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

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 condensation method, and functionalized with folic acid. 21
overcome the problems raised above is loading of TMZ onto Finally, TMZ-loaded SPION/PEG-PBA-PEG-folic acid (TMZ/
certain nanoplatforms that might be able to enhance the drug MNPs-FA) nanoparticles were synthetized according to the
stability and preserve its biological activity. 13 , 14 double emulsion solvent evaporation (DESE) method. 15 Then,
Application of an external magnetic field (EMF) or the properties of the fabricated nanoparticles such as size, zeta
conjugating the magnetic nanosystems with an active targeting potential, two- and three-dimensional shapes and superparamag-
ligand reportedly can improve the accumulation of nanoparticles, netic properties were determined.
as well as their cellular uptake in GBM sites. 11 , 15 Under the
In vitro drug release profile triggered by AMF
influence of EMF, the accumulation of nanoparticles can be
significantly increased based on the particle size and their EPR The release of TMZ from MNPs in water under AMF was
effect at tumor sites. 16 Furthermore, binding of specific ligands evaluated through the diffusion procedure. A dialysis bag was
to the surface of MNPs, such as folic acid (FA) can increase the filled with a 1 ml of TMZ-MNPs suspensions (5 mg/ml), and
overall cellular uptake through folate-receptor-mediated endo- then immersed in phosphate buffer solution (PBS). Prepared
cytosis pathways. 17 , 18 The major advantage of a folate receptor dialysis bag was placed in the glass holder and exposed to an
is that it is significantly expressed by glioma cells, as well as the AMF (13.56 MHz, 100 W) for 10 min. At predetermined
endothelial cells constituting the blood brain barriers. However, intervals, 2 ml of external PBS solution was collected and
it is normally down-regulated in intact brain tissues. 19 , 20 replaced with an equal volume of fresh PBS. The TMZ
Considering all of the limitations in the treatment of brain concentration was determined based on the absorbance of
cancers, and with a strict inclination toward the use of targeted samples at 329 nm using the UV-spectrophotometer. During the
drug delivery systems, we came up with a novel type of test, the temperature changes were measured by a copper–
combinatorial therapy based on magnetic hyperthermia and constantan thermocouple. Additionally, the release 37 and 43 °C
chemotherapy. Our method employs SPIONs encapsulated in (constant temperature) by incubator shaker was also investigated
polymer-based smart nanoparticles modified with FA-ligand as a control release.
under AMF (MH) and thermal-triggered TMZ release (heat-
mediated chemotherapy). Cell culture
In a previous work, TMZ-loaded magnetic-based polymer
C6 rat glioblastoma cells (ATCC CCL-107) were cultured in
coated nanoparticles modified with folic acid were synthetized
Ham's-F12 supplemented with 10% fetal bovine serum (FBS),
by a modified multiple emulsion method. 15 The characteristics
penicillin (100 U/mL) and streptomycin (100 mg/mL) at 37 °C,
of nanoparticles such as size, zeta potential, drug loading and
5% CO2, and 95% air humidity. C6 cell line was purchased from
encapsulation efficiency were then investigated, respectively. In
the Cell Bank of the Pasteur Institute of Iran and all reagents for
clinical trials, localized hyperthermia and controlled drug release
cell culture were purchased from Atocel Company (Budapest,
are critical challenges in brain cancer treatment. Intrigued by the
Hungary).
encouraging results of previous glioma cancer studies, in this
study, we tried to use the alternating magnetic field and thermo- In vivo allograft glioma tumor model
responsive nanoparticles to follow the Oncothermia method in
order to locally treat the glioma tumor in rats. To that end, Wistar The rats used in this study were Male Wistar rats (180-220 g)
rats with C6 glioblastoma tumor were selected for an in vivo and all were purchased from the Experimental Studies Center of
study. The nanoparticles functionalized with folic acid ligand Iran University of Medical Sciences (Tehran, Iran). All animal
were then administered to the rats afflicted with the disease, experiments were performed according to the guidelines of the
along with EMF (NdFeB, 1.3 T). The head of rats was placed Ethical Committee of Iran University of Medical Sciences in
under the coil (13.56 MHz, 40 Am −1) for magnetic hyperther- Animal Experiments (No. IR.IUMS.REC 1395). The animals
mia. In the end, treatment efficacy of AMF, magnetic were anesthetized, by injection of ketamine/xylazine solution
hyperthermia alone and in combination with TMZ-loaded mixture; then, 10 μl cell suspension containing one million in
thermoresponsive nanoparticles was evaluated. Then, the Ham's F-12 medium was slowly injected into the right prefrontal
expression levels of Bax and Bcl-2 proteins were investigated region of the brain (+2 mm anterior, +2 mm lateral, and 4 mm
by Western blotting method. 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
Methods imaging was performed using a 3 T scanner (MAGNETOM
Prisma, Siemens, Germany) with turbo-spin-echo protocol.
