Mitigation of Magnetic Particle Hyperthermia side effects

by magnetic field controls

A. R. Tsiapla1,2*, A. A. Kalimeri1,2, N. Maniotis1,2, E. Myrovali1,2,

T. Samaras1,2, M. Angelakeris1,2, O. Kalogirou1,2

1 School of Physics, Faculty of Sciences, Aristotle University, 54124 Thessaloniki, Greece

2 MagnaCharta, Center for Interdisciplinary Research and Innovation (CIRI-AUTH), 57001

Thessaloniki Greece

* Corresponding author: aitsiapl@physics.auth.gr

DOI: http://10.1080/02656736.2021.1899310
Journal: International Journal of Hyperthermia
Abstract

In magnetic particle hyperthermia, a promising least-invasive cancer treatment,

malignant regions in proximity with magnetic nanoparticles undergo heat stress, while
unavoidably surrounding healthy tissues may also suffer from heat either directly or
indirectly by the induced eddy currents, due to the developed electric fields as well. Here, we
propose a facile upgrade of a typical magnetic particle hyperthermia protocol, to selectively
mitigate eddy currents’ heating without compromising the beneficial role of heating in
malignant regions. The key idea is to apply the external magnetic field intermittently (in an
ON/OFF pulse mode), instead of the continuous field mode typically applied. The parameters
of the intermittent field mode, such as time intervals (ON time: 25-100 s, OFF time: 50-200 s,
Duty Cycle:16-100%) and field amplitude (30-70 mT) are optimized based on evaluation on
healthy tissue and cancer tissue phantoms. The goal is to sustain in cancer tissue phantom the
maximum temperature increase (preferably within 4-8oC above body temperature of 37oC),
while in the healthy tissue phantom temperature variation is suppressed far below the 4oC
dictating the eddy current mitigation. Optimum conditions of intermittent field (ON/OFF:
50/100 in s, Duty Cycle: 33%, magnetic field: 45mT) are then examined in ex-vivo samples
verifying the successful suppression of eddy currents. Simultaneously, a well-elaborated
theoretical approach provides a rapid calculation of temperature increase and, furthermore,
the ability to quickly simulate a variety of duty cycle times and field controls may save
experimental time. Eventually, the application of an intermittent field mode in a magnetic
particle hyperthermia protocol, succeeds in eddy current mitigation in surrounding tissues
and allows for the application of larger field amplitudes that may augment hyperthermia
efficiency without objecting typical biomedical applicability field constraints such as
Brezovich criterion.

Keywords: magnetic nanoparticles; magnetic hyperthermia; intermittently applied AMF; cancer

therapy;

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Introduction
Cancer is still the second largest cause of death worldwide with 9.6 million deaths in
2018 [1]. Magnetic particle hyperthermia (MPH) is an adjunct cancer treatment based on
biomedical nanotechnology that complements the established therapies (chemotherapy,
radiation therapy) increasing the therapeutic efficacy of the latter and at the same time
reducing their side effects [2, 3, 4].
The combination of magnetic fields (MF) and magnetic nanoparticles (MNPs) is
currently implemented in diagnosis (Magnetic Resonance Imaging, MRI) [5], in therapy (drug
deposition, magnetic hyperthermia) and for handling cancerous tissues (biolabeling,
bioseparation) [6]. MNPs can penetrate biological tissues, since they easily cross many
biological barriers and are considered relatively safe for humans [7]. Accordingly, MPH has
been proposed in a clinical trial together with radiation therapy in patients with recurrent
glioblastoma, reducing radiation session duration by 50% and increasing life expectancy by
100% [8, 9].
MPH is based on the heat production by MNPs (expressed by the Specific Loss Power,
SLP, in W/g) under alternating magnetic field (AMF) exposure in the radio frequency range
(100-1000 kHz) [10], due to magnetic hysteresis losses and/or relaxation mechanisms [11].
The result may be gradual apoptosis or even necrosis of cancer cells that are subjected to heat
shocks within the hyperthermia window of 41-45oC, while surrounding healthy tissues
typically sustain such temperature ranges [12, 13, 14].
The major challenges of MPH for its widespread clinical use against cancer are:
production of the appropriate AMF, accurate tissue thermometry, long-term biocompatible
behavior of the MNPs, spatially homogeneous distribution of the AMF and MNPs in the tumor,
the effect of biological matter on the MNPs heating capacity and finally tolerance and / or
safety of patient exposure to AMF, directly related to eddy current side effects [15, 16, 17].
MPH clinical studies are already under way in Europe in conjunction with radiotherapy [8, 18,
19], while recently, a new magnetic hyperthermia center specializing in brain tumors opened
at the Independent Public Clinical Hospital No. 4 in Lublin, Poland [20].
Eddy currents are induced by the AMF and it has been shown that they limit MPH
treatment efficacy in clinical trials [15, 16, 17]. The product Ho×f, typically known as the
Brezovich criterion (where Ho is the amplitude and f is the frequency of the magnetic field)
provides a safety threshold for magnetic field driven therapy. It was initially proposed in 1988
[21] as the upper limitation for major discomfort in patients undergoing AC magnetic field
driven therapies. For micron-sized magnetic implants, it was reported as

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Ho×f=4.85×108 A m-1 s-1, but since, magnetic carriers are shrinking in size, down to nanoscale
elements, nowadays it seems rather stringent, and requires reconsideration [22]. As Ho×f
increases, the desired heating rate in cancer tumor regions augments accordingly, yet such
increase is directly reflected to the heating of surrounding healthy tissues, due to eddy
currents, a direct side-effect. Thus, application of MPH should follow certain field constraints,
unless the effect of eddy currents is safely mitigated. Hergt et al. [23] revisited the Brezovich
criterion and proposed one order of magnitude greater value for the limit (Ho×f ≤ 5x109 A m-1
s-1) while Bellizzi et al. [24, 25] recently used a 3D realistic human head model and proposed,
numerically calculating, an optimization criterion by examining its reliability and feasibility. It
is worthwhile mentioning that in these cases [24, 25] the Ho×f product was two orders of
magnitude larger than the Brezovich criterion.
It is widely accepted that to prevent unwanted heating in normal tissue the field
strength frequency product H0×f should be limited. In MPH treatment where AMF coils are
employed for MNPs excitation, another crucial parameter that affects the strength of eddy
currents is the radial position r within the coil in which the phantom (or the tissue) exists and
defines the radius of the eddy current path within the tissue. The phantom can be placed at
any point inside the coil along the radial direction from 0 to R, where R is the radius of the
coil, while the absorbed power density at each point within the tissue, due to eddy currents,
changes as a function of r2 [26]. Thus, for large tissues or small coils, where r≈R (i.e. the
location of the tissue is close to coil surface), the undesirable eddy currents’ heating is
exacerbated. To sum up, the magnitude of eddy currents depends not only on the AMF source
(H0 and f), but also on the size of the tissue exposed to the field. However, according to
Herrera et al. the magnitude of eddy currents also depends on the size of the nanoparticles.
More specifically, they showed that small-sized MNPs (d <20 nm) as in our case have
negligible appearance of eddy currents in contrast to larger-sized MNPs, wherein the
occurrence of eddy currents is significant [27].
Alternative approaches to circumvent eddy current constraints during magnetically
driven theranostics schemes are on the way. For example, Ivkov et al. [28] proposed the
application of ON/OFF cycles, i.e. an intermittent exposure mode with an AMF of 153 kHz
frequency and 40-130 mT amplitude aiming to reduce the power of eddy currents in mice
through heat dispersion and dissipation during the OFF-time interval. They have shown that
reducing unwanted heating of healthy tissues is not only a function of the amplitude of the
applied field and the timing of the ON/OFF intervals, but also significantly depends on the
relative duration of the ON and OFF time, which is called "duty cycle" [28]. Another attempt to

