therapy. Borrás et al. [22] prepared GO/
SPION nanocomposite as contrast agents in MRI. Recently, the attractive
ability of GO to interact, degrade membrane and inhibit enveloped vi­
ruses like SARS-CoV-2 encourages researchers to use this material to
combat coronaviruses [23,24].
Functionalization is a versatile strategy to tackle shortcomings or
donate salient features to materials. For instance, attempt has been
made to improve colloidal stability and blood circulation time of SPION
via polymeric surface coating. Bach et al. [25] prepared SPION nano­
particles capped with polyethylene glycol (PEG) to reduce the agglom­
eration. Kazemzadeh et al. [26] enhanced SPION stability in water/oil
emulsion with the aid of chitosan. Dolatkhah et al. [27] developed
nanocomposites of GO loaded with PEGylated SPION nanoparticles and
grafted with methotrexate and stimuli-responsive linkers for photo­
thermal and chemotherapy of breast cancer. Ma et al. [28] synthesized a
multifunctional PEG functionalized GO/SPION composite to acquire
high stability in physiological solutions. This nanostructure also
exhibited strong optical absorbance which is beneficial for applications
in cancer therapy.
The novelty of current study involves following aspects: (i) since
biocompatibility is one of main issues of using materials in medical field
[29–35], cell behavior of PEG functionalized SPION decorated on GO
sheets (GO/SPION@PEG) and chitosan functionalized SPION decorated
on GO sheets (GO/SPION@CS) has been studied and the morphology of
cells with and without applying magnetic field has been observed, (ii)
the magneto thermal ability of GO/SPION@PEG and GO/SPION@CS to
kill cancerous cells is investigated through applying an external mag­
netic field, (iii) the ability of GO/SPION@PEG and GO/SPION@CS to
inhibit covid-19 activity is addressed.
2. Materials and methods
2.1. Materials
Polyethylene glycol (PEG, 6000 molecular weight), chitosan medium
molecular weight, iron (II) chloride tetrahydrate (FeCl2⋅4H2O, 99%),
iron (III) chloride hexahydrate (FeCl3.6H2O, 99%), sodium hydroxide
(NaOH, 99%), glutaraldehyde (C5H8O2, 99%), and acetic acid
(CH3COOH, 2% w/v) were purchased from Sigma-Aldrich. Graphene
Oxide was prepared using graphite powder, sodium nitrate (NaNO3,
99%), sulfuric acid (H2SO4, 98%), potassium permanganate (KMnO4,
99%), hydrochloric acid (HCl, 10%) and hydrogen peroxide (H2O2,
30%) which all were provided from Merck. Ammonia (NH4OH, 30%)
and trisodium citrate (Na3C6H5O7, 99%) were also purchased from
2
Diamond & Related Materials 127 (2022) 109149
Merck and used for the synthesis of GO/SPION.
2.2. Synthesis of graphene oxide
Graphene oxide was prepared by chemical oxidation and exfoliation
of natural graphite powder via the modified hummer's method [36].
Graphite powder (5 g) and NaNO3 (2.5 g) were mixed in 115 mL
concentrated H2SO4 solution in a beaker, kept under an ice bath with
continuous stirring. Then, KMnO4 (15 g) was added very slowly while
maintaining the temperature below 15 ◦ C for 2 h. Further, the suspen­
sion was transferred into a water bath and stirred for 3 h at 35 ◦ C until it
became pasty brownish. Then, 230 mL of deionized water was added
slowly to the above suspension and consequently, temperature rose to
98 ◦ C due to the exothermic reaction. After 30 min, the solution is finally
treated with 50 mL H2O2 to terminate the reaction by appearance of
yellow color. Further, the mixture was washed with HCL and deionized
water several times for purification followed by freeze drying.
2.3. Synthesis of SPION
FeCl3 and FeCl2 with mole ratios of 1:2 were dispersed in distilled
water and stirred for 30 min. Next, 300 mL of 0.5 M NaOH solution was
prepared and heated up to 70 ◦ C. Then, the mixture of FeCl2 and FeCl3
solution was dropped gradually during 1 h into the NaOH solution and
stirred for 4 h. The solution color changed from brown to black and iron
oxide nanoparticles precipitated at the bottom of the beaker. Finally,
nanoparticles were washed with ethanol and distilled water several
times, separated by a magnet and freeze-dried.
2.4. Synthesis of GO/SPION
0.1 g GO was ultrasonicated in 100 mL of deionized water to disperse
completely. Then, as prepared iron oxide nanoparticles were added to
the GO suspension under nitrogen atmosphere and the mixture was
heated up to 80 ◦ C and stirred for 1 h. To adjust the PH of the mixture, 5
mL of ammonia solution (30 %) was added. Finally, 1.0 g trisodium
citrate was added and the temperature was kept at 80 ◦ C for 24 h to form
black precipitate as GO/SPION. The final product was washed with
deionized water by centrifugation and dried in an oven at 60 ◦ C
overnight.
2.5. Synthesis of GO/SPION@CS
0.4 g chitosan was ultrasonicated in 200 mL acetic acid (2% w/v) for
2 h to dissolve completely. Then, 0.1 g SPION nanoparticles and 2 ml of
glutaraldehyde were added to the chitosan solution and the mixture was
stirred for 1 h to cover the surface of nanoparticles. Then, the precipi­
tated SPION@CS were washed and dried. In the next step, 0.3 g of GO
were dispersed in water, SPION@CS was added to the suspension and
the suspension was transferred to a water bath at 50 ◦ C and stirred for
90 min. To adjust the pH, 10 mL of 1 M NaOH was added. The final
product was obtained as black powder, washed with deionized water
and dried in an oven overnight.
