BASIC SCIENCE

Nanomedicine: Nanotechnology, Biology, and Medicine

31 (2021) 102319

Original Article nanomedjournal.com

Thermosensitive magnetic nanoparticles exposed to alternating magnetic

field and heat-mediated chemotherapy for an effective dual therapy in rat
glioma model
Reza Afzalipour, PhD a, b , Samideh Khoei, PhD b, c,⁎, Sepideh Khoee, PhD d ,
Sakine Shirvalilou, PhD c,⁎⁎, Nida Jamali Raoufi, PhD e , Manijeh Motevalian, PhD f, g ,
Mohammad Yahya Karimi, BS f
a
Student Research Committee, Iran University of Medical Sciences, Tehran, Iran
b
Department of Medical Physics, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
c
Finetech in Medicine Research Center, Iran University of Medical Sciences, Tehran, Iran
d
Department of Polymer Chemistry, School of Chemistry, College of Science, University of Tehran, Tehran, Iran
e
Department of Physiology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
f
Razi Drug Research Center, Iran University of Medical Sciences, Tehran, Iran
g
Department of Pharmacology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran
Revised 26 September 2020

Abstract

The goal of this study was to develop a new method based on Oncothermia with concomitant use of the temozolomide (TMZ)-loaded
magnetic nanoparticles conjugated with folic acid (TMZ/MNPs-FA) and alternative magnetic field (AMF) and evaluate its efficacy in the
treatment of C6 glioma in rats. TMZ/MNPs-FA were prepared and evaluated for their size, surface charge, magnetic saturation, hemolysis
and in vitro AMF-triggered release. The glioma rat models were treated with free TMZ, MNPs-FA and TMZ/MNPs-FA in the presence or
absence of AMF (43 °C). The results confirmed that a combinatorial therapy consisting of AFM hyperthermia and thermosensitive TMZ/
MNPs-FA could significantly suppress tumor growth, increase survival rate and promote apoptosis (P < 0.0001). Therefore, this treatment
strategy may be a powerful modality for treatment of cancer, as the thermal and mechanical effects of magnetic nanoparticles exposed to
AMF can increase the therapeutic efficacy of conventional chemotherapy.
© 2020 Elsevier Inc. All rights reserved.

Key words: Oncothermia; Temozolomide; Alternative magnetic field; Magnetic nanoparticle; Folic acid; Glioma

Today, alternating magnetic field hyperthermia (AMFH) or
simply “magnetic hyperthermia” (MH) is widely considered in
pre-clinical and clinical trials as an adjuvant therapy for
Funding statement: We acknowledge that this research was partially
funded by the School of Medicine, Iran University of Medical Sciences
(IUMS) (grant no. 29809) and partially by the Iran National Science
Foundation (INSF) (grants no. 96000733). Also, we thank the National Brain
Mapping Laboratory of Iran for providing the MR imaging.
Conflict of interest: Nothing to be declared.
⁎Correspondence to: S. Khoei, Finetech in Medicine Research Center,
Department of Medical Physics, School of Medicine, Iran University of
Medical Sciences, Tehran, Iran.
⁎⁎Correspondence to: S. Shirvalilou, Finetech in Medicine Research
Centre, Iran University of Medical Sciences.
E-mail addresses: Khoei.s@iums.ac.ir, skhoei@gmail.com, (S. Khoei),
shirvaliloo.s@tak.iums.ac.ir, sakine.shirvaliloo@gmail.com. (S. Shirvalilou).
https://doi.org/10.1016/j.nano.2020.102319
1549-9634/© 2020 Elsevier Inc. All rights reserved.
treatment of various types of solid tumors. 1 , 2 In MH, magnetic
nanoparticles (MNPs) are incorporated to act as nano-heaters,
and convert magnetic energy into a localized focus of heat under
an AMF. 3 , 4
It has been suggested that multi-modal treatments (MH and
chemotherapy, named onchothermia) are mostly beneficial for
patients with brain tumors. 5–7 Else, it has been frequently
suggested that heat can be used as a remote control for drug
release. 8 Also heat can enhance the toxicity of many chemo-
therapeutic agents by increasing the membrane permeability,
transmembrane transport and blood flow. 9 , 10
Despite the benefits of combination therapy, limitations such
as the short half-life of chemotherapy drugs, along with their
systemic toxicity and inability to cross the blood–brain barrier
have hindered many efforts in development of adjuvant
therapies. 11 Temozolomide (TMZ) has become a gold standard
for patients with glioma brain tumors. It has a half-life of