Preparation and characterization of smart nanoparticles Finally, the acquired MR images were analyzed by ITK-SNAP
The TMZ/MNP-FA smart nanoparticles were synthetized in 3.4. software for calculating glioma tumor volume. 22
the Department of Polymer Chemistry of Tehran University Ex vivo red blood cell hemolysis assay
(Tehran, Iran), using the protocol reported previously. 15 The
nanoparticles were prepared in three steps: First, superparamag- The biocompatibility of nanoparticles and their toxicity
netic iron oxide nanoparticles (SPIONs) were prepared by effects on rat erythrocytes (RBCs) were investigated by a
chemical co-precipitation procedure. 11 Second, triblock copol- hemolysis assay. Rat RBCs were extracted from blood samples
ymers (PEG-PBA- PEG) were synthesized by using a poly- 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 3

Table 1 temperature in the tumor area was extracted from each infrared
Different types of treatment groups in the present study. image. Also, the temperature of the tumor area and surrounding
Groups Treatment modalities tissue was obtained by the thermocouple probe. Due to the low
1 Sham (normal saline) depth of the tumor, the temperature measured with a thermo-
2 Free TMZ (3 mg/kg) couple probe in the tumor area (in the region of nanoparticle
3 MNPs-FA (44 mg/kg) accumulated) was the same as that of the maximum temperature
4 TMZ/MNPs-FA (44 mg/kg) obtained from the infrared images. Five rats were sacrificed from
5 MH (13.56 MHz, 40 A/m, 50 W, 20 min) each group and their brains were collected for Western blotting
6 Free TMZ (3 mg/kg) plus MH (13.56 MHz, 40 A/m, 20 min) assay and the other 5 rats were maintained for monitoring their
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)
life-span, body weight change and evaluating tumor volume with
MR imaging.

Western blot analysis

PBS. Then, TMZ/MNPs-FA suspension at different concentra-
tions (10 to 1000 μg/mL) was added to RBCs prepared samples, Two days after treatment, the expression of Bax and Bcl-2
and PBS and deionized water (without NPs) were used as a proteins in rat glioma tumor was assessed by western blotting
negative and positive control, respectively. All samples were assay. Total protein from homogenized glioma tissues was
incubated under shaking (37 °C, 2 h). After, the supernatant of a extracted with RIPA lysis buffer (Santa Cruz Biotechnology,
sample was collected and transferred to a 96-well plate and the Dallas, USA) and quantified by the Bradford protein assay. 26
quantitation of hemoglobin was carried out by a UV–visible The primary antibodies (rabbit anti-Bcl-2 polyclonal antibody (1/
spectrophotometer (BioTek, Winooski, USA) at 577 nm. 1000), rabbit anti-Bax polyclonal antibody (1/2000)) and HRP
Finally, the hemolysis percentage was calculated using the conjugated-secondary antibody (1/2000) were used for Western
following equation. Blotting. All the protein bands were visualized using ECL
Hemolysis (%) = [(sample 577 nm- negative control 577 Western Blotting Detection Reagent (ChemiDocXRS; Bio-Rad,
nm)/(positive control 577 nm-negative control 577 nm)] × 100. South San Francisco, USA) and quantified by densitometry
using ImageJ software and normalized to that of β-actin protein.
In vivo Onchothermia experiment All antibodies were purchased from Abcam Company (Cam-
bridge, MA, USA).
Ten days following the tumor cell injection, glioma-bearing
rats were monitored by MR imaging and rats with the mean Statistics analysis
tumor volume of 50-100 mm 3 were randomly divided into seven
treatment groups (n = 10), as shown in Table 1. All data were presented as mean ± standard deviation (SD).