2.6. Synthesis of GO/SPION@PEG
0.2 g of as prepared SPION nanoparticles were dispersed in 50 mL
deionized water by ultrasonication. Then 0.6 g of polyethylene glycol
(PEG) was added to the solution and stirred for 24 h. After stirring,
SPION@PEG nanoparticles were washed with deionized water and dried
at room temperature. In the next step, 0.04 g of GO was dispersed in 40
mL of deionized water. 0.04 g of SPION@PEG was added to the sus­
pension and stirred overnight at room temperature. Finally, GO/
SPION@PEG was washed with distilled water by centrifugation and
dried in an oven at 30 ◦ C.
S. Kohzadi et al.
2.7. Characterization
The size, morphology and distribution of SPION on the GO sheet
were studied using field emission scanning electron microscopy (FESEM,
TESCAN MIRA3). The hydrodynamic diameter of functionalized GO/
SPION nanocomposites was measured by dynamic light scattering (DLS)
(Nano S, Malvern, UK). The crystalline phase was evaluated by X-ray
diffraction (XRD, X'Pert PRO MDP, with Cu kα radiation λ = 0.15418
nm, 40 mA, 40 kV). Scanning was carried out within the range of 2θ
angle from 10◦ to 80◦ with a 0.02◦ step at room temperature. The
crystallite size of SPIONs was calculated from the XRD pattern using
Scherrer's equation: D = kλ/βcosθ, where k is the shape factor that can be
assigned a value of 0.89 if the shape is unknown, λ is X-ray wavelength of
Cu Kα radiation 1.54 Å. β is the full width at half maximum in radians
and θ is the Bragg angle [37].
Fourier transforms infrared spectroscopy (FTIR) (NicoletMAGNA-IR
560) was performed in the range of 400–4000 cm− 1 to identify func­
tional groups and chemical interactions between GO and bare/func­
tionalized SPION nanoparticles.
Assessment of magnetic properties of the samples was performed by
vibratory sample magnetometer (VSM) (Daghigh Meghnatis Kashan Co.,
Kashan, Iran) at room temperature, over the range of ±10 kOe.
2.8. Magnetic fluid hyperthermia
To assess the efficiency of SPION nanoparticles as well as bare and
functionalized GO/SPION for magnetic hyperthermia, the magnetic
heating was done using an AC magnetic induction system (Hyperther­
mia, nastyco, 1000 W output power, f = 400 KHz, HAC = 120 Oe). To
measure the specific absorption rate (SAR) and intrinsic loss of power
(ILP) of all magnetic colloids, 5 ml of each sample with the concentra­
tion of 3 mg/ml were placed into the center of the heating coil and the
fiber optic probe was placed in the center of the samples to record the
temperature rise at interval of 60 s. To calculate the SAR and ILP values,
the initial linear slope of ΔT/Δt curve was taken. SAR was determined
by calorimetric measurements using following equation:
ΔT Cp
SAR = (1)
Δt ϕ
where Cp is the heat capacity of liquid solvent (4.186 J/g K for water)
and φ = 3 × 103− is the mass of magnetic samples per unit mass of
deionized water.
The intrinsic loss power (ILP) is a heating transformation ability
which normalized SAR with respect to field strength and frequency and
is measured using following formula:
/
ILP = SAR ƒH2 (2)
where, H is the applied AC magnetic field and f is frequency of the coil.
2.9. In vitro studies
2.9.1. In vitro cytotoxicity assay
The cytotoxicity was assessed for samples using MTT assay [(3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) bromide].
Fibroblast cell line (L-929) was cultured in Dulbecco's Modified
Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) supplemented with
10% fetal bovine serum (FBS) and 1% antibiotic penicillin-streptomycin
solution followed by incubation (37 ◦ C, 5% CO2). The cells were tryp­
sinized and passaged up to 80–90% confluence, and third passage cells
were utilized for further investigations.
Sample sterilization was performed by exposure to UV light for 20
min. L-929 cells were seeded in 200 μl of culture medium at a density of
104 cells/well in a 96-well plate and incubated for 24 h at 37 ◦ C. Then,
culture media was aspirated and the cells were treated for another 24
and 48 h with 200 μl of media containing different concentrations of the
3
Diamond & Related Materials 127 (2022) 109149
following formulations: cells without nanoparticles (control), and five
synthesized samples including, SPION nanoparticles, GO, GO/SPION,
GO/SPION@PEG, and GO/SPION@CS at concentrations of 100, 300,
and 500 ppm. After completion of incubation time, cells were further
incubated for 3 h with 20 μl of MTT (5 mg/ml). After treatment, media
was discarded from each well, and subsequently, 200 μl of dimethyl
sulphoxide (DMSO) was added to solubilize the formazan crystals.
Readings were taken from a multi-plate reader (Thermo Fisher) at 540
nm. Percentage cell viability was calculated using following equation.
Relative cell viability (%) = (ODs /ODc ) × 100
where ODs and ODc represent the optical density of the sample and
control, respectively.
2.9.2. Flow cytometric analysis of apoptosis/necrosis
L-929 cells were incubated with GO/SPION@PEG and GO/
SPION@CS at concentration of 100 ppm for 24 h. The cells were washed
with PBS and then centrifuged at 1300 rpm for 5 min. Next, 5 μl of
Annexin V-fluorescein isothiocyanate (FITC) and 5 μl of propidium io­
dide (PI) were added to the cell suspension and they were incubated at
ambient temperature for 15 min in the dark. Finally, the samples were
analyzed using a flow cytometer instrument (BD Biosciences, USA).
2.9.3. Magnetic in vitro cytotoxicity study
To investigate the effect of applying magnetic field on fibroblast
cells, L929 cells were seeded in two cell culture plates at a density of 104
cells/well and incubated for 24 h at 37 ◦ C. Then the cells were exposed
to 100 ppm sterilized nanocomposites for 24 h. subsequently, static
magnetic field (350 mT) was placed under one of the cell culture plates
and both plates were incubated separately for 90 min (37 ◦ C, 5% CO2).