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introduce clinically applicable techniques in order to address the heating issue of healthy
tissues is reported by Stigliano et al. [26]. Firstly, the hyperthermia coil, i.e. a large electric
field region, is selectively displaced away from healthy tissues. Secondly, the coil is in constant
motion with respect to the tissue under treatment, so that successive heat losses due to eddy
currents are dispersed in a larger tissue volume. Studies on tissue models have shown that
displacement and motion reduce the maximum temperature by 74% and 19% in simulation
and 77% and 33% experimentally, respectively [26]. Moreover, Nieskoski and Trembly [29]
proposed an optimized AMF single-coil instead of a Helmholtz coil, to optimize the heating of
the MNPs which is limited by the current-induced heating. They determined a power ratio
which is the ratio of MNPs’ induced heat to surface eddy current heating in order to compare
the geometries of the coil and showed that it depends on the target depth of MNPs. Also, they
demonstrated that by increasing the power ratio, the concentration of MNPs necessary for
therapeutic efficacy in tissue, decreases [29]. Other reports [30, 31] have also suggested
improved design of AMF coils, while Kumar et al. [32] examined the performance of skin
surface cooling application to reduce maximum temperature, due to eddy currents. They used
a water jacket during small animals’ exposure to high AMF amplitude which almost had no
interaction with magnetic field and did not affect its desired amplitude [32].
In this manuscript, we evaluate the role of an intermittently applied AMF on the
thermal response of iron oxide (Fe3O4) MNPs with respect to field parameters and in direct
comparison with the corresponding continuously applied AMF. This work aims to reduce the
heating of healthy tissues, due to eddy currents, without compromising the beneficial role of
MPH in cancerous cells. Thus, applied field amplitude Ho may augment, even surpassing the
clinically acceptable constraint of Brezovich criterion, without worrying for side-effect of
regional potential heating of non-malignant sites. This may be achieved by intermittent field
application, multiple ON/OFFs, within an optimized time window, in phantom and ex-vivo
samples, where heating (i.e. temperature increase) is restricted to selected specific sites in
excellent agreement with the corresponding simulated MPH sequences.

Methods
Sample preparation
MNPs with a mean diameter of 10 nm were prepared by aqueous co-precipitation of
ferric and ferrous salts in alkaline conditions [33]. Details on sample synthesis and their
physical characterization (Figure S1) are presented in supporting information.

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 To proceed with phantom sample preparation, we followed the simple idea of gels,
specifically those made of agarose, which are routinely used as phantom models, while they
comprise the only transparent porous materials which successfully simulate animal tissues
[34]. More specifically, 3-4% agarose gel content mimics the microstructure of hard tissues
[35], while lower content gels have porosities similar to soft tissues such as the human brain
[34]. Accordingly, three different phantoms based on agarose solutions were synthesized: (1)
healthy tissue phantom: Ph0, (2) cancer tissue phantom Ph1, and (3) two layer tumor -
healthy tissue interface Ph2, composed of a bottom cancer tissue phantom layer (bottom) and
a healthy phantom tissue layer (top) as shown in Figures 1a, 1b and 1c, respectively.
Accordingly, three ex-vivo samples based on veal tissue were prepared: (1) veal tissue
sample, named Xv0 (Figure 1d), which is the reference one, (2) veal tissue sample with MNPs,
named Xv1 (Figure 1e), in two different concentrations 4mg/mL (Xv1a) and 8mg/mL (Xv1b),
and (3) two-region tumor-healthy tissue interface named Xv2 (Figure 1f), where the MNPs
(8mg/mL) were placed in the center of the beaker and dark area within the sample’s center
represents tumor region, while the surrounding red colored area healthy tissue. More details
on phantom sample fabrication are given in supporting information section synthesis and
properties.

Ph0 Ph1 Ph2

Xv0 Xv1 Xv2

Figure 1. Samples under study a) Ph0: Reference: agarose solution corresponding to healthy phantom, b)
Ph1: agarose with MNPs, corresponding to cancer phantom c) Ph2: Tumor- healthy tissue interface, d)

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Xv0: Reference: Veal tissue sample corresponding to healthy tissue, e) Xv1: Veal tissue sample with MNPs
(Xv1a: 4 mg/mL, Xv1b: 8 mg/mL) corresponding to cancer tissue, f) Xv2: Tumor (central region)- healthy
(peripheral region) tissue interface.

Magnetic particle hyperthermia experiment

Magnetic particle hyperthermia was carried out in a commercial AMF generator (1.2
kW Ambrell Easyheat 0112) under the frequency of 375 kHz and variable magnetic field
amplitude of 30-70 mT. Sample’s temperature was recorded in 0.4 s intervals for ample time
(i.e. 900 s) with an Optic Fiber Temperature sensor immersed in the central region of sample
under study. To start with, we record the MPH reference, which is a typical experimental
sequence performed in the MNPs aqueous solution with the same concentration and under
the same field conditions followed hereafter. Schematics of experimental protocol (Figure S2)
now appear in supporting information magnetic particle hyperthermia section together with
corresponding details on measuring protocol. A representative graph of aqueous solution
MNPs heating efficiency is depicted in Figure S3 at concentration 4 mg/mL and 60 mT/375
kHz, showing the facile entrance within the hyperthermia window at the first 50 s, thus
yielding a technologically exploitable SLP value of 546 W/g. Error bars in values of sample’s
temperature increase (ΔΤ), resulting from MPH experiments, are estimated by uncertainty
assessment that arises from the temperature recording method, such as the resolution and
sensitivity of optic fiber, and the not ideally uniform temperature distribution within the
sample [36].

Duty cycle
The crucial factor in the intermittent mode of applying the AMF is the so-called duty
cycle, which correlates the field ON / OFF time durations, and is expressed by the following
equation:
Field ON time (s)
Duty cycle = Field ON time (s) + Field OFF time (s) x 100% (1)

where “Field ON time (s)+Field OFF time (s)” is defined as the total duration time of one
operation cycle (period of an operation cycle). Table 1 gives the duty cycle (%) values and the
corresponding field ON/OFF field duration in s.

Table 1. Duty cycles and the corresponding field ON/OFF values (in s).

Duty cycle (%) ON/OFF (s)

16 50/250
20 50/200
25 50/150
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 33 50/100
50 50/50
100 50/0

In all samples, the intermittent modes with the duty cycles of Table 1 were examined,
while continuous operation was serving as the reference of a typical hyperthermia sequence.
Finally, the total duration of the phantom study measurement was 30 min (15 min for the
heating phase and 15 min for the cooling phase, when the sample returns to its initial
temperature in the absence of a field). In the case of the ex- vivo samples these intervals were
doubled to 60 min (30 min heating phase, 30 min cooling phase) in order to provide a
measurable signal.

Theoretical approach
The objective of the theoretical approach was the evaluation and validation of the
experimental optimum conditions by obtaining the numerical temperature increase curves
and comparing them with the corresponding experimental ones. So, a simple algorithm, based
on the work by Neufeld et al. [37], was chosen for the fast estimation of the temperature
development in the case of intermittent external field application. Thus, we follow a safety
assessment model to predict temperature increase during MRI. The model is based on
theoretical considerations like relating peak temperatures in the presence or absence of local
thermoregulation, as well as on data extracted from simulations involving anatomical models
to determine a characteristic time constant τ which is a characteristic parameter of the tissue
and ranges between 100 and 600 s, depending on the tissue type and the temperature
increase during MPH treatment [30]. The main assumption of Neufeld et al. is that the
temperature increase exhibits exponential behavior and eventually tends to equilibrium. This
increase is given by applying the following analytical relationship:

𝛥𝛵𝑖+1 = 𝛥𝑇𝑖 + (1 − 𝑒 −𝛥𝑡𝑖⁄𝜏 )(𝑇𝑖𝑛𝑐 − 𝛥𝑇𝑖 ) (2)

where
(2) 𝛥Τ𝑖 is the temperature increase at a specific time ti, ΔΤ𝑖+1 is the temperature increase

at the next time interval, Δ𝑡𝑖 is the time interval (step), 𝑇𝑖𝑛𝑐 is the maximum temperature
achieved during AMF application and τ is the time constant. In order to extract the
temperature sequence in the case of intermittent AMF application, we modified Equation (2)
by means of the following expression:

𝛥𝛵𝑖+1 = 𝛥𝑇𝑖 + (1 − 𝑒 −𝛥𝑡𝑖⁄𝜏 )(𝑇𝑖𝑛𝑐 × 𝜅 − 𝛥𝑇𝑖 ) (3)