One day later, the animals were injected intravenously into A Student's t test and ANOVA followed by post hoc
the tail vein with normal saline, free TMZ, MNPs-FA and TMZ/ comparisons were performed to determine the significant
MNPs under magnetic field condition. After 2 h magnetic difference between two and multiple groups, respectively,
targeting, rats were anesthetized using a ketamine/xylazine using SPSS statistical software (version 22) and GraphPad
injection. Then, for localized hyperthermia, head of rats was Prism (version 6). A value of P < 0.05 was defined to be
placed in an alternating magnetic coil (13.56 MHz, 40 A/m) for statistically significant.
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 Results
this experiments were chosen to fit the ethically accepted AMF Characterization of the TMZ-loaded magnetic nanoparticles
limits (Hf = 5.42× 10 8 Am −1 s −1 < 5× 10 9 Am −1 s −1). 4 , 23 conjugated with folic acid
Figure 4 shows the geometry of the coil system and AMF
radiation pattern prepared in COMSOL. We used software A summary of the properties of the synthesized TMZ-loaded
COMSOL Multiphysics 4.3b software for modeling coils MNPs-FA nanoparticles is presented in Figure 2. The TEM
system. 24 The exposed area to the alternating magnetic field image shows 40 nm spherical nanoparticles that appear to be
during the hyperthermia time was monitored by taking well dispersed (Figure 2, A). The results obtained from the DLS
photographic images using the Testo 875 thermal imager and Zetasizer also indicate a hydrodynamic size of 58.61 ±
(Testo 875-1i, Germany) with high infrared resolution and 1.12 nm and a Zeta potential of −29.85 ± 0.47 mV, respectively
thermocouple (Figure 5, B-C). During hyperthermia, the skin (Figure 2, B). Figure 1, C presents the saturation magnetization
and tumor temperatures of rats were controlled using an IR (Ms) values of SPIONs, MNPs-FA and TMZ-loaded MNPs-FA,
camera and a copper-constantan thermocouple, respectively. 25 which were 59.21, 38.75 and 36.54 emu/g, respectively. The
For this purpose, we removed a piece of skull (1 × 1 cm 2) and slight fall in the Ms value of TMZ/MNP-FA nanoparticles
placed the thermocouple probe inside the tumor and another compared to that of blank SPIONs or MNP-FA nanoparticles can
probe inside the normal tissue, and at the same time used the IR be attributed to the non-magnetic TMZ and folic acid molecules
camera. In this case, the results of the thermocouple and the that led to a fall in the magnetic field effect. In addition, all
camera did not differ much (0.2-0.6 °C), and this may be due to nanoparticles exhibited superparamagnetic properties without
the low depth of the tumor (Figure 5, A-E). The maximum 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 the accelerated TMZ release by the effect of magnetic
hyperthermia under AMF. The results clearly indicated that by
As reported in a previous study, MNPs-FA nanoparticles raising the temperature, drug release was increased. As shown in
reduced the release rate of TMZ and increased the release time Figure 2, A, a rapid release of TMZ occurred within the first
(in vitro and in vivo) by up to 15-fold. Here, the temperature- 8 min of AMF exposure; however, as the AMF was switched off,
responsive property and remote-control effect of the nanoparti- the release rate was significantly decreased. The TMZ release of
cles are put under the spotlight. To evaluate this hypothesis, we MNPs-FA nanoparticles after 8 min with AMF exposure was
investigated the cumulative release profiles of TMZ at 52.1 ± 2.3%, while the release at the same time without AMF
temperatures of 37 and 43 °C (using a heater stirrer), as well 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 5

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 time. However, when nanoparticles were absent, the temperature
shown in Figure 2, B. Figure 2, B clearly shows that, if smart rise was not localized and occurred over a longer period of time.
thermosensitive MNP nanoparticles exposed with AMF, they Infrared images of glioma rats treated with nanoparticles in the
would be able to significantly increase the heat. presence of AMF showed that during AMF hyperthermia, the
white area indicating the high temperature signal was greater at
Hemolytic toxicity the tumor site than the adjacent tissue (Figure 5, E). According to
To investigate whether our synthetized MNP-FA with/ the thermometric results for hyperthermia at 43 °C, the rats were
without TMZ could induce hemolysis, we incubated rat RBC placed in the magnetic coil, 20 min for AMF only and 8 min for
samples with different concentrations of nanoparticles for 2 h. AMF with magnetic nanoparticles.