Please cite this article as: Afzalipour R, et al, Thermosensitive magnetic nanoparticles exposed to alternating magnetic field and heat-mediated
chemotherapy for an effective dual therapy in rat glioma model. Nanomedicine: NBM 2021;31:102319, https://doi.org/10.1016/j.nano.2020.102319
2 R. Afzalipour et al / Nanomedicine: Nanotechnology, Biology, and Medicine 31 (2021) 102319
approximately 1.8 h in blood plasma. 12 An astonishing way to
overcome the problems raised above is loading of TMZ onto
certain nanoplatforms that might be able to enhance the drug
stability and preserve its biological activity. 13 , 14
Application of an external magnetic field (EMF) or
conjugating the magnetic nanosystems with an active targeting
ligand reportedly can improve the accumulation of nanoparticles,
as well as their cellular uptake in GBM sites. 11 , 15 Under the
influence of EMF, the accumulation of nanoparticles can be
significantly increased based on the particle size and their EPR
effect at tumor sites. 16 Furthermore, binding of specific ligands
to the surface of MNPs, such as folic acid (FA) can increase the
overall cellular uptake through folate-receptor-mediated endo-
cytosis pathways. 17 , 18 The major advantage of a folate receptor
is that it is significantly expressed by glioma cells, as well as the
endothelial cells constituting the blood brain barriers. However,
it is normally down-regulated in intact brain tissues. 19 , 20
Considering all of the limitations in the treatment of brain
cancers, and with a strict inclination toward the use of targeted
drug delivery systems, we came up with a novel type of
combinatorial therapy based on magnetic hyperthermia and
chemotherapy. Our method employs SPIONs encapsulated in
polymer-based smart nanoparticles modified with FA-ligand
under AMF (MH) and thermal-triggered TMZ release (heat-
mediated chemotherapy).
In a previous work, TMZ-loaded magnetic-based polymer
coated nanoparticles modified with folic acid were synthetized
by a modified multiple emulsion method. 15 The characteristics
of nanoparticles such as size, zeta potential, drug loading and
encapsulation efficiency were then investigated, respectively. In
clinical trials, localized hyperthermia and controlled drug release
are critical challenges in brain cancer treatment. Intrigued by the
encouraging results of previous glioma cancer studies, in this
study, we tried to use the alternating magnetic field and thermo-
responsive nanoparticles to follow the Oncothermia method in
order to locally treat the glioma tumor in rats. To that end, Wistar
rats with C6 glioblastoma tumor were selected for an in vivo
study. The nanoparticles functionalized with folic acid ligand
were then administered to the rats afflicted with the disease,
along with EMF (NdFeB, 1.3 T). The head of rats was placed
under the coil (13.56 MHz, 40 Am −1) for magnetic hyperther-
mia. In the end, treatment efficacy of AMF, magnetic
hyperthermia alone and in combination with TMZ-loaded
thermoresponsive nanoparticles was evaluated. Then, the
expression levels of Bax and Bcl-2 proteins were investigated
by Western blotting method.
Methods
Preparation and characterization of smart nanoparticles
The TMZ/MNP-FA smart nanoparticles were synthetized in
the Department of Polymer Chemistry of Tehran University
(Tehran, Iran), using the protocol reported previously. 15 The
nanoparticles were prepared in three steps: First, superparamag-
netic iron oxide nanoparticles (SPIONs) were prepared by
chemical co-precipitation procedure. 11 Second, triblock copol-
ymers (PEG-PBA- PEG) were synthesized by using a poly-
condensation method, and functionalized with folic acid. 21
Finally, TMZ-loaded SPION/PEG-PBA-PEG-folic acid (TMZ/
MNPs-FA) nanoparticles were synthetized according to the
double emulsion solvent evaporation (DESE) method. 15 Then,
the properties of the fabricated nanoparticles such as size, zeta
potential, two- and three-dimensional shapes and superparamag-
netic properties were determined.
In vitro drug release profile triggered by AMF
The release of TMZ from MNPs in water under AMF was
evaluated through the diffusion procedure. A dialysis bag was