After completion of incubation time, cells were further incubated for 3 h
with 20 μl of MTT (5 mg/ml). After treatment, media was discarded from
each well, and subsequently, 200 μl of DMSO was added to solubilize the
formazan crystals. Metabolic activity was determined for magnetic field
treated and non-treated L929 cells.
To estimate the magnetic cytotoxicity effect of GO/SPION@PEG and
GO/SPION@CS on human bladder carcinoma cancer cell, EJ138 cells
were seeded in cell culture at a density of 10− 4 cells/well and incubated
for 24 h at 37 ◦ C. Then the cells were exposed to 100, 300, and 500 ppm
sterilized nanocomposites for 24 h. subsequently, static magnetic field
(350 mT) was placed under the cell culture plate and was incubated for
90 min (37 ◦ C, 5% CO2). After completion of incubation time, cells were
further incubated for 3 h with 20 μl of MTT (5 mg/ml). After treatment,
media was discarded from each well, and subsequently, 200 μl of DMSO
was added to solubilize the formazan crystals.
2.9.4. Cell morphology
L929 cells were seeded on two cell culture plates at a density of 104
cells/well and incubated for 24 h at 37 ◦ C. After incubation period, the
cells were exposed to 100 ppm sterilized samples for 24 h. Subsequently,
static magnetic field (350 mT) was placed under one of the cell culture
plates and both plates were incubated separately for 90 min (37 ◦ C, 5%
CO2). After incubation, the media was discarded from each well, and
200 μl of 2.5% glutaraldehyde was added for 20 min to fix the cells.
Then, the samples were dehydrated in graded concentrations of ethanol
(10, 20, 30, 40, 50, 60, 70, 80, 90, and 100%). Eventually, the samples
were placed in a vacuum oven for 24 h. ultimately, the specimens were
gold sputter-coated for SEM analysis.
2.10. SARS-CoV-2 surrogate neutralization antibody ELISA
Neutralizing antibodies against SARS-CoV-2 was measured using
functional surrogate neutralization antibody ELISA designed based on
SARS-CoV-2 RBD and human angiotensin-converting enzyme 2 (ACE2)
interaction in the 96-well ELISA plate (Pishtaz Teb Diagnostics; cat.
S. Kohzadi et al.
numbers: PT-CoV2NT-96). In this assay, 50 μl of nanostructures
dispersed in PBS and 50 μl horseradish peroxidase conjugated ACE2
were mixed in the appropriate wells coated with SARS-CoV-2 RBD
protein and then incubated for 30 min at 37 ◦ C. After adding chromo­
genic substrate 3,3′ ,5,5′ -Tetramethylbenzidine (TMB) into the wells, the
absorbance of the developed colors was measured. The quantity of
neutralization was calculated based on a standard curve drawn against
humanized SARS-CoV-2 neutralizing monoclonal antibody. Percentage
of inhibition was calculated using following equation.
( / ) cessful coating of PEG on the iron oxide nanoparticles.
Relative virus inhibition (%) = ODstandard − ODsample ODstandard × 100
where OD sample and OD standard represent the optical density of sample
and standard with highest concentration, respectively.
2.11. Statistical analysis
Student t-test with a significance of P values <0.05 was utilized to
analyze the data. The SPSS software was used for statistical analysis (V
25, New York, US). The obtained data were reported as mean and
standard deviation values.
3. Results and discussion
3.1. Characterization
The morphology of the pristine GO as well as synthesized nano­
structure and decoration of SPION on the GO sheet are presented in
Fig. 1a. The FESEM images of GO display exfoliated sheet like
morphology which is consistent with 2D hexagonal lattice structure of
GO. The sheets have smooth surface and wrinkled edges with approxi­
mately 45 nm thick. FESEM images of GO/SPION and GO/SPION@CS
reveal that the GO sheet is decorated by a large quantity of isolated
SPION nanoparticles and is almost uniformly distributed on the surface
of GO without agglomeration. Moreover, the sizes of decorated SPION
nanoparticles are almost uniform and spherical in shape with diameter
of 20 ± 5.1 nm and 25 ± 7.4 nm for GO/SPION and GO/SPION@CS,
respectively. The FESEM results of PEG functionalized SPION-GO (par­
ticle size ~25 ± 12.2 nm) show that the SPION@PEG is fully covered the
GO sheets.
These results suggest that the SPION nanoparticles are decorated on
GO sheets which enable a good dispersion of these nanoparticles. Some
reports suffered from agglomeration and formation of clusters of SPION
on the surface of GO sheets [38,39]. Good distribution of SPION on GO
sheets is a crucial aspect to obtain uniform properties in the
nanostructures.
