Annals of R.S.C.B., Vol. XXI, Issue 1, 2016, pp. 11 – 21
Received 14 November 2016; accepted 23 November 2016.
doi: 10.ANN/RSCB-2016-0017:RSCB
Hyperthermia effects on normal and tumor skin cells
MACAVEI SERGIU GABRIEL (1),(2), SUCIU MARIA (2),(3),*, CRACIUNESCU IZABELL (3),
BARBU-TUDORAN LUCIAN (2),(3), TRIPON SEPTIMIU CASIAN (2),(3), BALAN RADU (1)
1
Departament of Mechatronics and Machine Dynamics, Faculty of Mechanical Engineering,
Technical University of Cluj-Napoca
2
National Institute of Research and Development for Isotopic and Molecular Technologies, Cluj-Napoca, Romania
3
Faculty of Biology and Geology, Babes-Bolyai University, Cluj-Napoca, Romania
*Corresponding author
Maria Suciu PhD.
5-7 Clinicilor Str., Cluj-Napoca, Email:[email protected].
Keywords. Cellular organelles, Structure and Function, Cell Biology.
Summary
tumors in magnetic resonance imaging, to
targeting and treating tumors with cytostatic
adsorbed on nanoparticles or hyperthermia.
Due to the superparamagnetic properties of
iron oxide nanoparticles, hyperthermia is
becoming a real alternative to general
cytostatic administration in cancer treatments
(Hyperthermia in Cancer Treatment, 2016).
Hyperthermia is a method of treating cancer
by heating the tumor. Today there are three
main types of treatments: localized, regional
and general (whole body heating). The
method using magnetic fluid hyperthermia
consists of a combination of an inductive
applicator and magnetic fluid. In this
combination, there is the benefit of heating
only the tumor at a field intensity 1000 times
bigger than using whole body hyperthermia,
and so, the effects lead to a controlled
apoptosis of tumor cells more quickly and
with no discomfort to the patient (Fannin,
2002; Rosenwig, 2002).
Superparamagnetic iron oxide nanoparticles
(SPIONs) are widely used today in multiple
medical applications, one of them being
hyperthermia. When SPIONs are placed into a
controlled alternating magnetic field, they
start to heat up and transfer this caloric energy
to the adjacent media. This property of
SPIONs is very desirable in case of localized
tumor
treatment.
We
used
normal
keratinocytes cell line and melanoma cell line
to compare the effects of 50 nm naked and
polyethylene glycol(PEG)-coated SPIONs in
a 48 hours' time range after a hyperthermia
treatment. SPIONs were produced using the
oil mini-emulsion method and their effects
were analyzed by colorimetric assays
reflecting the mitochondrial and membrane
integrity status of the cells, and by electron
microscopy analyses. Results indicate that
PEG-coated SPIONs have a delayed but more
pronounced effect on melanoma cells than on
normal cells.
Introduction
Materials and methods
Survival rates for cancers have increased from
49% to 69% in recent statistics (Cancer Facts
and Figures, 2016). These improvements are
possible due to developments in medical
technologies, such as nanotechnology. Iron
nanoparticles were found to have many
benefits in medicine, starting from visualizing
Nanoparticle synthesis. For the synthesis of
Fe3O4 magnetic nanoparticles, with size
around 10 nm, the co-precipitation method of
ferric and ferrous salts under the presence of
Ar gas was used (Turcu et al., 2015). In a
typical experiment 5.4 g of FeCl3 (0.1 M) and
The Romanian Society for Cell Biology ©, Annals of R. S. C. B., Vol. XXI, Issue 1, 2016, Maria Suciu, pp. 11 – 21
11
Annals of R.S.C.B., Vol. XXI, Issue 1, 2016, pp. 11 – 21
Received 14 November 2016; accepted 23 November 2016.
doi: 10.ANN/RSCB-2016-0017:RSCB
1.99 g of FeCl2 (0.05 M) were dissolved into
200 mL of distilled water. After stirring for 60
minutes, chemical precipitation was achieved
at 700 °C under vigorous stirring by adding
200 ml of NH3 solution (25 %) under
presence of Ar gas. The reaction system was
kept at 700 °C for 2 h and the pH of the
solution was maintained around 12. After the
system was cooled to room temperature, the
precipitates were separated by a permanent
magnet and washed three times with distilled
water until neutral pH. Finally, Fe3O4
magnetic nanoparticles are washed with
acetone and dried in oven at 60-70 °C.