filled with a 1 ml of TMZ-MNPs suspensions (5 mg/ml), and
then immersed in phosphate buffer solution (PBS). Prepared
dialysis bag was placed in the glass holder and exposed to an
AMF (13.56 MHz, 100 W) for 10 min. At predetermined
intervals, 2 ml of external PBS solution was collected and
replaced with an equal volume of fresh PBS. The TMZ
concentration was determined based on the absorbance of
samples at 329 nm using the UV-spectrophotometer. During the
test, the temperature changes were measured by a copper–
constantan thermocouple. Additionally, the release 37 and 43 °C
(constant temperature) by incubator shaker was also investigated
as a control release.
Cell culture
C6 rat glioblastoma cells (ATCC CCL-107) were cultured in
Ham's-F12 supplemented with 10% fetal bovine serum (FBS),
penicillin (100 U/mL) and streptomycin (100 mg/mL) at 37 °C,
5% CO2, and 95% air humidity. C6 cell line was purchased from
the Cell Bank of the Pasteur Institute of Iran and all reagents for
cell culture were purchased from Atocel Company (Budapest,
Hungary).
In vivo allograft glioma tumor model
The rats used in this study were Male Wistar rats (180-220 g)
and all were purchased from the Experimental Studies Center of
Iran University of Medical Sciences (Tehran, Iran). All animal
experiments were performed according to the guidelines of the
Ethical Committee of Iran University of Medical Sciences in
Animal Experiments (No. IR.IUMS.REC 1395). The animals
were anesthetized, by injection of ketamine/xylazine solution
mixture; then, 10 μl cell suspension containing one million in
Ham's F-12 medium was slowly injected into the right prefrontal
region of the brain (+2 mm anterior, +2 mm lateral, and 4 mm
deep) using a stereotaxic apparatus. Rats were then transferred to
the respective cages (four rats in a cage) and monitored by MR
imaging for tumor growth (every 5 days). T2-weighted MR
imaging was performed using a 3 T scanner (MAGNETOM
Prisma, Siemens, Germany) with turbo-spin-echo protocol.
Finally, the acquired MR images were analyzed by ITK-SNAP
3.4. software for calculating glioma tumor volume. 22
Ex vivo red blood cell hemolysis assay
The biocompatibility of nanoparticles and their toxicity
effects on rat erythrocytes (RBCs) were investigated by a
hemolysis assay. Rat RBCs were extracted from blood samples
and diluted with PBS so that 4 U of RBCs was added to 1 U of
 R. Afzalipour et al / Nanomedicine: Nanotechnology, Biology, and Medicine 31 (2021) 102319
Table 1
Different types of treatment groups in the present study.
Groups Treatment modalities
1 Sham (normal saline)
2 Free TMZ (3 mg/kg)
3 MNPs-FA (44 mg/kg)
4 TMZ/MNPs-FA (44 mg/kg)
5 MH (13.56 MHz, 40 A/m, 50 W, 20 min)
6 Free TMZ (3 mg/kg) plus MH (13.56 MHz, 40 A/m, 20 min)
7 MNPs-FA (44 mg/kg) plus MH (13.56 MHz, 40 A/m, 8 min)
8 TMZ/MNPs-FA (44 mg/kg) plus MH (13.56 MHz, 40 A/m, 8 min)
PBS. Then, TMZ/MNPs-FA suspension at different concentra-
tions (10 to 1000 μg/mL) was added to RBCs prepared samples,
and PBS and deionized water (without NPs) were used as a
negative and positive control, respectively. All samples were
incubated under shaking (37 °C, 2 h). After, the supernatant of a
sample was collected and transferred to a 96-well plate and the
quantitation of hemoglobin was carried out by a UV–visible
spectrophotometer (BioTek, Winooski, USA) at 577 nm.
Finally, the hemolysis percentage was calculated using the
following equation.
Hemolysis (%) = [(sample 577 nm- negative control 577
nm)/(positive control 577 nm-negative control 577 nm)] × 100.
In vivo Onchothermia experiment
Ten days following the tumor cell injection, glioma-bearing
rats were monitored by MR imaging and rats with the mean
tumor volume of 50-100 mm 3 were randomly divided into seven
treatment groups (n = 10), as shown in Table 1.
One day later, the animals were injected intravenously into
the tail vein with normal saline, free TMZ, MNPs-FA and TMZ/
MNPs under magnetic field condition. After 2 h magnetic
targeting, rats were anesthetized using a ketamine/xylazine
injection. Then, for localized hyperthermia, head of rats was
placed in an alternating magnetic coil (13.56 MHz, 40 A/m) for