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where κ is a constant that equals to 1, when the AMF is ON and to 0, when the AMF is OFF,
resulting to the local heating and cooling of the phantom, respectively.
To start with, we performed a fitting function based on Equation (2) to the
experimental temperature increase curve for the case of continuously applied AMF to
estimate the maximum temperature 𝑇𝑖𝑛𝑐 , for a specific duration of time, and the
corresponding time constant τ. The time step Δ𝑡𝑖 was set to 0.1 s. This method of limited time
interval selecting ensures that the fitting parameters are accompanied with a reasonably
small standard deviation [38].
After substituting the estimated 𝑇𝑖𝑛𝑐 and τ values in Equation (3) we got the temporal
evolution of temperature during the intermittent application of the AMF. Initially,
temperature increase 𝛥𝛵0 is zero. At each time step, 𝛥𝑇𝑖 is updated by Equation (3) offering a
rapid calculation of 𝛥𝑇𝑖 and, furthermore, the ability to quickly simulate a large number of
different duty cycles. The above technique was applied in both Ph1 and Ph2 phantoms and
also in Xv1 and Xv2 samples.
In order to outline the validity of our approach, we employed a combined
electromagnetic-thermal model based on COMSOL Multiphysics simulations of the MPH set up
and phantom samples. This model is analyzed and presented in the supporting information of
the manuscript, where the numerical results of electromagnetic and heat transfer simulations
are shown in Figures S6-S10. The geometry of the model, illustrated in Figure S6, is following
the experimental MPH apparatus. Simulations were conducted for two samples. The first is
the healthy tissue phantom (Ph0) and the second is the cancer tissue phantom (Ph1).
Throughout the COMSOL simulations, we utilized a concise model that offers a combination of
the heat transfer equations with Maxwell’s time-harmonic equations, the definition of
dynamic heating and electromagnetic boundary conditions, and the employment of a robust,
fully coupled solver ensuring: a) an accurate estimation of the magnetic field shown in Figures
S7, S8 b) a detailed description of the heating process shown in Figure S9 revealing the not
ideally homogeneous temperature spatial distribution within the sample c) a precise
estimation of the temperature increase curves for both Ph0 and Ph1 when continuous or
intermittent AMFs are applied as depicted in Figure S10.
In Figure S10 (a) the validation of Equation 2 (continuously applied AMF) is achieved
after the comparison of the temperature increase curves obtained by this approximation with
the ones obtained numerically after the full simulation with COMSOL. This comparison is
employed for both Ph0 and Ph1 samples and the maximum temperature increase deviation
between the two methods (approximation and simulation) is found equal to ΔΤmax ~ 0.5 oC.

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This small deviation clearly illustrates the validity of Equation 2. Similarly, Figure S10 (b)
depicts the validation of Equation 3 (intermittently applied AMF), for Ph0 and Ph1 samples.
The maximum temperature increase deviation between this equation and the numerical
solution is even lower than in the case of continuously applied AMF and equals to ΔΤmax ~ 0.3
oC, a value that safely validates Equation 3.
The whole procedure of the theoretical approach followed is depicted in the flow chart
shown in Figure S5 of supporting information.

Results and Discussion

The proof of principle was verified in the phantom study, where the parameters
applied were optimized, and subsequently evaluated in ex vivo samples. As far as we know,
the in vivo and ex vivo studies, conditions and effect may differ dramatically because of
biological interactions with the nanoparticles and also perfusion cannot be neglected in in
vivo models, as in ex vivo [39]. One of the major challenges is to reduce heating, due to eddy
currents during MPH. Thus, we demonstrate agarose models namely phantoms (Ph0, Ph1,
Ph2) and ex vivo models in veal tissues (Xv0, Xv1 and Xv2) in order to study the impact of
operation cycle, duty cycle and magnetic field amplitude on field-induced eddy currents.
Finally, a theoretical approach was undertaken to check the thermal response, in order to
compare our experimental results in both models. Consequently, the results of this approach
may be implemented in any stage of biomedical application as means of fine tuning
(augmenting) MPH heating efficiency.

Α. Phantom Study
The aim of this study was to find the optimum conditions, in which the heating of
healthy tissues, due to eddy currents is reduced, when an intermittent AMF is applied. At the
same time, the appropriate MNPs’ heat release must be maintained at such levels to guarantee
hyperthermia temperatures. For these reasons three different agarose phantoms
(Ph0, Ph1, Ph2) were used. Initially, we wanted to determine the right operation cycle, then
the appropriate duty cycle and, finally, the proper magnetic field amplitude for Ph0 and Ph1
phantoms. From these three studies, the optimal conditions were verified via a theoretical
approach and tested in the Ph2 phantom. Τhe latter phantom experiment took place to study
how healthy tissues may be affected by surrounding heating imposed by MNPs on malignant
tissues.

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 Α.1. Operation Cycle
Firstly, it was crucial to study the operation cycle in phantoms by varying the duration
of time and applying an intermittent mode ON/OFF (25/50, 50/100, 75/150, 100/200 in s),
while the duty cycle remained stable at 33%. Figure 2 shows the ΔΤ curves as a function of
time for Ph0 and Ph1 phantoms. The black line corresponds to continuous operation of AMF
(ON/OFF: 900/0). For the case of intermittent mode, ΔΤ coincides with the maximum
temperature increase observed, i.e. the highest ΔΤ peak. Field discontinuity is readily
visualized with the zigzag form of the heating curves and is illustrated by the different colored
lines. In all cases, the magnetic field and the frequency were kept constant at 60 mT and 375
kHz, respectively. Also, in order to have a better understanding of Figure 2 the maximum ΔΤ
for each experiment in the two phantoms is summarized in Table 2.

Figure 2. Experimental ΔΤ curves for all operation cycles used in a) Ph0: reference: healthy tissue and b)
Ph1: cancer tissue, phantoms. The black line represents the continuous mode of AMF ON/OFF: 900/0 (in
s) and purple, red, blue and dark cyan curves depict the intermittent mode ON/OFF: 25/50, 50/100,
75/150, 100/200 (in s), respectively. The applied experimental conditions were: AMF amplitude: 60
mT/375 kHz and duty cycle: 33%; the shaded bands illustrate the hyperthermia window (ΔΤ = 4-8°C).

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Table 2. Operation cycle experiments in phantoms (Ph0: reference: healthy tissue, Ph1: cancer tissue).
ON OFF Ph0 Ph1
(s) (s) ΔΤ (oC) ΔΤ (oC)
25 50 4.1±0.3 11.1±0.7
50 100 3.8±0.2 10.4±0.6
75 150 4.3±0.3 11.4±0.7
100 200 4.4±0.3 12.4±0.8
900 0 11.2±0.7 21.8±1.3

As it is obvious, considering a body temperature equal to 37oC, a temperature increase

of 4-8oC (shaded area with red color) appears sufficient to enter the MPH window (41-45oC) a
prerequisite for subsequent clinical application. In the curves shown in Figure 2, the
measured ΔΤ in the Ph0 phantom is a result of the eddy currents, while in the Ph1 phantom
the increase is also due to the presence of the MNPs. Observing Table 2, for both phantoms the
period of each operation cycle was not a critical factor as, differences between ΔΤ values were
not significant. On the contrary, the intermittent application of the magnetic field as compared
to continuous application led to a smaller ΔΤ (reduction of approximately 60%) not only in
the Ph0, but also in the Ph1 phantom. In Ph1 the goal was to achieve the maximum possible
reduction of ΔΤ due to eddy currents, while at the same time, maintaining a satisfactory heat
release from the MNPs, so that the treatment of hyperthermia could be effectively applied.
Based on this principle, the time duration of one operation cycle ON/OFF:50/100 (in s) was
chosen as the optimum in order to move on with the duty cycle study.

Α.2. Duty cycle

Another important parameter which can significantly influence the heating rate is the
duty cycle. Thus, the “ON” time was kept constant at 50 s, according to the preceding results,
while the time “OFF” was varied from 50 to 250 s. Figure 3 shows the ΔΤ curves of
intermittently applied AMF with different duty cycles, resulting from varying the “OFF” time.
In all cases, the magnetic field and the frequency were kept constant at 60 mT and 375 kHz,
respectively. For a better understanding of the curves depicted in Figure 3, the maximum ΔΤ
for each duty cycle experiment in both phantoms is presented in Table 3.