Fig. 1b shows the FT-IR spectra of GO, SPION, GO/SPION, GO/
SPION@CS and GO/SPION@PEG. The FTIR spectrum of GO has a peak
at 1029 cm− 1 which is ascribed to the C–O bond, confirming the
presence of oxide functional groups after the oxidation process [40]. The
peak at 1635 cm− 1 shows that the C– – C bond still remains before and
after the oxidation process [41,42]. The peak at 1245 cm− 1 is ascribed to
C-OH stretching [43,44]. The absorbed water in GO is shown by a broad
peak at 3434 cm− 1, contributed by the O–H stretch of H2O molecules
[41,44]. In the spectrum of SPION nanoparticles, the peak at 578 cm− 1 is
characteristic of Fe–O vibrations. Moreover, the peaks at 869 cm− 1 and
the broad region absorption band around 3345 cm− 1 are both attributed
to the hydroxyl stretching vibrations of OH groups [45,46]. The FTIR of
GO/SPION reveals a peak centered at 559 cm− 1 corresponding to the
stretching vibration of the Fe–O bonds in the tetrahedral crystal lattice
of SPION [47,48]. The peaks at 1572 cm− 1, 2921 cm− 1 and 3395 cm− 1
are assigned to the skeletal vibration of graphitic chain, C–H stretching
of (CH2) and hydroxyl (OH) functional group, respectively [48]. In
addition, the peaks at 1110 cm− 1 can be attributed to ether (C-O-C)
group [45,48]. For GO/SPION@CS, the peaks at 571 cm− 1, 1647 cm− 1,
2926 cm− 1 and 3447 cm− 1 are ascribed to Fe–O stretching, N–H
4
Diamond & Related Materials 127 (2022) 109149
vibration of NH2 in chitosan [49], stretching vibration of C–H in chi­
tosan and O–H stretching of hydroxyl group [50], respectively. Addi­
tionally, the peaks at 1044 cm− 1 and 1442 cm− 1 are corresponded to the
stretching vibration of C-O-C in the glucose circle of chitosan as well as
CH3 and CH2 symmetrical bending, respectively [49,50]. For GO/
SPION@PEG, the characteristic peaks appear at 585 cm− 1, 1052 cm− 1,
1427 cm− 1, 1625 cm− 1, 2924 cm− 1, and 3421 cm− 1 corresponded to
Fe–O stretching, C–O stretching, C-O-C bending, C– – C, C–H and O–H
stretching, respectively [44,47,51,52]. This result confirms the suc­
The particle size of functionalized GO/SPION nanocomposites was
also measured using DLS. The particle size of GO/SPION@CS was found
to be ~396.5 with proper polydispersity index (PDI) (0.347), as shown
in Fig. 1c. The mean particle size of 730.9 nm was obtained for GO/
SPION@PEG, with PDI (0.214) (Fig. 1c). Since DLS method measures
the hydrodynamic diameter of the particle including the layer solvation,
the obtained values were larger than the diameters measured by FESEM
images. Moreover, The PDI values of both samples indicated that the
nanocomposites had a narrow size distribution.
Fig. 1d displays magnetization hysteresis curves of the synthesized
samples. According to the sigmoid shape of magnetization curve, all the
synthesized nanostructures have superparamagnetic character. The
saturation magnetization (Ms) of SPION, GO/SPION, GO/SPION@PEG
and GO/SPION@CS were estimated around 41, 20, 19 and 8 emu/g,
respectively. The highest saturation magnetization is attributed to
SPION nanoparticles which is in harmony with reported results in lit­
eratures [48,53]. GO/SPION hysteresis curve presents higher magnifi­
cation among nanostructures. The decrement in the magnetization of
functionalized GO/SPION compared with GO/SPION could be attrib­
uted to the presence of non-magnetic polymer species surrounding iron
oxide nanoparticles which plays an adverse effect on the performance of
magnetization expression [54].
The crystalline nature and average crystallite size of synthesized
SPION as well as GO/SPION were evaluated using XRD. As can be seen
in Fig. 1e, the absence of peak at 26◦ corresponding to graphite and
presence of the peak at 11.16◦ in the XRD pattern of GO show that the
product is oxidized after the chemical oxidation and exfoliation, indi­
cating an increase in d-spacing [52]. XRD spectrum of SPION nano­
particles indicates several diffraction peaks at 2θ = 30.56◦ , 35.71◦ ,
43.26◦ , 53.81◦ , 57.21◦ , and 62.28◦ which indexed to (220), (311), (400),
(422), (511), and (440) planes of SPION respectively, according to
JCPDS card # 019–0629 identifying a cubic spinel structure [48]. The
crystallite size of SPION was estimated to be 11.8 nm using the most
intense peak (311) in XRD pattern. No impurities were found in the
sample. Moreover, XRD pattern of GO/SPION also confirms the presence
of SPION nanoparticles on the surface of GO sheets.
3.2. Magnetic hyperthermia
In order to evaluate the magnetic-heating ability of synthesized
samples, superparamagnetic samples (SPION, GO/SPION, GO/
SPION@PEG, and GO/SPION@CS) at the concentration of 3 mg/ml in
deionized water were subjected to applied magnetic field (AMF) (400
KH, 120 Oe) for 900 s with 60 s intervals.
Fig. 2a demonstrates a time dependent gradual increase in temper­
ature of all samples under AC magnetic field generated by hyperthermia
system. It has been reported that the heating efficiency of super­
paramagnetic nanomaterial is originated from Néel and/or Brownian
relaxation which refers to the rotation of magnetic moment and physical
rotation of magnetic nanoparticles, respectively [55,56]. The compari­
son of the ΔT for different samples suggests that SPION has the highest
amount of ΔT, and as such, the amount of ΔT follows the order SPION>
GO/SPION> GO/SPION@PEG> GO/SPION@CS. No significant tem­
perature rising is observed after 660 s for GO/SPION@PEG and GO/
SPION@CS.
SAR values of superparamagnetic samples were calculated from the
S. Kohzadi et al. Diamond & Related Materials 127 (2022) 109149

Fig. 1. (a) The FESEM images of synthesized GO, GO/SPION, GO/SPION@CS, GO/SPION@PEG and the measured particle size distribution. (b) FTIR spectra of
synthesized SPION nanoparticles, GO, GO/SPION, GO/SPION@CS, and GO/SPION@PEG. (c) DLS analyses of the size distribution of GO/SPION@CS and GO/
SPION@PEG. (d) Magnetization curves of synthesized SPION, GO/SPION, GO/SPION@PEG and GO/SPION@CS. (e) XRD pattern of synthesized SPION nano­
particles, GO and GO/SPION.