a specified period (8 or 20 min). The current passed through a
custom-made four-turn water-cooled copper coil of 40 mm in
internal diameter. The frequency and field range of coil used in
this experiments were chosen to fit the ethically accepted AMF
limits (Hf = 5.42× 10 8 Am −1 s −1 < 5× 10 9 Am −1 s −1). 4 , 23
Figure 4 shows the geometry of the coil system and AMF
radiation pattern prepared in COMSOL. We used software
COMSOL Multiphysics 4.3b software for modeling coils
system. 24 The exposed area to the alternating magnetic field
during the hyperthermia time was monitored by taking
photographic images using the Testo 875 thermal imager
(Testo 875-1i, Germany) with high infrared resolution and
thermocouple (Figure 5, B-C). During hyperthermia, the skin
and tumor temperatures of rats were controlled using an IR
camera and a copper-constantan thermocouple, respectively. 25
For this purpose, we removed a piece of skull (1 × 1 cm 2) and
placed the thermocouple probe inside the tumor and another
probe inside the normal tissue, and at the same time used the IR
camera. In this case, the results of the thermocouple and the
camera did not differ much (0.2-0.6 °C), and this may be due to
the low depth of the tumor (Figure 5, A-E). The maximum
3
temperature in the tumor area was extracted from each infrared
image. Also, the temperature of the tumor area and surrounding
tissue was obtained by the thermocouple probe. Due to the low
depth of the tumor, the temperature measured with a thermo-
couple probe in the tumor area (in the region of nanoparticle
accumulated) was the same as that of the maximum temperature
obtained from the infrared images. Five rats were sacrificed from
each group and their brains were collected for Western blotting
assay and the other 5 rats were maintained for monitoring their
life-span, body weight change and evaluating tumor volume with
MR imaging.
Western blot analysis
Two days after treatment, the expression of Bax and Bcl-2
proteins in rat glioma tumor was assessed by western blotting
assay. Total protein from homogenized glioma tissues was
extracted with RIPA lysis buffer (Santa Cruz Biotechnology,
Dallas, USA) and quantified by the Bradford protein assay. 26
The primary antibodies (rabbit anti-Bcl-2 polyclonal antibody (1/
1000), rabbit anti-Bax polyclonal antibody (1/2000)) and HRP
conjugated-secondary antibody (1/2000) were used for Western
Blotting. All the protein bands were visualized using ECL
Western Blotting Detection Reagent (ChemiDocXRS; Bio-Rad,
South San Francisco, USA) and quantified by densitometry
using ImageJ software and normalized to that of β-actin protein.
All antibodies were purchased from Abcam Company (Cam-
bridge, MA, USA).
Statistics analysis
All data were presented as mean ± standard deviation (SD).
A Student's t test and ANOVA followed by post hoc
comparisons were performed to determine the significant
difference between two and multiple groups, respectively,
using SPSS statistical software (version 22) and GraphPad
Prism (version 6). A value of P < 0.05 was defined to be
statistically significant.
Results
Characterization of the TMZ-loaded magnetic nanoparticles
conjugated with folic acid
A summary of the properties of the synthesized TMZ-loaded
MNPs-FA nanoparticles is presented in Figure 2. The TEM
image shows 40 nm spherical nanoparticles that appear to be
well dispersed (Figure 2, A). The results obtained from the DLS
and Zetasizer also indicate a hydrodynamic size of 58.61 ±
1.12 nm and a Zeta potential of −29.85 ± 0.47 mV, respectively
(Figure 2, B). Figure 1, C presents the saturation magnetization
(Ms) values of SPIONs, MNPs-FA and TMZ-loaded MNPs-FA,
which were 59.21, 38.75 and 36.54 emu/g, respectively. The
slight fall in the Ms value of TMZ/MNP-FA nanoparticles
compared to that of blank SPIONs or MNP-FA nanoparticles can
be attributed to the non-magnetic TMZ and folic acid molecules
that led to a fall in the magnetic field effect. In addition, all
nanoparticles exhibited superparamagnetic properties without
magnetic hysteresis at 25 °C (Figure 1, C).
4 R. Afzalipour et al / Nanomedicine: Nanotechnology, Biology, and Medicine 31 (2021) 102319