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Figure 3. ΔΤ curves of each duty cycle experiment in a) Ph0: reference: healthy tissue and b) Ph1: cancer
tissue phantoms. The black, red, blue, dark cyan and pink curves depict the intermittent mode ON/OFF:
50/50, 50/100, 50/150, 50/200 and 50/250 (in s), respectively. The applied experimental conditions
were: AMF: 60 mT/375 kHz and the hyperthermia window is illustrated with a shaded band.

Table 3. Duty cycle experiments in a) Ph0: reference: healthy tissue and b) Ph1: cancer tissue phantoms.
ON OFF Duty cycle Ph0 ΔΤ Ph1 ΔΤ
(s) (s) (%) (oC) (oC)
50 250 16 1.8±0.1 5.7±0.3
50 200 20 1.9±0.1 7.1±0.4
50 150 25 1.9±0.1 8.7±0.5
50 100 33 3.8±0.2 10.4±0.6
50 50 50 6.2±0.4 16.4±1.0
50 0 100 11.2±0.7 21.8±1.3

Comparing Figures 3a and 3b, there are differences between the Ph0 and Ph1
phantoms. In general, the ΔΤ of Ph0 did not enter the hyperthermia window except of 50%
duty cycle, i.e., ON/OFF: 50/50 (in s). On the contrary, the ΔΤ in all duty cycles of Ph1 reached

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it, and this was due to the presence of MNPs. The 33% duty cycle (red line) was considered as
the optimum one, since it maintained the ΔΤ at a satisfactory level (10.4oC, corresponding to a
clinical body temperature of 47.4 oC [40]). Despite the relative high ΔΤ, surpassing the MPH
window, such a duty cycle may be exploited clinically, since ΔΤ naturally undergo different
decrease levels due to perfusion [41, 42].

Α.3. Magnetic field amplitude

Τhe next step of this study was the selection of the optimal magnetic field by examining
four different AMF amplitudes: 30, 45, 60 and 70 mT. The following experiments were carried
out using the optimal operation cycle and duty cycle conditions (ON/OFF: 50/100 in s and
33%, respectively). Figure 4 displays the ΔΤ curves of the two optimum magnetic field
amplitude values (45 mT and 60 mT at constant frequency of 375 kHz) in Ph0 and Ph1.
Moreover, ΔΤ curves for 30 mT and 70 mT are presented in Figure S4 (supporting
information). Table 4 provides the maximum ΔΤ for each magnetic field amplitude
experiment in both phantoms.

Figure 4. ΔΤ curves of the two optimum magnetic

field amplitude values [a) 45mT/375kHz, b)
60mT/ 375kHz] in Ph0 (reference: healthy tissue)
and Ph1 (cancer tissue) phantoms. Dark cyan and
red curves represent the intermittent mode
ON/OFF: 50/100 (in s) in Ph1 and Ph0,
respectively, while blue and black curves illustrate
the continuous mode ON/OFF: 900/0 (in s) in Ph1
and Ph0, respectively. Shaded bands denote the
hyperthermia window.

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Table 4. Magnetic field experiments in a) Ph0: reference: healthy tissue and b) Ph1: cancer tissue,
phantoms.
Field ON OFF Duty cycle Ph0 ΔΤ Ph1 ΔΤ
(mT) (s) (s) (%) (oC) (oC)
50 100 33 0.4±0.02 1.1±0.1
30
900 0 100 3.4±0.2 5.3±0.3
50 100 33 2.9±0.2 7.2±0.4
45
900 0 100 9.1±0.6 17.0±0.6
50 100 33 3.8±0.2 10.4±1.0
60
900 0 100 11.2±0.7 21.8±1.3
50 100 33 3.6±0.2 12.6±0.8
70
900 0 100 12.3±0.7 28.2±1.7

As expected, an increase in magnetic field amplitude results in the increase of the

maximum temperature reached. From Table 4 at 30 mT, the ΔΤ of Ph0 phantom decreased
considerably, the heating due to eddy currents also reduced almost to zero. Nevertheless, the
corresponding value in Ph1 phantom did not managed to reach to the required ΔΤ value to
enter the hyperthermia window. At 70 mT except that the field is rather high, during
continuous mode AMF application resulting to excessive ΔΤ values (>70oC), the Ph0 and Ph1
agarose phantoms began to decompose. So, these two magnetic fields amplitudes were
rejected for subsequent evaluation. On the contrary, the applied magnetic field amplitude of
45 mT suppresses ΔΤ from 9.1 to 2.9 oC in Ph0 (healthy phantom), when the field mode was
changed from continuous to intermittent. Accordingly, in Ph1 (cancer phantom), ΔΤ
decreased from 17.9 to 7.2 oC yet sustained within the MPH window. In general, ΔΤ ranging
from 4 to 8 oC outlines the MPH window by considering an initial body temperature equal to
37 oC. Thus, the ΔΤ of 7.2 oC would result to temperature to 44.2 oC, rendering this MPH,
effective. Similar features were observed also for the 60mT, where ΔΤ in Ph0 and Ph1 were
3.8 and 10.4 oC, respectively.
From Table 4, we see that ΔΤ of Ph0 (health phantom) was 2.9 and 3.8 oC for 45 and
60mT, respectively. Thus, 45 mT leads to a larger reduction of eddy currents’ heating. These
values compare relatively well with the Ph0 reference ΔΤ for continuous mode of 30 mT
which is 3.4 oC. On the contrary, the therapeutic capacity of the MNPs was enhanced, since the
Ph1 (cancer phantom) ΔΤ of 5.3 oC by the 30mT continuous mode was much lower than the
corresponding ΔΤs of 7.2 oC and 10.4 oC observed when 45mT and 60mT were applied,

~ 14 ~
respectively. Thus, we may surmise that doubling the field from 30 to 60 mT the ΔΤ in Ph1
(cancer phantom) also doubled (from 5.3 to 10.4 oC), without increasing the side effects on
healthy tissues, ΔΤ in Ph0 increased from 3.4 to 3.8 oC. From the above analysis it is clear that
with the utilization of intermittent exposure AMF, it is possible to exceed the limit set by
Brezovich (Ho×f was equal to 1.35×1010 A m-1 s-1 when Ho=45 mT and 1.79×1010 A m-1 s-1 when
Ho=60 mT) and yet to attain milder side effects and efficient treatment. More specifically, by
using an intermittent mode AMF we managed to increase the applied AMF amplitude and at
the same time to achieve a reduction in eddy currents’ heating and an increase in the
therapeutic capacity of the MNPs.

Α.4. Theoretical approach

After the experimental results in phantoms Ph0 and Ph1, a theoretical approach was
attempted in order to verify the choice of the optimal conditions (magnetic field amplitude,
duty cycle) by estimating numerically the temperature rise curves. Following the framework,
described in methods, the curves for both cases of continuous and intermittent magnetic field
mode were occurred. Figure 5 depicts the theoretical and experimental curves for Ph0 and
Ph1 phantoms in the case of continuous (900/0 in s) and intermittent mode (50/100 in s) for
the two best magnetic field amplitudes (45 and 60mT). Experimental (blue, dark cyan) and
theoretical (black, red) curves were found to be in good agreement for both phantoms and
magnetic field conditions.

~ 15 ~
Figure 5. Theoretical approach of Ph0: reference: healthy tissue phantom a) 45mT and c) 60mT under
continuous and intermittent mode, and Ph1: cancer tissue phantom b) 45mT and d) 60mT under
continuous and intermittent mode. In continuously applied AMF mode theoretical and experimental
curves were in good agreement. In the case of intermittent mode, the peaks of theoretical and
experimental curves were very close for both Ph0 and Ph1 phantoms. The hyperthermia window is
depicted with the shaded bands.

For the continuous mode and field amplitudes of 45 mT and 60 mT and after 900 s of
treatment, a comparison was made between theoretical, obtained from Equation 2, and
experimental temporal evolution of the ΔΤ in both phantoms (Ph0 and Ph1). At 45 mT the
estimated time constant τ and maximum temperature 𝑇𝑖𝑛𝑐 were estimated at 580 s and 9oC,
respectively for Ph0, and 495 s and 16oC, respectively for Ph1. At 60 mT the ΔΤ was evaluated
to be 10.5 oC and 20.6 oC for Ph0 and Ph1, respectively, with the corresponding time constants
being equal to 533 and 456 s, respectively. In the case of intermittent mode magnetic field, the
ΔΤ curves in Ph0 and Ph1 were obtained theoretically by substituting in Equation 3 the values
of time constant and 𝑇𝑖𝑛𝑐 obtained for the continuous mode. The theoretical curves calculated
by Equation 3 were in good agreement with the corresponding experimental curves. This
outcome verifies that there is a reliable way of numerically predicting the temporal evolution

~ 16 ~
of temperature in the intermittent mode, when it is possible to assess the respective ΔΤ
curves for the continuous mode. In this way, such an approach may not only evaluate an
experimental sequence but gain significant experimental time to quickly optimize treatment
protocol parameters of the intermittent mode.