The relevant chemical reaction can be
expressed as follows:
Fe2+ + 2Fe3+ + 8OH- →Fe3O4 + 4H2O
The as prepared magnetic nanoparticles
were covered in a second reaction step with
polyethylene glycol (PEG) in order to obtain a
uniform biocompatible shell on the magnetic
nanoparticles’ surfaces. The polyethylene
coated
magnetic
nanoparticles
were
synthesized in distilled water by dissolving 20
g (10 wt. %) of PEG and 1 wt. % of magnetic
nanoparticles and the reaction mixture was
left to react at room temperature, under
vigorous magnetic stirring overnight. The
product was magnetically separated and
washed 3 times with distilled water and redispersed in water (10 ml). Stable colloidal
suspensions of magnetic nanoparticles coated
with polymer in water have been obtained.
For the synthesis of 50 nm magnetic
SPIONs, magnetic clusters were prepared
using the oil in water mini-emulsion method
(Craciunescu et al., 2016). Toluene based
ferrofluid (0.5 wt% Fe3O4) was added to an
aqueous solution containing the surfactant
(1.795 g). The presence of PEG molecules
resulted in the formation of micelles, where
the
surfactant
molecules
organized
themselves with the polar end in the water
phase and the non-polar end in the oil phase.
The as created droplets contained the
magnetic nanoparticles dispersed in toluene.
To obtain a stable mini-emulsion, the twophase mixture was homogenized using an
ultrasonic finger U.P. 400S, for 2 minutes. In
the second step the organic phase, toluene,
was evaporated under magnetic stirring (500
rpm), at 1000 C in an oil bath. The magnetic
clusters were subsequently washed with
methanol-water mixture (50 ml) to remove
any excess of reactants and then dispersed in
distilled water.
A schematic representation of the multisteps synthesis procedure of magnetic clusters
coated with PEG is presented in Fig. 1. Stable
colloidal suspensions of magnetic clusters
coated with polymer in water have been
obtained.
Fig. 1. Synthetic route to prepare magnetic clusters
coated with PEG (adapted from Craciunescu et al.,
2016; with permission).
Nanoparticles characterization by TEM
and EDS. Naked and PEG-coated SPIONs
were applied onto carbon coated 300 mesh
copper grids. Images were taken on a Hitachi
STEM HD-2700 electron microscope at
200kV acceleration voltage, using an
Enfinium camera from Gatan. 100 particles
were measured to determine the mean
distribution of nanoparticles.
Hyperthermia induction method. The
Resistor-Inductor-Capacitor (RLC) circuit is
powered by a sinusoidal signal obtained from
an Arbitrary Waveform Generator type
WW2571A and a custom-made power wide
band amplifier with following features:
frequency range 100kHz-100MHz, power
range RF 1-200W.
The cell culture plates were placed in to
the center of the alternating magnetic field for
20 min, at 100 Oe and 1,5MHz, and then
returned to the incubator. The cell media was
replaced with new one to insure the removal
The Romanian Society for Cell Biology ©, Annals of R. S. C. B., Vol. XXI, Issue 1, 2016, Maria Suciu, pp. 11 – 21
12
Annals of R.S.C.B., Vol. XXI, Issue 1, 2016, pp. 11 – 21
Received 14 November 2016; accepted 23 November 2016.
doi: 10.ANN/RSCB-2016-0017:RSCB
of excess nanoparticles that would otherwise
be sitting on the cell culture layer.
Cell culture. We used a normal human
keratinocyte cell line (HaCaT), which was a
gift from dr. Alina Sesarman, ICEI ClujNapoca, and human melanoma cell line A375
from ATCC. HaCaT cells were cultured on
plastic 25 cm2 dishes in DMEM
supplemented with 10% fetal calf serum, 1%
penicillin-streptomycin and 1% L-glutamine.
A375 cells were cultured according to the
producer recommendations in DMEM
supplemented to 4.5 g/l glucose, 10% fetal
calf serum, 1% penicillin-streptomycin and
1% L-glutamine. Cells were kept in a
humidified incubator at 37 ºC and in a 5%
CO2 atmosphere. When reaching 80%
confluency, cells were detached from the
culture plate using trypsin and were plated to
the required culture surfaces.