Figure 1. Characterization of nanoparticles. (A) Representative TEM micrograph of TMZ/MNPs-FA nanoparticles synthetized by multiple emulsion method.
(B) Hydrodynamic size distribution of TMZ/MNPs-FA nanoparticles. (C) Magnetization curve of SPIONs, MNPs-FA and TMZ/MNPs-FA samples.

Figure 2. (A) Cumulative release profiles of TMZ from TMZ/MNP-FA vs time at 37 and 43 °C using a water bath up to 34 °C due to heat-triggered release using
an AMF for 8 min (after 8 min AMF radiation was switched off). The percentage of TMZ released reached 50% after 8 min under AMF and 30% and 2% at 43
and 37 °C, respectively. (B) Heat profile of TMZ/MNP-FA nanoparticles measured with an infrared thermal camera for 12 min treatment under AMF. The AMF
was applied at a power of 100 W and frequency of 13.56 MHz.

In vitro TMZ controlled release
As reported in a previous study, MNPs-FA nanoparticles
reduced the release rate of TMZ and increased the release time
(in vitro and in vivo) by up to 15-fold. Here, the temperature-
responsive property and remote-control effect of the nanoparti-
cles are put under the spotlight. To evaluate this hypothesis, we
investigated the cumulative release profiles of TMZ at
temperatures of 37 and 43 °C (using a heater stirrer), as well
as the accelerated TMZ release by the effect of magnetic
hyperthermia under AMF. The results clearly indicated that by
raising the temperature, drug release was increased. As shown in
Figure 2, A, a rapid release of TMZ occurred within the first
8 min of AMF exposure; however, as the AMF was switched off,
the release rate was significantly decreased. The TMZ release of
MNPs-FA nanoparticles after 8 min with AMF exposure was
52.1 ± 2.3%, while the release at the same time without AMF
was 30% and less than 2% at 43 and 37 °C, respectively. The
 R. Afzalipour et al / Nanomedicine: Nanotechnology, Biology, and Medicine 31 (2021) 102319
Figure 3. In vitro hemolysis results of the different concentrations of MNPs-FA with/without loading TMZ after incubation (2 h) with diluted red blood cells of
rats. (Data were presented as the mean ± SD, n = 3, statistical significance was presented with (*) P < 0.05, (**) P < 0.01 and (***) for P < 0.001,
respectively.)
temperature variations in the different AMF exposure times are
shown in Figure 2, B. Figure 2, B clearly shows that, if smart
thermosensitive MNP nanoparticles exposed with AMF, they
would be able to significantly increase the heat.
Hemolytic toxicity
To investigate whether our synthetized MNP-FA with/
without TMZ could induce hemolysis, we incubated rat RBC
samples with different concentrations of nanoparticles for 2 h.
The results showed that by increasing the concentration of
nanoparticles (10 to 4000 μg/ml), the hemolysis rate in rat blood
samples was elevated (Figure 3). At concentrations above 250,
the increase in hemolysis was more significant for TMZ/MNP-
FA compared to that of MNP-FA nanoparticles (P < 0.05).
According to ASTM E2524-08, 25 a hemolysis rate above 5% can
damage erythrocytes. Accordingly, both nanoparticles (MNPs-
FA and TMZ/MNPs-FA) appear to be homo-compatible at
concentrations below 3000 μg/ml.
In vivo temperature variations of magnetic hyperthermia
(MNPs-FA + AMF)
After successful growth of glioma tumors (50-100 mm 3), the
rats were divided into two groups. One group received
nanoparticles (44 mg/kg) and was kept under a magnetic field
for 2 h. The other treatment group received saline control.
Finally, both groups were exposed to AMF (20 min,
13.56 MHz, 50 W) and body temperature was measured during
hyperthermia. We used the alternating magnetic coils
(13.56 MHz, 40 A/m) system for generating homogeneous
magnetic field. As shown in Figure 4, A, the density of the
magnetic field is uniform inside the coil. Figure 5, D shows the
temperature variations in different treatment groups. As can be
seen in Figure 4, A, the application of in the presence of magnetic
nanoparticles caused a significant time-dependent increase in
temperature, especially in the tumor area at the shortest possible
5
time. However, when nanoparticles were absent, the temperature
rise was not localized and occurred over a longer period of time.
Infrared images of glioma rats treated with nanoparticles in the
presence of AMF showed that during AMF hyperthermia, the
white area indicating the high temperature signal was greater at
the tumor site than the adjacent tissue (Figure 5, E). According to
the thermometric results for hyperthermia at 43 °C, the rats were
placed in the magnetic coil, 20 min for AMF only and 8 min for
AMF with magnetic nanoparticles.
In vivo Onchothermia efficacy
The antitumor effects of the different treatment modalities (TMZ,
MNPs-FA, and TMZ/MNPs-FA under AMF exposure
(13.56 MHz, 40 A/m)) were evaluated in rat glioma by monitoring
tumor volume and survival time (Figure 6). Tumor volume in the
saline group (control) exhibited a 300% and 500% increase in 17 and
24 days after the initial cell injection, respectively. Tumor volume
changes and survival curves (Figure 6, C & D) show that only
MNPs-FA and TMZ injections or AFM alone could not lead to
tumor suppression. After treatment with TMZ or AFM alone,
although the tumor size increased slowly relative to the saline and
MNPs-FA group, but tumor volume increased again after 24 days of
cell injection. TMZ combined with AMF increased survival to
67 days (P < 0.05), and remarkably decreased the tumor size by
about 3-fold compared to that of the saline group on day 24. TMZ-
loaded nanoparticles had similar results and increased survival to
71 days and decreased tumor size 3.6-fold compared to controls on
day 24 (P < 0.01, Figure 5, C & D). MNPs-FA and TMZ-loaded
MNPs-FA nanoparticles in combination with AMF exhibited the
best response; they could decrease tumor volume 7-10 folds on day
24 and increase the survival of rats from 97 to 106 days (P < 0.001,
Figure 6, C & D). However, there was no significant difference
between these groups (P > 0.05), which indicates the major role for
MH therapy (MNPs-FA+ AFM), leading to a significant enhance in
treatment efficiency (P < 0.001). MR images also showed that the
6 R. Afzalipour et al / Nanomedicine: Nanotechnology, Biology, and Medicine 31 (2021) 102319