Α.5. Tumor- healthy tissue system interface (Ph2)

The above experimental and theoretical results were obtained inside homogeneous
phantoms mimicking either healthy or MNPs loaded tumor tissue. It is important to examine
how healthy tissues are affected by local heating of MNPs after magnetic field application.
Thus, a phantom of the tumor-healthy tissue interface (Ph2) was examined in two different
operating modes, namely continuous mode (900/0 in s) and intermittent mode with 33%
duty cycle (50/100 in s). The magnetic field and the frequency were kept constant at 45 mT
and 375 kHz, for both operating modes. Measurements were taken at three different points
(P.1, P.2, P.3) as shown in Figure 6 (a). More specifically, P.1 was in the surface of the healthy
tissue layer, P.2 was at the interface of the two layers and P.3 was in the cancer tissue layer.
Figure 6 (b) illustrates the ΔΤ curves taken at the three aforementioned points. For the
continuous mode (900/0 in s) of P.1, P.2, P.3 the curves are depicted with dark cyan, blue and
pink, respectively. Also, for the intermittent mode (50/100 in s) of P.1, P.2, P.3 the curves are
portrayed with red, black and olive, respectively. Aditionally, Table 5 summarizes the results
obtained from the Ph2 phantom at the three aforementioned positions (P.1, P.2, P.3).

Figure 6. (a) The three measuring points (P.1, P.2, P.3) inside the Ph2 phantom, (b) ΔΤ curves measured
at these points (P.1, P.2, P.3) for continuous and intermittent operating modes. Hyperthermia window is
denoted with a shaded band.

~ 17 ~
Table 5. Continuous and intermittent mode magnetic fields experiments in Ph2 phantom.

Ph2 ΔΤ (oC)

Measuring points
ON/OFF (s) P.1 P.2 P.3
50/100 4.0±0.2 4.9±0.3 7.2±0.4
900/0 10.5±0.6 12.5±0.8 17.0±1.0

Figure 6 (b) shows that there was a ΔT increase in all positions (P.1, P.2, P.3) for the
continuous application of AMF. For the intermittent exposure with a duty cycle of 33%
(50/100 in s), the measurements at positions P.1 and P.2 were very different from all the
experiments performed above. Their behavior was neither that of the continuous one, nor
that of the intermittent mode magnetic field. In fact, in position 2 (black curve) the peaks that
appeared were not at defined positions and distances from each other as they were in our
previous experiments. In position P.1 (red curve) the peaks were almost non-existent. Finally,
comparing P.3 with P.1 and P.2, ΔΤ increased faster in both operating modes, a phenomenon
that is clearly due to the presence of MNPs.
Results collected in Table 5 demonstrate clearly the proof of principle of this study
since at P.1 (normal tissue phantom) the ΔΤ due to eddy currents decreased substantially,
from 10.5oC to 4.0oC by exchanging the field mode from continuous to intermittent one. On
the other hand, at P.3 (cancer tissue phantom), the ΔΤ of 7.2oC resided within the
hyperthermia temperature window contrary to the corresponding much greater decrease of
17.0oC in the continuous field mode. The most important finding from the Table 5 was that the
ΔΤ in intermittent mode at P.2 was minimal (from 4.0 to 4.9oC), so we may surmise that the
healthy -cancer tissue interface is not practically affected by the released heat.

Β. Εx vivo study
Until now, only a few studies [43, 44] have assessed MNPs thermal behavior inside
biological tissues to achieve the translation from laboratory setups to clinical application. In
order to proceed further, phantom study was followed by an ex-vivo study. In this stage,
experiments on veal tissues were conducted to determine whether the previous optimum
operating parameters (ON/OFF: 50/100 in s, duty cycle: 33%, magnetic field: 45mT) were
applicable in ex vivo conditions. Thus, we examined: (a) a reference veal tissue in order to
simulate healthy tissue, Xv0, (b) veal tissue with MNPs to simulate cancer tissue, Xv1, (c)

~ 18 ~
tumor-healthy tissue interface, Xv2. Finally, a theoretical approach took place only in the Xv2
system in order to be compared with our experimental results.

B.1. Application of optimum conditions

After finding the ideal operating parameters from the agarose phantoms, the
application of them in ex vivo tissues came next. Figure 7 is intended to represent the ΔΤ
curves in healthy veal tissue, Xv0, and cancer tissue, Xv1. Two different concentration values
of MNPs were utilized for the Xv1 phantom leading to Xv1a, which contained 4 mg/mL, and
Xv1b, containing 8mg/mL to acquire measurable signals in the ex-vivo studies in accordance
with in-vivo studies [45]. It should be noted that the magnetic field and the frequency were
kept constant at 45 mT and 375 kHz, respectively. Table 6 summarizes the experimental
results obtained from the ΔΤ curves.

Xv1: 4mg/mL

Xv1: 8mg/mL

Figure 7. ΔΤ curves of continuous and intermittent operating modes in ex vivo veal tissues without MNPs
(Xv0) and with MNPs a) Xv1: 4mg/mL MNPs, and b) Xv1: 8mg/mL MNPs. The hyperthermia window is
illustrated by the shaded bands.

~ 19 ~
Table 6. Continuous and intermittent mode magnetic fields in ex vivo veal tissues (without MNPs: Xv0,
with MNPs: Xv1).
Field ON OFF Xv0 ΔΤ Xv1 (4mg/mL) ΔΤ Xv1 (8mg/mL) ΔΤ
(mT) (s) (s) (oC) (oC) (oC)
45 50 100 3.4±0.2 4.2±0.3 6.6±0.4
900 0 10.8±0.6 15.0±0.9 20.7±1.2

In general, a similar behavior with the Ph0 healthy phantom was observed. However,
considering the first results, the duration time for the ex vivo experiments was doubled in
order to raise the ΔΤ and enter the hyperthermia window, since tissue-like samples were
involved. Figure 7 shows that in the intermittent operating mode and the phantom with 4
mg/mL, ΔΤ approached the hyperthermia window in the last seconds (1200-1800 s). On the
contrary, in the case of 8 mg/mL, this happened after the first 350 seconds (350-1800 s).
From Table 6, following the same selection criteria as for the other operating
parameters, the concentration of 8 mg/mL was chosen as the most appropriate one. Not only
a significant ΔΤ decrease in temperature due to eddy currents was observed, but also a
sufficiently high ΔΤ for hyperthermia treatment was maintained via the intermittent field
mode application.

Β.1. Ex vivo Tumor-healthy tissue interface system

The last step of this work involved an ex vivo system that was designed in such a way
that the core of the sample represents a putative tumor (Xv2 P.1), i.e., tissue loaded with
MNPs, surrounded by pure veal tissue that would imitate the healthy tissues (Xv2 P.2). The
purpose of this system was to investigate not only the response of this particular system to
the different modes of AMF application, but also to examine if healthy tissues (Xv P.2) are
affected by the neighboring heating of the MNPs (Xv2 P.1) located in the tumor area. Ιt should
be mentioned that in this experiment, the magnetic field and the frequency were kept
constant at 45 mT and 375 kHz, respectively. Figure 8 shows the ΔΤ curves in the ex vivo Xv2
and Table 7 summarizes the experimental results.

~ 20 ~
Figure 8. a) Points (P.1, P.2) where magnetic hyperthermia measurements were performed, b) ΔΤ curves
measured at these points (P.1, P.2) in continuous and intermittent mode of magnetic field application in
the ex vivo tumor-healthy tissue-like interface system Xv2. Hyperthermia window appears as a shaded
band.

Table 7. Continuous and intermittent mode magnetic field in Xv2 system.