The results showed that by increasing the concentration of
nanoparticles (10 to 4000 μg/ml), the hemolysis rate in rat blood In vivo Onchothermia efficacy
samples was elevated (Figure 3). At concentrations above 250, The antitumor effects of the different treatment modalities (TMZ,
the increase in hemolysis was more significant for TMZ/MNP- MNPs-FA, and TMZ/MNPs-FA under AMF exposure
FA compared to that of MNP-FA nanoparticles (P < 0.05). (13.56 MHz, 40 A/m)) were evaluated in rat glioma by monitoring
According to ASTM E2524-08, 25 a hemolysis rate above 5% can tumor volume and survival time (Figure 6). Tumor volume in the
damage erythrocytes. Accordingly, both nanoparticles (MNPs- saline group (control) exhibited a 300% and 500% increase in 17 and
FA and TMZ/MNPs-FA) appear to be homo-compatible at 24 days after the initial cell injection, respectively. Tumor volume
concentrations below 3000 μg/ml. changes and survival curves (Figure 6, C & D) show that only
In vivo temperature variations of magnetic hyperthermia MNPs-FA and TMZ injections or AFM alone could not lead to
(MNPs-FA + AMF) tumor suppression. After treatment with TMZ or AFM alone,
although the tumor size increased slowly relative to the saline and
After successful growth of glioma tumors (50-100 mm 3), the MNPs-FA group, but tumor volume increased again after 24 days of
rats were divided into two groups. One group received cell injection. TMZ combined with AMF increased survival to
nanoparticles (44 mg/kg) and was kept under a magnetic field 67 days (P < 0.05), and remarkably decreased the tumor size by
for 2 h. The other treatment group received saline control. about 3-fold compared to that of the saline group on day 24. TMZ-
Finally, both groups were exposed to AMF (20 min, loaded nanoparticles had similar results and increased survival to
13.56 MHz, 50 W) and body temperature was measured during 71 days and decreased tumor size 3.6-fold compared to controls on
hyperthermia. We used the alternating magnetic coils day 24 (P < 0.01, Figure 5, C & D). MNPs-FA and TMZ-loaded
(13.56 MHz, 40 A/m) system for generating homogeneous MNPs-FA nanoparticles in combination with AMF exhibited the
magnetic field. As shown in Figure 4, A, the density of the best response; they could decrease tumor volume 7-10 folds on day
magnetic field is uniform inside the coil. Figure 5, D shows the 24 and increase the survival of rats from 97 to 106 days (P < 0.001,
temperature variations in different treatment groups. As can be Figure 6, C & D). However, there was no significant difference
seen in Figure 4, A, the application of in the presence of magnetic between these groups (P > 0.05), which indicates the major role for
nanoparticles caused a significant time-dependent increase in MH therapy (MNPs-FA+ AFM), leading to a significant enhance in
temperature, especially in the tumor area at the shortest possible 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 that the tumor cells generally did not undergo apoptosis. TMZ
successfully suppressed the tumor growth for approximately and AMF (43 °C) alone exhibited a significant increase in Bax
80 days, after which the tumors recurred again (Figure 6, B). expression (P < 0.05), while the Bcl-2 protein remained
In addition, a signaling pathway involving the activation of approximately constant relative to the control and MNPs-FA
cancer cells is related to a remarkable increase in the expression group (P > 0.05, 1.3 < ratio ≤ 1.4). Treating glioma bearing
level of Bax protein, and repression of Bcl-2. As can be inferred rats with the TMZ-loaded MNPs-FA nanoparticles and TMZ
from Figure 7, A & B, rats treated with FA conjugated MNPs (43 °C) revealed a significantly higher Bax protein production
showed almost no Bax or Bcl-2 protein expression, as shown in than that of TMZ or AMF alone (P < 0.05), whereas the Bcl-2
Figure 7, C & D. The value of Bax/Bcl-2 ratio was ≤1, meaning 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 energy is wasted when the magnetic moments of the particles
in Bax and decrease in Bcl-2 expression, after combinatorial relaxes toward its equilibrium orientation (Neel relaxation). 30 As
therapy of AMF plus MNPs-FA with/without TMZ (P < 0.01 a result of this waste energy causes hyperthermia (Figure 4, B).