Figure 4. Generation of uniform magnetic field by coil system. (A) Model of the coil and the distribution of the magnetic field intensities (yellow arrows indicate
the direction of the magnetic flux). (B) The mechanism of heat production by nanoparticles under alternating magnetic field (AMF) due to Neel relaxation time.

Figure 5. MNP-FA nanoparticles as an effective in vivo heat mediator under AMF. (A) Yellow arrow indicates a piece removed from a rat skull (1 × 1 cm 2). (B
and C) Experimental setup and animal positioning inside the coil and thermometry with thermocouple and IR camera. (D) The profile of temperature variations
the C6 tumor-bearing rat treated with intravenous injection of 44 mg/kg MNP-FA in the presence or absence of AMF (13.56 MHz, 40 Am −1, 20 min) as a
function of time. (E) Infrared images of the glioma rats during 20 min of Onchothermia. Data are presented as mean ± SD (n = 6, (*) P < 0.05, (**) P < 0.01
and (***) for P < 0.001)).

combination treatment group of AMF and TMZ/MNPs-FA
successfully suppressed the tumor growth for approximately
80 days, after which the tumors recurred again (Figure 6, B).
In addition, a signaling pathway involving the activation of
cancer cells is related to a remarkable increase in the expression
level of Bax protein, and repression of Bcl-2. As can be inferred
from Figure 7, A & B, rats treated with FA conjugated MNPs
showed almost no Bax or Bcl-2 protein expression, as shown in
Figure 7, C & D. The value of Bax/Bcl-2 ratio was ≤1, meaning
that the tumor cells generally did not undergo apoptosis. TMZ
and AMF (43 °C) alone exhibited a significant increase in Bax
expression (P < 0.05), while the Bcl-2 protein remained
approximately constant relative to the control and MNPs-FA
group (P > 0.05, 1.3 < ratio ≤ 1.4). Treating glioma bearing
rats with the TMZ-loaded MNPs-FA nanoparticles and TMZ
(43 °C) revealed a significantly higher Bax protein production
than that of TMZ or AMF alone (P < 0.05), whereas the Bcl-2
protein level showed a slight decrease (P > 0.05, 1.5 < ratio ≤
 R. Afzalipour et al / Nanomedicine: Nanotechnology, Biology, and Medicine 31 (2021) 102319