Field ON OFF Xv2 P.1 ΔΤ Xv2 P.2 ΔΤ

(mT) (s) (s) (oC) (oC)
50 100 5.1±0.3 3.0±0.2
45
900 0 15.6±0.9 12.5±0.7

Observing image 8 (b), it seems that the hyperthermia curves in the continuous mode,
of P.1 rose faster than P.2. As for the multiple one, P.1 entered the hyperthermia limit, while
P.2 did not. With the help of the Table 7, it is clear what happened when a continuous and
intermittent mode was applied. Once again, ΔΤ due to eddy currents in P.2 decreased quite a
bit from 12.5 to 3.0 oC when intermittent mode, was applied. Concurrently, MNPs’ therapeutic
efficacy was maintained, as ΔΤ in P.1 reached the hyperthermia range. Τhe reduction noted
here was from 15.6 oC in continuous mode to 5.1 oC by applying intermittent mode. This latest
experiment reaffirms the advantages of intermittent operation mode over the continuous one
as not only the side effects in healthy tissues are reduced but at the same time the therapeutic
ability of the MNPs is retained.

~ 21 ~
 Β.2. Theoretical approach
The purpose here is to bestow a direct quantitative evaluation and validation of the
experimental optimum conditions by the direct comparison of the numerically calculated
values with the experimental ones. Under this framework, a comparison between theoretical
(red and black curves) and experimental (black and dark cyan curves) data, took place for the
optimum duty cycle (33%). The good agreement between the theoretical approach and the
experimental findings is outlined in Figure 9. This outcome constitutes an additive proof of
the suitability and validity of the optimum conditions emerged from the experimental
procedure.

Figure 9. Theoretical approach of the ex vivo model mimicking a tumor-healthy tissue interface a) point
Xv2 P.2 without MNPs, b) point Xv2 P.1, with MNPs, for both application modes of the AMF. Hyperthermia
window is shown with a shaded band.

For the continuous magnetic field mode of 45 mT field amplitude and after 1800 s of
treatment, a comparison between theoretical and experimental temperature temporal
evolution in both samples (Xv2 P.2 and Xv2 P.1) was performed. The estimated time constant
τ and maximum temperature increase 𝑇𝑖𝑛𝑐 were found equal to 556 s and 12οC, respectively,
~ 22 ~
for Xv2 P.2 and 525 s and 15.5οC, respectively, for Xv2 P.1. ΔΤ curves obtained theoretically
using Equation 3 in the case of intermittent AMF application were in good agreement with the
corresponding experimental ones, indicating again the suitability of the numerical
methodology introduced for predicting the temporal evolution of temperature under
discontinuous application of AMFs.
The application of intermittent operation mode resulted in induced eddy currents with
very short duration and so in mitigating the undesired healthy tissue heating, even when
reaching Ho×f values much higher than Brezovich limit (1.35×1010 A m-1 s-1 for Ho=45 mT).
This outcome permits the use of higher AMF amplitudes and thus allowing for a significant
enhancement of the MNPs’ treatment efficacy.
Finally, in this work, based on the size of the 8-turn coil (inner diameter of 2.54 cm),
the phantom diameter is restricted to 3 cm, while future goal concerns the use of larger
diameter coils and thus the use of larger diameter phantoms (i.e. 10 cm), for subsequent in-
vivo studies.

Conclusions
In a MPH protocol, the intermittent magnetic field mode is superior to the continuous
magnetic field mode with respect to side-effect overheating naturally field application. The
reason is that smaller temperature variation, due to eddy currents is obtained and at the same
time MPH efficiency is practically maintained, resulting in a much more successful, effective
treatment with milder side-effects. This outcome was confirmed by phantoms and ex vivo
MPH experiments. In addition, the numerically validated theoretical approach, proposed in
this study, could be easily and rapidly applied for the establishment of an optimum protocol.
The optimal conditions for achieving this goal were ON/OFF: 50/100 (in s), duty cycle 33 %,
magnetic field amplitude 45 mT in both agarose phantoms and ex vivo experiments. For the
latter, the optimal MNPs concentration was 8 mg/mL. Furthermore, it has been shown that by
doubling the magnetic field amplitude, the ΔΤ in the cancer tissue phantom was also doubled,
thus having better therapeutic potential, sparing side effects in healthy tissues. Therefore, we
could argue that by applying an intermittent mode magnetic field it is possible to overcome
the Brezovich criterion.

Disclosure statement
The authors report no conflicts of interest.

~ 23 ~
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 Mitigation of Magnetic Particle Hyperthermia side effects
by magnetic field controls

A. R. Tsiapla1,2*, A. A. Kalimeri1,2, N. Maniotis1,2, E. Myrovali1,2,

T. Samaras1,2, M. Angelakeris1,2, O. Kalogirou1

Supporting information

MNPs synthesis and properties

Magnetic nanoparticles (MNPs) with a mean diameter of 10 nm were
prepared by aqueous co-precipitation of ferric and ferrous salts in alkaline
conditions. MNPs were synthesized following a procedure with a three necked
round bottom flask, which was placed in a water bath with mechanical stirring
and was heated to 90oC, as described in detail in a previous publication
[1].Firstly, 40 mL of water containing NaNO3 and NaOH were used, in order to
obtain final concentration values of 0.2 M NaNO3 and 0.4 M of NaOH. Secondly,
20 mL of FeSO4 H2O were added in the bottom flask to obtain a solution of 0.2 M,
which was dissolved in 10-2 M H2SO4. At this point, ethanol and distilled water at
a proportion, EtOH/H2O=1, were added in the reaction vessel. When the
precipitation was complete, the solution was heated to 90oC for three hours.
After that, the solution was left to naturally cool down to room temperature, in
order to reach conditions near equilibrium. Finally, the solid was separated by
magnetic decantation and washed several times with distilled water.

~1~
 80

60

40

Magnetization (emu/g)
20

-20

-40

-60
20 nm
-80
-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6
Magnetic Field (T)

Figure S1: Room temperature hysteresis loop of Fe3O4 MNPs. Inset is TEM imaging
outlining nanoparticle morphology.

TEM imaging showing the morphology of MNPs (inset figure), together

with room temperature hysteresis loop appear in Figure S1. MNPs exhibit
saturation magnetization of 73 emu/g, a value close to the one of bulk magnetite
despite their small average size of 10 nm, a hint for their good crystallinity.

Phantom and ex-vivo samples

Gels, especially those made of agarose, are routinely used as phantom
material, since they are transparent, porous and have properties similar to those
of animal tissues [2]. A 3-4% w/w agarose gel content mimics the microstructure
of hard tissues [3], while lower content gels have porosities similar to soft tissues
such as the human brain [2]. Accordingly, three different phantoms based on
agarose solutions were synthesized. Agarose solutions were prepared using
agarose, sodium chloride and distilled water. Sodium chloride (NaCl) was added
in the solution to increase the electrical conductivity (higher than the normal
tissue conductivity, >1 S/m) in order to provide a reliable measurement of
temperature increase (i.e. heating), due to eddy currents. Initially, the liquid
solution was transferred into a beaker at room temperature until it turned into a
gel and was marked as a reference sample corresponding to a soft human tissue.
Subsequently, following the same procedure, MNPs (0.02 g) were added in the
beaker with agarose and NaCl. These were mixed and stirred with a magnetic
stirrer at 85οC until they were completely dissolved. After that, the beaker was
placed directly in an ultrasonic bath to maintain the homogeneous dispersion of
the MNPs and was left to cool down in room temperature, in order to create the

~2~
phantom mimicking cancer tissue loaded with MNPs. The diameter of this layer
was 3 cm, while the thickness was 0.7 cm. Finally, a healthy/cancerous tissue
interface was prepared, in order to investigate how healthy tissues are affected
by cancer tissue’ heating during MPH. This interface was achieved between a
first layer including the MNPs solution and mimicking the cancerous tissue, and a
second layer made solely of the agarose solution simulating healthy tissue, both
prepared with the methods described above. Thus, three different phantoms
based on agarose solutions were synthesized, namely (1) phantom healthy
tissue: Ph0, (2) phantom cancer tissue Ph1, and (3) tumor- healthy tissue
interface Ph2, as shown in Figures 1a, 1b and 1c, respectively. Accordingly, three
ex-vivo samples based on veal tissue were prepared: (1) veal tissue sample,
named Xv0 (Figure 1d), which is the reference one, (2) veal tissue sample with
MNPs, named Xv1 (Figure 1e), in two different concentrations 4mg/mL (Xv1a)
and 8 mg/mL (Xv1b), and (3) tumor-healthy tissue interface, named Xv2 (Figure
1f), where the MNPs (8mg/mL) were placed in the center of the beaker.