10 μl of naked or PEG-coated SPIONs was
added to the cell media in final concentrations
ranging from 0.1 to 500 μg/ml. At 24 and 48
hours after the SPIONs treatment, cells were
analyzed by the methods described below.
MTT method. Cells were plated in 96
wells plate, using 12*103 cell/well, and left to
grow for 24 hours. SPION's were added to the
culture media, and 24 hours later the
mitochondrial activity was assessed by 3-(4,5Dimethylthiazol-2-yl)-2,5Diphenyltetrazolium Bromide (MTT) method.
For hyperthermia analysis, SPIONs were left
in contact with the cells for 24 hours, then the
cell media was replaced with fresh one and
the cells were placed in to the magnetic field.
After 24 hours, the cells were analyzed by
MTT. Briefly, the MTT compound was added
to each well in a final concentration of 0.5
mg/ml. Cells were returned to the incubator
for 1.5 hours, after which the cell media was
removed and the cells were lysed in acidified
iso-propanol. The absorbance of MTT was
read at 550 nm and the background at 630 nm
using BioTek Synergy HT plate reader and
Gen5 Plate Reader Program (Riss et al.,
2016). Each concentration was tested five
times and each plate contained untreated cells
as positive control, vehicle controls, and
negative controls (cells treated with Tween 20
2%). Data refers to mean ± standard error
from at least three independent experiments.
Comparison between groups was performed
with student’s t-test and values of p<0.05
were considered significant; all calculations
were realized in Microsoft Excel.
LDH method. From the cell cultures plated
for MTT, 50 μl of culture media was
subtracted for the LDH analysis. The
following components of the LDH test were
added to a new 96 wells plate: 50 μl cell
media, 50 μl 50 mM lithium lactate solution,
50 μl 200 mM tris solution at pH 8, and 50 μl
NAD
solution
(a
mixture
of
ionitrotetrazolium
violet,
phenazine
metosulphate, and nicotinamide dinucleotide).
The absorbance of LDH was read at 490 nm
and the background at 690 nm using BioTek
Synergy HT plate reader and Gen5 Plate
Reader Program (Chan, Moriwaki and De
Rosa, 2013). Each concentration was tested
five times and each plate contained untreated
cells as positive control, vehicle controls and
negative controls (cells treated with Tween 20
2%). Data refers to mean ± standard error
from at least three independent experiments.
Comparison between groups was performed
with student’s t-test and values of p<0.05
were considered significant; all calculations
were realized in Microsoft Excel.
Nanoparticle uptake analysis by TEM.
Cells were plated on 6 mm glass cover slips
in a 12 wells plate. 100 μg/ml SPIONs treated
cells and hyperthermia treated cells were
prepared for TEM analysis as follows: cells
were fixed with 2.7% glutaraldehyde and
post-fixed with 1% osmium tetroxide,
dehydrated in increasing concentrations of
ethanol and embedded in Epon resin. Samples
were polymerized at 60 ºC until hard.
Samples were trimmed and sectioned into
ultrathin 50 nm sections using a Diatome
diamond knife on the Leica UC6
ultramicrotome. Sections were recovered on a
carbon coated 200 mesh copper grids, stained
with uranyl acetate and lead citrate, and
The Romanian Society for Cell Biology ©, Annals of R. S. C. B., Vol. XXI, Issue 1, 2016, Maria Suciu, pp. 11 – 21
13
Annals of R.S.C.B., Vol. XXI, Issue 1, 2016, pp. 11 – 21
Received 14 November 2016; accepted 23 November 2016.
doi: 10.ANN/RSCB-2016-0017:RSCB
analyzed in a TEM Jeol type JEM 1010 with
MegaView II CCD Camera.
other hand, the hysteresis losses can be
estimated as it has been proposed by Yamada
et al. (2006), where heat capacity Q generated
by magnetite can be calculated by the formula
Q= km f Dw B2 [W/ml],
where:
km= 2.4-10-3[W/Hz/(mgFe/ml)/T2/ml],
f – exciting frequency of applied field [Hz],
B - external magnetic field [T],
Dw - weight density of magnetic fluid [mg
Fe/ml].
As for relaxation mechanisms of
ferrofluids there are two physical processes
responsible for the power dissipation: Néel
and Brownian relaxations (Rosenweig, 2002).