Figure 6. The anti-tumor effects of various treatments against glioma tumor-bearing rat after intravenous injection of 3 mg/kg TMZ or TMZ-loaded MNP-FA in the presence or absence of AMF (13.56 MHz, 40 Am−1, 43 °C) on
the 10th day of C6 cell injection. (A) Axial section of T2-weighted MR images. (B) Glioma tumor volume in different treatment groups up to the 21st day. (C) Kaplan–Meier survival curve. Data are presented as mean ± SD (n =
5 rat per group, (*) P < 0.05, (**) P < 0.01 and (***) for P < 0.001)).
7
8 R. Afzalipour et al / Nanomedicine: Nanotechnology, Biology, and Medicine 31 (2021) 102319

Figure 7. The effect of TMZ, MNP-FA, and TMZ-MNP-FA with or without AMF exposure (13.56 MHz, 40 Am −1, 43 °C) on the (A) relative protein expression
of Bax and Bcl-2, and (B) Bax/Bcl-2 protein ratio detected by Western blot in C6 glioma tumor tissue. Data are presented as mean ± SD (n = 3, (*) P < 0.05,
(**) P < 0.01, (***) P < 0.001), (****) P < 0.0001) and ( Δ) for P < 0.05 significant respect to the controls of Bax and Bcl-2 proteins, respectively).