and P < 0.05, ratio > 3, Figure 7) was applied. 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
Discussion MNPs in the presence of an AMF and at physiological
temperature is presented in Figure 2. The results show that the
Oncothermia is a new type of combinational therapy that TMZ/MNPs-FA nanoparticles were stable at 37 °C (body
couples hyperthermia and chemotherapy together to treat deeply temperature), but when subjected to AMF, the resultant heat
located tumors such as brain malignancies, where localized heat increased the release rate of TMZ by affecting the polymer
deposition and controlled drug release still remain the most coating. As the AMF was cut off and the heat dissipated, drug
formidable challenges in treatment of brain tumors. 1 , 27 The aim release decreased, indicating the thermal sensitivity of the
of this study was to introduce an elegant thermos-sensitizing nanoparticles that allowed the nanoparticles to be released in a
nano-platform specialized for carrying TMZ that can be controlled manner. Minaei et al measured the TMZ release rate
incorporated by Oncothermia for treatment of gliomas. In this from FA-conjugated magnetic nanoparticles. They showed drug
method, an alternating magnetic field (13.56 MHz, 40 Am −1) is release rates of 48.3 ± 1.7% and 74.43 ± 0.77% within 4 h and
used as the external source of energy for magnetic hyperthermia. 48 h, respectively. 31 Mao et al showed that Tf-PLGA-TMZ
As shown in Figure 1, A & B, the synthesized TMZ-loaded released about 60% of loading TMZ in less than 2 h. 32 Similar
superparamagnetic FA-conjugated (TMZ/MNPs-FA) nanoparti- results have been reported for TMZ drug release by Yu et al 33
cles had a size below 50 nm and a negative charge of 30 mV. and Zeng et al. 14 So comparing our results with past studies
Hysteresis loops recorded at room temperature for different shows that magnetic hyperthermia offers great potential as it can
nanoparticles show the superparamagnetic properties of all be remotely controlled drug release.
nanoparticles (Figure 1, C). Therefore, TMZ/MNPs-FA nano- To confirm the role of AMF in the rise of temperature to
particles were only capable of producing heat through the Neel 43 °C in glioma models, after the intravenous injection of the
relaxation mechanism (due to the flipping motion of the SPIONs conjugated MNPs (Fe = 9 mg/kg) and application of an external
magnetic moment 28) when exposed to the AMF, because of zero magnetic field (2 h), the glioma rats were placed inside the
magnetic hysteresis (Figure 4, B). The absorption of radiofre- magnetic coil (13.56 MHz, 20 min) and their body temperature
quency energy by biological tissue results in higher tissue measured. Since an adequate concentration of magnetic
temperatures as a function of time, which eventually causes cell nanoparticles is required to be transported to the tumor
death due to hyperthermia. 29 In fact, an external AMF provides microenvironment, it has been suggested to use folic acid ligand
energy, and helps rotate the magnetic moments in overcoming and external magnetic fields to accomplish this goal. However,
the energy barrier (E = KV), where K is the anisotropy constant this cannot be easily accomplished. Ohtake et al reported that
and V is the volume of the magnetic core such as SPIONs. This since they failed to inject sufficient concentrations of Fe(Salen)
 R. Afzalipour et al / Nanomedicine: Nanotechnology, Biology, and Medicine 31 (2021) 102319 9

nanoparticles into the mice brain, they used a subcutaneous developed thermo-responsive TMZ/MNPs-FA (44 mg/kg) in
tumor model in the leg. 34 Figure 5 demonstrates that the MNPs- combination with an AMF hyperthermia (13.56 MHz, 40 Am −1)
FA nanoparticles can properly target the tumor cells, and was confirmed by a considerable tumor growth suppression and
produce heat under the AMF especially in the tumor site. Similar significant induce apoptosis. Accordingly, this combinatorial
results were reported by Liu et al, 35 Balivada et al and Basel et therapy could serve as a promising Oncothermia procedure for
al 36 , 37 with the exception that they injected magnetic nanopar- the treatment of glioma.
ticles directly into the tumor area. In another study, Shirvalilou et
al reported that they used 20 mg of SPIO nanoparticles for References
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modalities (MF and acid folic ligand) can effectively reduce Reguera E, Barber DF, et al. Flower-like Mn-doped magnetic
injectable drug doses, which can reduce systemic toxicity. 38 To nanoparticles functionalized with α v β3-integrin-ligand to efficiently
our surprise, the results of the hemolysis experiments presented induce intracellular heat after alternating magnetic field exposition,
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