5
S. Kohzadi et al.
Fig. 2. (a) AMF dependent temperature kinetics of 300 ppm concentration of samples at 400 kHz frequency and 9 KA/m AC magnetic field amplitude. (b)
comparative diagrams of SAR and ILP values using initial linear slop method of samples in suspension (c) SAR and Ms. values, and (d) ILP and Ms. values of
the samples.
ΔT versus time graph using the initial linear slope method at fixed fre­
quency of 400 kHz and with an AMF of 120 Oe (~9.55 kAm− 1). The SAR
values are found to be 594, 305, 283 and 199 W/g for SPION, GO/
SPION, GO/SPION@PEG, and GO/SPION@CS, respectively (Fig. 2b).
This result coincides with the Ms value of these compositions obtained
from VSM technique (Fig. 2c). The higher the Ms value, the heating ef­
ficiency is more pronounce. It has been reported that the following
phenomena may affect SAR value: (1) fluctuation in the flip direction of
magnetic moments of SPION during Neel relaxation due to the alteration
in barrier heights of magnetic anisotropy and/or inter-particle and inter-
aggregate dipolar interactions; (2) deflection from log-normal distribu­
tion of magnetic dipole moments and twinning defects [55]. Among
hybrid nanostructures, GO/SPION shows a higher SAR value, which
indicates its superior heating profile. Kumar et al. reported that deco­
ration of SPION on GO sheets increases the colloidal stability which in
turn, enhances the heating properties of the synthesized compositions
[21]. The decrement of SAR values of GO/SPION@PEG and GO/
SPION@CS than GO/SPION could be ascribed to the polymeric coating,
which consequently influences the inter-particle dipolar interactions
[56]. The estimated SAR values of GO/SPION and functionalized GO/
SPION nanocomposite are in accordance with the data reported by
Sugumaran et al. [57] for GO/SPION and PVP-functionalized SPION
nanoparticles.
Since many parameters such as particle shape, size, distribution,
nanoparticles concentration, frequency and magnitude of external AMF
6
Diamond & Related Materials 127 (2022) 109149
affect SAR value, the ILP is considered as an alternative value to evaluate
the heating transformation ability [21,55–60]. ILP is independent of
frequency and field strength of AMF, giving the opportunity of
comparing the heating efficiency of different nanomaterials synthesized
in other laboratories [57]. System-independent ILP values are found to
be 18.3, 9.4, 8.7, and 6.2 nHm2kg− 1 for SPION, GO/SPION, GO/
SPION@PEG, and GO/SPION@CS, respectively (Fig. 2a). Similar to the
SAR value, there is a direct relationship between the Ms and ILP value of
compositions (Fig. 2d). It is recommended that the ILP values of higher
than 3 nHm2kg− 1 are desirable for magnetic hyperthermia therapy [21].
It is worth mentioning that our samples have ILP values even higher than
the best available commercial materials [57]. Although both GO/
SPION@PEG and GO/SPION@CS have the potential to use in cancer
therapy, GO/SPION@PEG with higher SAR and ILP values is preferred.
3.3. In vitro studies
3.3.1. In vitro cytotoxicity assay
It is necessary to investigate the safety level of the synthesized
nanomaterials for in vivo application. The first step of this kind of
investigation is in vitro analyses, e.g., the mitochondrial activity.
Therefore, the cytotoxicity of the synthesized GO, SPION as well as
hybrid GO/SPION was evaluated on L929 cells using MTT assay after 24
and 72 h (Fig. 3). The biocompatibility results show the differences in
mitochondrial activity of L929 cells that depend on the type of
S. Kohzadi et al.
Fig. 3. Relative cell viability of synthesized samples against L-929 cells at different concentrations after (a) 24 h and (b) 72 h of treatment,* = p < 0.05. (c) Flow
cytometric cytotoxicity assay performed by dual parameter analysis of PI and Annexin-V dyes. L-929 cells were treated with 100 ppm of GO/SPION@PEG and
GO/SPION@CS.
nanomaterial and concentration (0,100, 300, 500 ppm).
As can be seen in Fig. 3a and b, SPION nanoparticles exhibit no
cytotoxicity in time periods of 24 and 72 h (p > 0.05). Shundo et al.
demonstrated that even higher concentration of iron oxide nanoparticles
up to 1000 μg/mL does not reduce the cell viability [58]. This can be
associated with low uptake of SPION by the cells [38]. Low toxic effect
of SPION on Cos-7 monkey kidney cells and GH3 pituitary tumor cells
was also reported [59,60]. Merely a slight decrease in cell viability was
noted at a dose of 100 and 300 ppm in GO sample after 72 h. The low
toxicity of GO can be assigned to the presence of oxygen containing
functional groups at the edges of the graphene sheet, which folds the
sharp edges [45]. The effects of GO on mouse fibroblast cells depend on
GO dose as well as the culture time [38]. No significant cytotoxicity is
detected for all three hybrid GO/SPION samples after 24 and 72 h, even
at high concentrations but all samples show decreases in viabilities in a
dose dependent manner. The cell viability of hybrid composites at 24
and 72 h incubation at 500 ppm concentration was calculated and found
to be 92 ± 0.3% and 87 ± 1.8% respectively for GO/SPION, 77.6 ± 1%
and 77 ± 0.66% respectively for GO/SPION@PEG, 85 ± 1.8% and 71 ±
7
Diamond & Related Materials 127 (2022) 109149
1.5% respectively for GO/SPION@CS. The cell viability of GO/
SPION@PEG does not change considerably even with increasing incu­
bation time from 24 to 72 h. However, the cell viability of GO/SPION
and GO/SPION@CS shows descending trend with time increment.
Khafaji et al. [61] reported more than 90% cell viability of L929 cell line
against PEGylated silica coated iron oxide nanoparticles after 24, 48,
and 72 h. Liu et al. [62] reported that PEG molecular weight could in­
fluence the cell viability. Gul et al. research revealed that less chitosan
de-acetylation increases its degradability and degradation products
which have a harmful effect on cell viability [63].