1.7). Ultimately, the present study detected a stronger increase
in Bax and decrease in Bcl-2 expression, after combinatorial
therapy of AMF plus MNPs-FA with/without TMZ (P < 0.01
and P < 0.05, ratio > 3, Figure 7) was applied.
Discussion
Oncothermia is a new type of combinational therapy that
couples hyperthermia and chemotherapy together to treat deeply
located tumors such as brain malignancies, where localized heat
deposition and controlled drug release still remain the most
formidable challenges in treatment of brain tumors. 1 , 27 The aim
of this study was to introduce an elegant thermos-sensitizing
nano-platform specialized for carrying TMZ that can be
incorporated by Oncothermia for treatment of gliomas. In this
method, an alternating magnetic field (13.56 MHz, 40 Am −1) is
used as the external source of energy for magnetic hyperthermia.
As shown in Figure 1, A & B, the synthesized TMZ-loaded
superparamagnetic FA-conjugated (TMZ/MNPs-FA) nanoparti-
cles had a size below 50 nm and a negative charge of 30 mV.
Hysteresis loops recorded at room temperature for different
nanoparticles show the superparamagnetic properties of all
nanoparticles (Figure 1, C). Therefore, TMZ/MNPs-FA nano-
particles were only capable of producing heat through the Neel
relaxation mechanism (due to the flipping motion of the SPIONs
magnetic moment 28) when exposed to the AMF, because of zero
magnetic hysteresis (Figure 4, B). The absorption of radiofre-
quency energy by biological tissue results in higher tissue
temperatures as a function of time, which eventually causes cell
death due to hyperthermia. 29 In fact, an external AMF provides
energy, and helps rotate the magnetic moments in overcoming
the energy barrier (E = KV), where K is the anisotropy constant
and V is the volume of the magnetic core such as SPIONs. This
energy is wasted when the magnetic moments of the particles
relaxes toward its equilibrium orientation (Neel relaxation). 30 As
a result of this waste energy causes hyperthermia (Figure 4, B).
One of the amazing benefits of thermos-sensitizing nanopar-
ticles is that their release temperature can be easily set up by a
remote control. The drug release profile from the conjugated
MNPs in the presence of an AMF and at physiological
temperature is presented in Figure 2. The results show that the
TMZ/MNPs-FA nanoparticles were stable at 37 °C (body
temperature), but when subjected to AMF, the resultant heat
increased the release rate of TMZ by affecting the polymer
coating. As the AMF was cut off and the heat dissipated, drug
release decreased, indicating the thermal sensitivity of the
nanoparticles that allowed the nanoparticles to be released in a
controlled manner. Minaei et al measured the TMZ release rate
from FA-conjugated magnetic nanoparticles. They showed drug
release rates of 48.3 ± 1.7% and 74.43 ± 0.77% within 4 h and
48 h, respectively. 31 Mao et al showed that Tf-PLGA-TMZ
released about 60% of loading TMZ in less than 2 h. 32 Similar
results have been reported for TMZ drug release by Yu et al 33
and Zeng et al. 14 So comparing our results with past studies
shows that magnetic hyperthermia offers great potential as it can
be remotely controlled drug release.
To confirm the role of AMF in the rise of temperature to
43 °C in glioma models, after the intravenous injection of the
conjugated MNPs (Fe = 9 mg/kg) and application of an external
magnetic field (2 h), the glioma rats were placed inside the
magnetic coil (13.56 MHz, 20 min) and their body temperature
measured. Since an adequate concentration of magnetic
nanoparticles is required to be transported to the tumor
microenvironment, it has been suggested to use folic acid ligand
and external magnetic fields to accomplish this goal. However,
this cannot be easily accomplished. Ohtake et al reported that
since they failed to inject sufficient concentrations of Fe(Salen)
 R. Afzalipour et al / Nanomedicine: Nanotechnology, Biology, and Medicine 31 (2021) 102319
nanoparticles into the mice brain, they used a subcutaneous
tumor model in the leg. 34 Figure 5 demonstrates that the MNPs-
FA nanoparticles can properly target the tumor cells, and
produce heat under the AMF especially in the tumor site. Similar
results were reported by Liu et al, 35 Balivada et al and Basel et
al 36 , 37 with the exception that they injected magnetic nanopar-
ticles directly into the tumor area. In another study, Shirvalilou et
al reported that they used 20 mg of SPIO nanoparticles for
glioma drug delivery. They only used an external magnetic field
to target the drug. This suggests that the use of two drug delivery
modalities (MF and acid folic ligand) can effectively reduce
injectable drug doses, which can reduce systemic toxicity. 38 To
our surprise, the results of the hemolysis experiments presented
in Figure 3 suggest that injection of this amount of nanoparticles
(44 mg/kg) in rats did not cause any blood toxicity. The
hemolysis results of our synthetized nanoparticles for TMZ
delivery showed less toxicity than previously reported studies
such as Jain et al (Tf-conjugated SLNs) 39 and Sharma et al
(PAMAM-chitosan). 40
In the present study, the effect of different therapeutic methods on
glioma rat models was evaluated by monitoring of tumor volume,
survival and apoptosis rate by Western blotting. Our results indicated
that combinational therapy comprising TMZ/MNPs-FA nanoparti-
cles and AMF hyperthermia (43 °C) exhibited the strongest
antitumor effect in a treatment of glioma, compared to other
methods. This procedure successfully inhibited the proliferation rate
of tumor, and increased the survival rate and Bax/Bcl-2 protein ratio
(P < 0.001, Figures 6 & 7), which were in accordance with the
results of Ohtake et al 34 and Chen et al. 41 Although they had
performed their experiments on subcutaneous tumors, this is still a
major step forward over their studies. Comparison of the survival
rate of TMZ-treated gliomas rats in our study (TMZ/MNPs-FA,
75 days) with similar studies such as Mao et al (Tf-PLGA-TMZ,
34 days) 32 and Towner et al (OKN + TMZ, 60 days) 42 showed that
these nanoparticles might be suitable folic acid-modified magnetic
nanoplatforms for TMZ targeted drug-delivery to solid tumors under
external magnetic field. On the other hand, comparing tumor volume
changes of glioma rats treated with Onchothermia in our study (TMZ
and magnetic hyperthermia) with similar studies such as Babincová
et al (TMZ and photothermal therapy) 27 and Zeng et al (DOX and
magnetic hyperthermia) 14 showed that these nanoparticles were
remarkably capable of inhibiting tumor growth. This enhanced
antitumor effect can be attributed to several parameters related to the
concomitant effects of chemotherapy and hyperthermia. Our results
did indicate prominent antitumor effects for single treatment
modality, TMZ or AMF hyperthermia, which may be due to the
low dose of TMZ and thermal dose (Figures 6 & 7). In contrast,
when AMF hyperthermia was combined with TMZ, or the TMZ was
loaded onto the conjugated nanoparticles, the antitumor effects were
significantly increased (P < 0.01). Previous studies have shown that
an increase in tumor temperature may lead to an increase in tissue
perfusion, and thereby improve the distribution of chemotherapy
drugs 5,43 and decrease drug resistance through sensitizing the tumor
cells to the drug. 41
Therefore, in the present study, for the first time, we have
combined the concepts from active drug targeting, controlled
release, and localized magnetic hyperthermia for glioma cancer
therapy. The potential therapeutic efficacy of the newly
9
developed thermo-responsive TMZ/MNPs-FA (44 mg/kg) in
combination with an AMF hyperthermia (13.56 MHz, 40 Am −1)
was confirmed by a considerable tumor growth suppression and
significant induce apoptosis. Accordingly, this combinatorial
therapy could serve as a promising Oncothermia procedure for
the treatment of glioma.
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