Néel relaxation is connected with the
fluctuation of magnetic moment direction
across an anisotropy barrier and the
characteristic
relaxation
time
τNa
nanoparticle system is given by the ratio of
the anisotropy energy KV to the thermal
energy kT as follows: τN=τoexp[KV/(kT)],
where (τo≈ 10-9s).
In the case of the second relaxation
mechanism, the time associated with the
rotation diffusion is the Brownian relaxation
time: τB=4𝝅ηrh3, where rh3is the
hydrodynamic radius which due to particle
coating may be essentially larger than the
radius of the magnetic particle core.
But there are limits of the current
approaches in the context of the state of the
art in the field. The alternating magnetic field
does not only damage cancerous tissue but
also causes an unwanted non-selective heating
of healthy tissue due to eddy currents.
Atkinson, Brezovich, and Chakraborty (1984)
proposed more than 20 years ago, based on
patient discomfort, that for a loop diameter of
about 30 cm the maximum limit on the
product H×f=4.85×108Am−1Hz. In this
product, H is the magnetic field in A/m and f
is the frequency in Hz. Their test was based
on the patient withstanding the treatment for
more than one hour without any major
discomfort.
The power density in the cancer layer is
about 8000 times higher with magnetic fluid
Results and discussions
Nanoparticles characterization by TEM and
EDS
The combined TEM and EDS analyses
confirmed the presence of naked and PEGcoated iron oxide clusters with diameters with
a mean distribution of 53±14.6nm (naked
SPIONs) and 56 ±18.15 nm (PEG-coated
SPIONs) respectively. Naked SPIONs have a
spherical appearance and the 10 nm units of
nanoparticles can be clearly distinguished.
PEG-coated SPIONs, also form a shell of
PEG that is visible in TEM images.
Fig. 2. Naked SPIONs form stable spherical clusters
with a median distribution dimension of 53±14.6 nm
(A); PEG-coated SPIONs with a median distribution
dimension of 56 ±18.15 nm (B); C - EDS analysis of
naked SPIONs; D - EDS analysis of Peg-coated
SPIONs.
Hyperthermia induction method
Magnetic losses in an alternating magnetic
field responsible for power dissipation e.g. to
be utilized for heating arise from hysteresis
and/or Néel or Browian relaxation. Hysteresis
losses may be determined by integrating the
area of hysteresis loops, a measure of energy
dissipated per cycle of magnetization. It
depends on the field amplitude, the magnetic
prehistory as well as the magnetic particle
size domain (Herget et al., 2006). On the
The Romanian Society for Cell Biology ©, Annals of R. S. C. B., Vol. XXI, Issue 1, 2016, Maria Suciu, pp. 11 – 21
14
Annals of R.S.C.B., Vol. XXI, Issue 1, 2016, pp. 11 – 21
Received 14 November 2016; accepted 23 November 2016.
doi: 10.ANN/RSCB-2016-0017:RSCB
than without it. That means that eddy
currents effects are completely negligible
regarding the heating of the injected magnetic
fluid (Fanin, 2002).
To test the effects of 50 nm SPIONs on
normal and cancerous cells we employed the
MTT and LDH release methods and electron
microscopy analyses.
Mitochondrial activity assay by MTT
method.
We used HaCaT cells due to the close
phenotype of this cell line to the keratinocytes
in the human skin (Boukamp et al., 1988) and
A375 cells due to the aggressiveness of these
cells that mimic the real behavior of the
melanoma when it reaches stage 3 of tumor
development and starts to migrate to lymph
nodes (Riker et al., 2008).
According to Garcia-Lopez et al. (2014)
following the ISO 10993-5, using the MTT
cell assay to test the viability of cells we can
interpret the results as follows: above 80% no
cytotoxicity,
within
80%–60%
weak
cytotoxicity,
60%–40%
moderate
cytotoxicity, below 40% strong cytotoxicity.
In the light of these findings, our MTT
analyses results indicate that naked and PEGcoated SPIONs affect the viability of both cell
types but in different ways and hyperthermia
determines different results depending on cell
type and the time point of the analysis.