Magnetic particle hyperthermia

Magnetic particle hyperthermia (MPH) was carried out with an external
alternating magnetic field (AMF) at a frequency of 375 kHz; four different
amplitudes of AMF were examined: 30, 45, 60 and 70 mT. Samples were placed
on top of a water-cooled induction 8-turn coil (inner diameter of the solenoid
was 2.54 cm and its height 4.3 cm) connected to an AC generator (1.2 kW
Ambrell Easyheat 0112). The cooling water temperature ranged from 8 to 10 °C
and the initial temperature was room temperature. The temperature inside the
samples was measured using a calibrated GaAs-based fiber optic probe (OTG-Q
Fiber Optic Temperature Sensor, OpSens) with temperature operating range:-
40°C to+250°C, resolution 0.01 °C, length: 1.5 m, diameter: 1.4 mm (including
cable sheathing) and total instrument accuracy: ± 0.8°C. The tip of the fiber was
immersed in samples at a height of 0.35 cm above the beaker bottom at the
center of the sample. In MPH, each measurement cycle includes a heating and a
cooling phase. Temperature data with acquisition rate of 0.4 s were collected via
the SoftSens software. Schematics of the experimental protocol (with device
photo bottom-left) are shown in Figure S2.

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Figure S2. The outline of an in vitro magnetic hyperthermia setup: Left is the
schematic representation, where the cross-section of coil, the thermometer probe
(optic fiber) and the vessel containing the sample are shown. The magnetic field
controller is shown in the leftmost picture. Top Right scheme outlines the formation
of AC magnetic field in a coil by alternating current polarity. Bottom Right graph is
a typical magnetic hyperthermia curve together with photo shortcuts with a
thermal camera to verify temperature measurements. Curve consists of a heating
part (~ up to 700 s), where the temperature increases under the application of an
AC magnetic field, and a subsequent cooling part (from 700 s to 1200 s) (AC field is
turned off). The last part of curve allows us to model the heat exchanges with the
environment naturally occurring also during heating, due to non-adiabatic
conditions.

The heat transfer between the AMF coil and the sample under study
cannot be fully avoided in a typical non-adiabatic MPH setup, such as the one we
used. We typically record not only the heating sequence (Field ON), but also the
cooling sequence (Field OFF) of the temporal variation of temperature.
Therefore, we are able, by using the modified law of cooling, to directly estimate
the heat exchange mechanisms between coil and sample and remove their
contribution from the specific loss power (SLP) estimation, which provided a
quantifiable measure of heating efficiency (W/g). Moreover, by routinely
measuring reference MPH curves, we can isolate magnetic from non-magnetic
origin heating contributions. Eventually, SLP values are free from different heat
exchange contributions of origin other than MNP heating and the magnetic field
heating effect is properly evaluated [Σφάλμα! Δεν έχει οριστεί
σελιδοδείκτης.]. The initial temperature of the experiment was room
temperature. All experiments were repeated twice to validate the temperature

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variations occurring, due to eddy currents. In each case, the choice criterion for
the applied protocol was to have the maximum effect (ΔTmax) in cancer tissue
phantom together with the minimum effect (ΔΤmin) in healthy tissue phantom.
In principle, iron oxide MNPs are hydrophobic and whether they are
stable and safe depends on their concentration. In general, MNPs with size <10–
20 nm, have very low toxicity and are used in many biomedical applications [4].
In our experiments MNPs were stable, because they were dispersed in agarose
gel, so they had not precipitated to the bottom of the beaker. Also, the MNPs that
were used in this work, produced high SLP: 546W/g (Figure S3 depicts the MPH
experiment on the MNPs under study in aqueous solution form; it can be seen
that in a very short time MNPs managed to reach the hyperthermia limit in less
than 50 s). Therefore, these MNPs are ideal for use in magnetic hyperthermia. All
measurements and corresponding SLP estimations were performed, following a
protocol we recently published, where the possible uncertainty sources during
experiments and estimations are included in the overall uncertainty of SLP
magnitude and provide reliable SLP values considering type B uncertainties
during measurements and considering all possible heat exchange free of
experimental (Type B) and estimation uncertainties [5].

Figure S3. A typical MPH two-stage experiment of a 4 mg/mL Fe3O4 MNPs aqueous
solution. Temperature increases during field application and surpasses hyperthermia
window within the first 30 s. The estimated SLP, at 60mT/375 kHz is found equal to 546
W/g indicating an excellent heating efficiency.

Magnetic field amplitude

The following experiments were carried out using the optimal operation cycle
and duty cycle conditions (ON/OFF: 50/100 in s and 33%, respectively). Figure

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S4 exhibits the ΔΤ curves of 30 mT and 70 mT magnetic field amplitude values at
constant frequency of 375 kHz in Ph0 and Ph1 phantoms.

Figure S4. ΔΤ curves of the two magnetic field amplitude values [a) 30mT/ 375kHz, b)
70mT/ 375kHz] in Ph0 (reference: healthy tissue phantom) and Ph1 (cancer tissue
phantom), phantoms. Dark cyan and red curves represent the intermittent mode ON/OFF:
50/100 (in s) in Ph1 and Ph0 respectively, while blue and black curves illustrate the
continuous mode ON/OFF: 900/0 (in s) in Ph1 and Ph0, respectively. Shaded bands
demonstrate the hyperthermia window.

Theoretical approach
Theoretical approach provides a direct quantitative evaluation and
validation of the experimental optimum conditions by the direct comparison of
the numerically calculated values with the experimental ones. Thus, we regard it
as an additive proof that our experimental findings of temperature variations are
correct and in excellent agreement with what is expected from theory. Such a
theoretical approach was chosen, because it provides a rapid calculation of
temperature increase and, furthermore, the ability to quickly simulate a large
number of different duty cycles and protocols in general, which is a sine qua non

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prerequisite for translation of the presented technique into clinical practice in
the future. A flow chart of our methodology follows in Figure S5.

Figure S5. Flow chart of the theoretical approach

Validation of the theoretical approach

In order to validate numerically the theoretical approach of our work, we
utilized a coupled electromagnetic-thermal model, based on COMSOL
Multiphysics full simulations that provides an accurate estimation of the
magnetic field and temperature distribution within Ph0 and Ph1 samples for the
cases of continuous and intermittent field mode. This simulation method is
taking into account all the physical (thermal, electric and magnetic) properties of
the materials used in experiments and thus bestows rational results. We first
design the geometry of the experimental setup and materials projected in the 2-
D axial symmetry model assuming that the coil, the vial and the phantom
geometries approximate axial symmetry, since the 2-D axisymmetric model is
the most convenient approach to ensure minimization of computational costs.
The 8-turn coil used in experiments for MPH treatment follows the same
design as the experimental one, while we also designed the 3 cm diameter
beaker, placed on the coil, with the 5 mL of agarose gel in the vial. A point data

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set was also created within the phantom at the position where the optic fiber
was placed for temperature recording. The model geometry is illustrated in
Figure S6 (a), where a scale bar is used to visualize the elements dimensions. An
extremely fine mesh was employed throughout the model geometry. The mesh
consisted of 13,860 domain elements counting for the 8-turn squared coil, the
cooling water flowing inside the coil, the vial, the phantom and the surrounding
air and finally 1,956 boundary elements were used. The size distribution of mesh
elements is illustrated in Figure S6 (b).

Figure S6. (a) Geometry design of the Figure S7 (a) Magnetic flux density spatial
experimental setup. The black circle at the distribution. Black arrows indicate the
center is the point where temperature was magnetic field vector direction. (b) Qelec
measured. (b) Color legends of mesh distribution within the phantom tissue
element sizes for the model used. volume. Qelec is the oncometric heat
generation rate due to eddy currents.