The in vitro studies are a valuable method to evaluate the interaction
of nanomaterials with the living cells and the possibility of their usage in
biological environment. L929 fibroblasts are recommended by ISO
10993-5 for cytotoxicity tests. Actually, the cytotoxicity of the nano­
materials is usually inconclusive between researchers as it is highly
dependent on various factors such as particle size, shape, chemical
composition, functional groups, synthesis techniques, cell lines, incu­
bation time and concentration [64]. Size of nanomaterial plays a key
role in interactions with the biological system and many biological-
S. Kohzadi et al.
nanomaterial related mechanisms such as cellular uptake depending on
it [65]. Nanomaterial sizes larger than 1 μm (e. g. GO) cannot easily
enter the cell, but they interact with proteins absorbed in the cells [65].
Furthermore, the optimal size for internalization inside the cells is
strongly related to the nanomaterial's surface chemistry [66,67]. In
general, Van der Waals or electrostatic forces are critical in the nano­
material interactions. Surface modification is a well-known strategy to
decrease toxicity, increase stability, and to control and modulate cellular
internalization in biomedical applications. Surface functionalization is
predominantly comprised by PEG, carboxyl group, hydroxyl group and
amine groups [67,68]. Moreover, interaction of nanomaterial with
biomolecules such as proteins upon contact with the biological envi­
ronment, known as protein corona, may also affect the nanomaterial-cell
interactions due to alteration of nanomaterial size or surface chemistry
[65–71]. Regarding the effects of GO and hybrid GO/SPION on cell
viability, the mechanism is not well explained and still requires further
analysis. Many in vivo studies of GO-based systems have been also
conducted in recent years. Rodrigues et al. demonstrated that lateral
dimensions play a fundamental role in the pulmonary response to GO
after intranasal instillation in mice [72]. Yang et al. reported that
PEGylated GO did not induce appreciable toxicity in mice after intra­
venous administration in a period of 3 months [73]. Karthika et al.
performed in vivo toxicity assay on zebrafish and the result showed the
high biocompatibility of the rGO/Fe3O4/CS without inducing significant
abnormalities [74]. Zan et al. indicated that graphene/Fe3O4 nano­
composite can be cleared from the body through the metabolic processes
of macrophages and therefore it is harmless to the living body [75].
Here, we clearly demonstrate that bare, PEG and chitosan func­
tionalized GO/SPION shows more than 70% cell viability in the time
period of 72 h even at 500 ppm concentration. Therefore, we believe
that such hybrid nanomaterials with high cytocompatibility would be
desirable for the application in biomedicine, e.g., as a drug carrier and/
or in hyperthermia.
8
Diamond & Related Materials 127 (2022) 109149
3.3.2. Flow cytometric analysis
The flow cytometry was performed to quantitatively evaluate live,
necrotic, early, and late apoptotic cells. The type of cell death (apoptotic
or necrotic) was analyzed by double staining with FITC and PI. Cells
which were PI and Annexin V negative were considered healthy, the
cells which were PI negative and Annexin V positive were considered
early apoptotic, cells which were positive for both PI and Annexin V
were considered late apoptotic and those which were PI positive and
Annexin V negative were considered necrotic. The cells with no treat­
ment were considered as the control group.
According to the flow cytometry results, a lower number of early,
late apoptotic as well as necrotic cells are found in GO/SPION@PEG and
GO/SPION@CS. This result reveals that the developed nanomaterials in
this study do not have a harmful effect on cell division. These findings
are in concurrence with the MTT assay and demonstrate that function­
alized GO/SPION nanocomposites have very low cytotoxicity to the
tested L929 cells at the concentration of 100 ppm. Kooti et al. reported
that the magnetic GO inlaid with silver nanoparticles (10 ppm) had not
led to cytotoxicity and cellular apoptosis of eukaryotic cells, and the
cells were being continuously proliferated [76].
3.3.3. In vitro magnetic cytotoxicity analysis
To determine the effect of magnetic field on the cytocompatibility of
normal cells, the samples (concentration of 100 ppm) were exposed to L-
929 cells under the applied constant magnetic field (350 mT) for 90 min.
Fig. 4a shows that the cell viability of SPION nanoparticles treated
L929 cells is almost 100%. However, a substantial decrease in cell
viability (~53.36%) is seen in the presence of a constant magnet. The
cell viability shows descending order from 97 ± 0.5% to 72 ± 3% for
GO/SPION, 90 ± 2% to 75 ± 0.6% for GO/SPION@PEG and 83 ± 2.1%
to 81 ± 0.5% for GO/SPION@CS under the applied magnetic field
(Fig. 4a). Although, the cell viability of PEG and chitosan functionalized
GO/SPION is lower than bare GO/SPION in the absence of the magnet,
Fig. 4. (a) Relative cell viability of SPION
nanoparticles as well as GO/SPION hybrid
composites against L-929 cells at the con­
centration of 100 ppm after 24 h of treat­
ment in the presence and absence of
constant magnet, * = p < 0.05. (b) Relative
cell viability of GO/SPION@PEG and GO/
SPION@CS against EJ138 cells at the con­
centrations of 100, 300 and 500 ppm after
24 h of treatment in the presence of constant
magnet, * = p < 0.05. (c) Relation between
relative cytotoxicity of GO/SPION@PEG and
GO/SPION@CS (concentration ~ 300 ppm)
and their saturation magnetization.
S. Kohzadi et al.
their magnetic lethality subsided. Because polymer coating reduces the
magnetic impact of nanoparticles on cell killing under the applied
magnetic field and therefore intercepts cell destruction [54]. Following
the recommendation of the ISO-10993-5 in which viabilities higher than
70% are not considered cytotoxic, we proposed the in vitro use of
functionalized GO/SPION in biomedical application which is necessary
to utilize the external magnetic field.
To investigate the magneto thermal effect of functionalized GO/
SPION on EJ138 human bladder cancer cells, GO/SPION@PEG and GO/
SPION@CS were exposed to a static magnetic field (350 mT) for 90 min
at the concentrations of 100, 300 and 500 ppm. Fig. 4b shows the cell
viability of cancer cells treated with functionalized nanocomposites. As
can be seen, the cell viability shows a descending trend under the
applied magnetic field in both samples. The presence of magnetic SPION
nanoparticles leads to the focus of the static magnetic field on the cells
and provides vulnerable conditions for cell viability [55]. The relation
between magnetic property and relative magnetic cytotoxicity of sam­
ples is shown in Fig. 4c. The results demonstrate that with decreasing
sample magnetization, the relative magnetic cytotoxicity percentage
shows a downward trending. As it is expected, the ability to kill cancer
cells is more pronounced for the GO/SPION@PEG than GO/SPION@CS
due to higher magnetization. Moreover, at higher concentrations (300
and 500 ppm), GO/SPION@PEG shows the ability of killing even more
than 50% of cancer cells which could be effective for cancer therapy. To
conclude, when targeted magnetic cytotoxicity is considered as a matter
of necessity, such as magnetic cancer therapy, GO/SPION@PEG could
be beneficial. Due to the relative biocompatibility of GO/SPION@CS, it
could play an operative role in biological targeting without the need for
tangible cytotoxicity such as magnetic drug delivery carriers.
3.3.4. Cell morphology
Optical microscopy has been used to evaluate changes in cell
morphology induced by magnetic compositions with and without a
constant magnet. Black and red arrows represent normal and abnormal
cells, respectively (Fig. 5). As can be seen, the cells retain their normal
shape when exposed to SPION nanoparticles and merely a few numbers
of cells have changed their morphology, exposing nanostructures
without magnetic field. Furthermore, alive cells exhibit spindle and
elongated morphology, while dead cells are more likely to exhibit round
morphology. It has been confirmed that cells usually tend to show
elongated shapes in favorable environments, which could be a sign of
biocompatibility of specific surfaces toward cell attachment [14]. By
applying an external magnet, in accordance with MTT assay results, cells
undergo deformation and change their morphology and such cellular
deformation is more prevalent for SPION and GO/SPION with higher
magnetization. It is obvious that magnetic SPION nanoparticles can
effectively deform cell appearance and consequently influence cell
Fig. 5. Optical microscopic images of L-929 cells exposed to the SPION nanoparticles as well as GO/SPION nanocomposites in the presence and absence of a constant
magnet. Black and red arrows mark the normal and abnormal cells, respectively.
9
Diamond & Related Materials 127 (2022) 109149
survival and proliferation under strong magnets. This could be attrib­
uted to DNA damage which caused by magnetic nanomaterials under the
applied magnetic field [77].
The cellular morphology and cytoskeleton structure of L-929 cells
induced by magnetic samples in the presence and absence of a magnet
were analyzed by SEM micrographs. As shown in Fig. 6, control cells
display their characteristic spindle-shaped morphology (filopodia) and
cover the glass substrate. It seems that they reflect mostly the healthy
phenotype of fibroblast cells [78]. No significant changes in morphology
occurred in the absence and presence of a magnetic field.
L-929 fibroblasts in the vicinity of SPION and GO/SPION retain their
original shape, and their membranes are not ruptured in the absence of a
magnet (Fig. 6). The cells adhere well to the substrate and flatten [79].
Round shape cells are rarely observed. Based on the MTT results, we
would not speculate that the cells undergo severe damage at these
samples. Although flatten cells can be distinguished in exposure to GO/
SPION@PEG, and GO/SPION@CS, cell attachment had been severed in
some areas and cells became more rounded due to loss of their polar
orientation [80]. The cell morphology has changed specifically for GO/
SPION@CS.
On the contrary under a magnet, the L-929 morphology in the vi­
cinity of SPION nanoparticles as well as GO/SPION and GO/SPION@­
PEG has changed. The cells lost their original, elongated shape, and the
flatten cells had no interconnectedness. Such observation is compatible
with the MTT results in the presence of a magnet. However, no note­
worthy differences in cell morphology were noticed between magnetic
and non-magnetic conditions for GO/SPION@CS, and this nano­
structure was not able to cause notable magnetic morphological
changes, which could be originated from its lower magnetization
compared with other nanostructures.
3.3.5. Antiviral analysis
The inhibitory effect of GO, SPION, GO/SPION, GO/SPION@PEG
and GO/SPION@CS on SARS-CoV 2 RBD and its receptor ACE2 inter­
action at the concentration of 100 ppm was evaluated using SARS-CoV-2
surrogate neutralization antibody ELISA method (Table 1).
According to the results, GO neutralizes 43% of RBD-ACE2 binding,
which nearly equals to inhibition observed at standard B. The strong
interaction of GO with spike, ACE2 receptor and spike-ACE2 complex
was reported by Unal et al. [23]. Maio et al. stated the complete SARS-
CoV-2 inhibition in GO functionalized face mask materials [81]. No sign
of antiviral activity is observed for SPION nanoparticles. GO/SPION
reveals merely 23% viral inhibition, which lessens than non-decorated
GO. This could be ascribed to the decoration of GO sheets with SPION
nanoparticles which hampers the connection of SARS-Cov-2 RBDs to GO
surfaces, confirming by FESEM images (Fig. 1a). GO/SPION@PEG
shows no neutralizing effect of SARS-CoV-2 virus specific anti-spike
S. Kohzadi et al. Diamond & Related Materials 127 (2022) 109149

Fig. 6. FESEM images of cell morphology of L929 cells exposed to the SPION nanoparticles as well as GO/SPION nanocomposites in the presence and absence of
constant magnet.