For the AMF conditions modelling we also need the dielectric properties
of phantom which are taken from [6], where the effect of background tissue
electrical conductivity on RF-induced heating was studied in a controlled system
of agar phantoms with various NaCl concentrations at 500 kHz, which is close to
the frequency used in our experiments (375 kHz). From the various input values

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of the alternating current (AC) amplitude we found the corresponding magnetic
flux density distribution. The special case of 45 mT magnetic flux density value at
the measurement point is depicted in Figure S7 (a). The heating due to induced
eddy currents, 𝑄𝑒𝑙𝑒𝑐 , is estimated by equation S1 after employing the magnetic
field distribution that derived from the solution of the electromagnetic problem:
1 1
𝑄𝑒𝑙𝑒𝑐 = 𝜎𝑬2 = 𝜎(𝜋𝜇0 𝑯𝑓𝑟)2 (S1)
2 2
where σ is the electrical conductivity, r is the radial position within the coil in
which the phantom exists and E and H are the electric and magnetic field,
respectively. Qelec distribution within the phantom tissue volume is depicted in
Figure S7 (b).
The magnetic flux calibration for the various AC amplitude values is
depicted in Table S1. The dielectric properties of the phantom tissue are shown
in Table S2. The targeted electric conductivity is estimated according to [6] and
is found equal to 1.2 S/m. This value is twice as high as (0.6 S/m [7]) the one of a
healthy tissue and thus gives rise to induced heating due to eddy currents. For
the relative permittivity we assume that the addition of NaCl will not change its
value which is taken from [7].

Table S1. Magnetic flux density values at the experimental measuring point as observed
from four different AC amplitudes.
AC amplitude Magnetic flux density
(A) (mT)
82.5 30
123.5 45
160 60
187.5 70

Table S2. Dielectric properties of the phantom tissue

Tissue Relative Targeted electric
permittivity conductivity
εr σ (S/m)
Phantom 1000 1.2

For the ON/OFF magnetic field mode, we imported an intermittently

applied alternating current expression with the proposed ON/OFF signal
resulting to an intermittent AMF as shown in Figure S8. In Figure S8 (a) the
temporal evolution of magnetic flux density amplitude B is shown at the

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 measuring point within the phantom (where B0=0.045 T) after utilizing with
COMSOL an intermittently applied alternating current. In order to find the
applied frequency of such a signal, a spectral analysis for the optimum proposed
ON/OFF signal (50/100 s) was followed. By employing a fast Fourier
transformation (FFT) on the magnetic flux density function, depicted in Figure S8
(a) as a time domain graph, we obtain the power spectral density of the FFT of
the applied signal. This graph is illustrated in Figure S8 (b) where the frequency
of magnetic field signal was found equal to the carrier frequency (375 kHz).

(a)

(b)
Power spectral density (a.u.)

Frequency (kHz)
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Figure S8. (a) Magnetic flux density amplitude B after 900 s operation of intermittently
applied alternating current for B0=0.045 T. B0 is measured at the defined point in the
phantom tissue. The ON/OFF duration time of the intermittently applied alternating
magnetic field condition is 50/100 s, respectively. (b) FFT power spectrum of the
intermittently applied magnetic field signal. The frequency of the generated magnetic field
pulses coincided with the carrier frequency of the AMF signal and was equal to 375 kHz.

Subsequently, for the heat transfer problem solution, the phantom

thermal properties are used, which are taken by previous works in phantom
MPH [8], [9] and presented in Table S3.

Table S3. Thermal properties of phantom tissue

Tissue Thermal Density Heat Viscosity Heat
conductivity (kg/m3) Capacity at (Pas) transfer
k ρ constant n Coefficient
(W/mK) pressure h
(J/kgK) (W/m2K)
cp
Phantom 0.52 1079 3540 0.8 3.3

In order to estimate the temperature increase both in the cancer tissue

phantom (Ph1) and healthy tissue phantom (Ph0) for the two modes of AMF
exposure (continuous and intermittent) we solve with COMSOL the heat transfer
module by substituting the total heat dissipation Q with Q = Qelec +Qmag. Where
Qmag is the MNPs power dissipation in the magnetic fluid. Since the MNPs system
studied consists of 10 nm superparamagnetic MNPs, Qmag is given by
Rosensweig’s theory [10] by taking into account the Néel and Brown relaxation
times and is given by equation (S2), where 𝜒0 is the equilibrium magnetic
susceptibility, H0 is the AMF amplitude, f the applied frequency and τ the
effective relaxation time
2𝜋𝑓𝜏
𝑄𝑚𝑎𝑔 = 𝜋𝜇0 𝜒0 𝐻02 𝑓 (S2)
1 + (2𝜋𝑓𝜏)2

Temperature spatial distributions within the model geometry are shown

in Figure S9 for Ph1, Figure S9 (a), (b), and Ph0, Figure S9 (c), (d), tissue
phantoms exposure on continuously and intermittently applied AMF. Those
solutions are obtained after 900 s of MPH treatment for 𝜇0 H0 and f values equal
to 45 mT and 375 kHz respectively. For the case of the intermittently applied

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AMF we used the ON / OFF: 50/100 (in s.) condition which has been proven to
be the optimum one.
Mild inhomogeneity in temperature distribution within all the samples is also
revealed by Figure S9 giving rise to uncertainties in ΔΤ nominal values. Thus, in
the main manuscript, the presentation of ΔΤ results is accompanied by their
corresponding errors for all the systems used. Those errors may also arise from
other sources that have been thoroughly examined in our recently published
study [5].

Figure S9. Temperature spatial distribution after 900 s for (a) cancer tissue phantom
exposure on continuously applied AMF (b) cancer tissue phantom exposure on
intermittently applied AMF (c) healthy tissue phantom exposure on continuously applied
AMF (d) healthy tissue phantom exposure on intermittently applied AMF.

Full simulated temperature increase (ΔΤ) curves, at the measurement

point depicted in Figure S5 (a)., are shown in Figure S10 along with the
corresponding approximate ΔΤ curves obtained by equation 2 (for the
continuously applied AMF) and equation 3 (for the intermittently applied AMF)
of the main manuscript and read respectively:

𝛥𝛵𝑖+1 = 𝛥𝑇𝑖 + (1 − 𝑒 −𝛥𝑡𝑖⁄𝜏 )(𝑇𝑖𝑛𝑐 − 𝛥𝑇𝑖 ) (𝑒𝑞. 2)

𝛥𝛵𝑖+1 = 𝛥𝑇𝑖 + (1 − 𝑒 −𝛥𝑡⁄𝜏 )(𝑇𝑖𝑛𝑐 × 𝜅 − 𝛥𝑇𝑖 ) (𝑒𝑞. 3)

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 18
(a)
16

14

12
ΔΤ (oC)

10

6
Ph1 (simulation)
4
Ph1 (approximation)
2 Ph0 (simulation)
Ph0 (approximation)
0
0 150 300 450 600 750 900
Time (s)
10
Ph1 (simulation) (b)
Ph1 (approximation)
8
Ph0 (simulation)
Ph0 (approximation)
ΔΤ (oC)

0
0 150 300 450 600 750 900
Time (s)
Figure S10. Comparison between the temperature increase obtained numerically after full
simulation with COMSOL (simulation curve) for 900 s of MPH treatment and (a) the
temperature increase obtained by our estimation method (approximation curve)
introduced by equation 2 and (b) the temperature increase obtained by employing
equation 3 of the main manuscript. Both methods are employed for the healthy (Ph0) and
cancer (Ph1) tissue phantoms. Simulation ΔΤ curves are taken at the measuring point
depicted in Figure S5 (a).

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 The agreement between our approximation, and the numerical data is
evident. More specifically, in Figure S10 (a) the validation of equation 2 is
achieved for both Ph0 and Ph1 samples since the maximum temperature
increase deviation between this equation and COMSOL simulation is found equal
to ΔΤmax ~ 0.5 oC. Similarly, Figure S10 (b) depicts the validation of equation 3 for
the same samples with the maximum temperature increase deviation being
equal to ΔΤmax ~ 0.3 oC. Those results render the estimation method, proposed in
this work, suitable for the establishment of an optimum protocol for a rapid and
accurate estimation of ΔΤ curves when experimental and numerical techniques
could get quite time consuming. In this way, someone can use this approach
either before or after experimental measurements to quickly optimize treatment
protocol parameters of either the continuously or the intermittently applied
magnetic field mode.

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