monoclonal antibodies. As shown in Fig. 1a, the surface of GO sheets is

Table 1
fully covered by SPION@PEG, which does not allow any access to SARS-
The inhibitory effect of GO, SPION, as well as bare and functionalized GO/
CoV-2 RBDs. The highest level of inhibition is related to GO/SPION@CS
SPION on SARS-CoV 2 virus.
with more than 86% viral inhibition.
Standard (μg/ Virus inhibition Samples Virus inhibition The SARS-CoV-2 virus, the causative agent of coronavirus-19, is a
ml) (%) (%)
single-stranded RNA with an envelope [82]. The virus contains several
A (0) 0 GO 43 structural proteins, including envelope protein (E), spike protein (S),
B (1) 46 SPION 0
nucleocapsid protein (N) and membrane protein (M) (Fig. 7a) [83,84]. S
C (2.5) 62 GO/SPION 26
D (5) 80 GO/SPION@CS 86.5 protein of coronaviruses subdivided to the N-terminal S1 subunit, which
E (10) 87 GO/ 0 forms the bulbous head of the S protein, and the C-terminal S2 subunit
F (40) 100 SPION@PEG that forms the stalk region of the protein and is directly embedded into
the viral envelope [85]. Upon interaction with a potential host cell, the
S1 and S2 subunits are responsible for receptor recognition/binding and

10
S. Kohzadi et al.
Fig. 7. (a) Schematic representation of the structural proteins of SARS-CoV-2 virus and its interaction with host cell receptor ACE2 through spike protein. Receptor
binding domain (RBD) plays a key role during this binding, (b) GO/SPION@CS and its interaction with RBD inhibits RBD-ACE2 binding.
membrane fusion, respectively [85]. S1 subunit contains a RBD that
identifies and binds to the receptor at the cellular level of ACE2, which
leads to endocytosis into lung cells and virus replication [86]. Infection
with SARS-CoV-2 virus induces acquired immune responses and as a
result, specific antibodies against viral antigens are found in patients'
circulatory system [84]. Therefore, specific antibodies against RBD can
play a protective and neutralizing role by inhibiting RBD-ACE2 binding.
Understanding the mechanism of nanomaterial-virus interaction is
somehow difficult due to the low size of viruses (2–300 nm) [85].
Several mechanisms include the blockade of viral proteins (polymerases
or enzymes) responsible for entry into human cells, or inhibition of
important viral enzymes responsible for genome replication or viral
assembly, and some target host cellular proteins that are involved in
virus activity [23,87]. Some research endeavors suggested that gra­
phene and its derivatives (i.e. GO) exhibited the ability to counter a
variety of human viral pathogens [85,88,89]. They proposed that GO
could directly interact with viruses via electrostatic interactions,
hydrogen bonding and redox reactions [90]. For instance, simulations
conducted by Raval et al. [91] revealed a strong binding efficiency be­
tween pristine multilayer exfoliated graphene and RBD of SARS-CoV-2
virus. They also found that the surface reactivity of the graphene ma­
terial was enhanced with increasing the number of carbon layers [90].
Unal et al. revealed that GO had affinity toward the ACE2 receptors,
spike protein and spike-ACE2 complex [23]. Furthermore, GO can
absorb charged lipids and destroy membranes, suggesting possibility of
interaction with enveloped viruses like SARS-CoV-2 virus [81].
The high surface area of GO provides an outstanding opportunity to
be functionalized or to be used as a substrate for loading other antiviral
agents [92]. It has been reported that chitin and chitosan can fight
against viral infection [87]. The proposed mechanisms of chitosan
antiviral activity are as follows; (i) destruction of virus structure through
electrostatic interactions between positively charged chitosan and
negatively charged capsid protein S; (ii) inactivation of viruses by
attaching to its tail fiber and dissociation of a virus; (iii) interfering with
11
Diamond & Related Materials 127 (2022) 109149
the viral replication process; (iv) binding to the viral ligands and pre­
venting their interaction with the host cell receptors [87]. Herein, GO/
SPION@CS shows superb antiviral performance due to the synergistic
effect of GO and chitosan to fight against corona viruses (Fig. 7b). The
well distribution of SPION@CS on GO surfaces makes it a promising
candidate to be used as an antiviral platform nanomaterial.
4. Conclusions
In summary, bare, PEG and chitosan functionalized SPION nano­
particles were uniformly decorated on graphene oxide sheets. GO sheets
decorated with SPION nanoparticles as well as GO/SPION@PEG and
GO/SPION@CS possessed a super-paramagnetic behavior. Due to
polymer coating, Ms values of functionalized GO/SPION were lower
than GO/SPION but not pronounced for GO/SPION@PEG. The devel­
oped GO/SPION demonstrated low toxicity on L929 cells even up to 500
ppm of concentration. High SAR value of 305 W/g and ILP value of 9.4
nHm2kg− 1 for GO/SPION@PEG make this composition a good candi­
date for cancer hyperthermia treatment. This issue was further sup­
ported by MTT assay by killing of cancer cells exposed to the GO/
SPION@PEG under the applied magnetic field. Meanwhile, GO/
SPION@CS was capable of neutralizing SARS-CoV-2 virus effectively
and could be considered as a promising material against Covid-19.
CRediT authorship contribution statement
Shaghayegh Kohzadi: Investigation, Methodology, Writing – orig­
inal draft. Najmeh Najmoddin: Supervision, Conceptualization,
Writing – review & editing. Hadi Baharifar: Supervision, Methodology,
Writing – review & editing. Mahdi Shabani: Methodology, Writing –
review & editing.
S. Kohzadi et al.
Declaration of competing interest
The authors declare no potential conflicts of interest concerning the
research, authorship, and publication of this article.
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