In the case of naked SPIONs melanoma
cells are slightly more affected than normal
keratinocytes at all tested concentrations at 24
hours. Both cell types position themselves in
the 60-80% mitochondrial activity, which
means that naked SPIONs induce a weak
toxicity in melanoma and keratinocytes after
24 hours of exposure (Fig. 3A). After 48
hours exposure keratinocytes have an above
80% mitochondrial activity at all tested
concentrations, which is equivalent to no
cytotoxicity. Melanoma cells remain in the
weak cytotoxicity range even at 48 hours of
contact with the naked SPIONs (Fig. 3B).
Fig.3. A - Normal keratinocytes and melanoma
exposed for 24 hours to 50 nm naked SPIONs; B Normal
keratinocytes and melanoma exposed for 48 hours
to 50 nm naked SPIONs; C - Normal keratinocytes and
melanoma exposed for 24 hours to 50 nm naked
SPIONs, then treated by hyperthermia, MTT analysis
at 24 hours post-hyperthermia. Dotted line represents
IC50 for mitochondrial activity; D - Normal
keratinocytes and melanoma exposed for 24 hours to
50 nm naked SPIONs, then treated by hyperthermia.
MTT analysis at 48 hours post-hyperthermia; E Normal keratinocytes and melanoma exposed for 24
hours to 50 nm PEG-coated SPIONs; F - Normal
keratinocytes and melanoma exposed for 48 hours to
50 nm PEG-coated SPIONs; G - Normal keratinocytes
and melanoma exposed for 24 hours to 50 nm naked
SPIONs, then treated by hyperthermia, MTT analysis
at 24 hours post-hyperthermia; H - Normal
keratinocytes and melanoma exposed for 24 hours to
50 nm naked SPIONs, then treated by hyperthermia,
MTT analysis at 48 hours post-hyperthermia (*
indicate statistical significance, p≤0.05).
After 24 hours of exposure to SPIONs (to
ensure nanoparticle endocytosis - Calero et
al., 2015; Osman et al., 2012) cells were
placed in an alternating magnetic field, then
left to recover for another 24 or 48 hours in
the incubator. To ensure that the effects of
hyperthermia will not be determined by
SPIONs that are not in direct contact with the
cells, and to offer fresh nutrients for the next
48 hours, the cell media was changed two
The Romanian Society for Cell Biology ©, Annals of R. S. C. B., Vol. XXI, Issue 1, 2016, Maria Suciu, pp. 11 – 21
15
Annals of R.S.C.B., Vol. XXI, Issue 1, 2016, pp. 11 – 21
Received 14 November 2016; accepted 23 November 2016.
doi: 10.ANN/RSCB-2016-0017:RSCB
hours before the hyperthermia treatment. 24
hours after the hyperthermia treatment with
the naked SPIONs the most affected were the
normal keratinocytes which reached the 50%
drop in mitochondrial activity at all tested
concentrations after the hyperthermia
treatment.
mean at this point that almost all cells died or
that these concentrations and time point
determines a loss of cell adhesion proteins
that leads to failure of cells to attach to
substrates. This can determine loss of cell
number during the steps of the MTT assay.
Melanoma cells had a statistically relevant
reduced mitochondrial activity at all tested
concentrations, with a dose-dependent
response (Fig. 3C). At 0.1 and 1 μg/ml naked
SPIONs concentrations, melanoma cells can
be included in the no cytotoxicity range,
dropping in the mild cytotoxicity range for the
remaining concentrations tested (10-500
μg/ml naked SPIONs).
At 48 hours after the hyperthermia
treatment keratinocytes recover close to
normal values, placing themselves in the no
cytotoxicity percent range, but melanoma
cells present a dose-dependent drop
in mitochondrial activity in the weak
cytotoxicity
range,
reaching
60%
mitochondrial activity at the 500 μg/ml naked
SPIONs concentration (Fig. 3D).
When using PEG-coated SPIONs,
keratinocytes presented an inverse response to
the
tested
concentrations,
increasing
mitochondrial activity with the increased
concentrations, reaching 110% activity at the
100 μg/ml concentration. Melanoma cells
were kept around the 80% mitochondrial
activity, in the no cytotoxicity to weak
cytotoxicity ranges, at all concentrations after
24 hours of exposure to PEG-coated SPIONs
(Fig. 3E).
After 48 hours of exposure to PEG-coated
SPIONs, keratinocytes had around 80%
mitochondrial activity, throughout the tested
concentrations, in the no cytotoxicity - weak
cytotoxicity range. Melanoma cells had a dose
response drop in their mitochondrial activity,
with values between 50-80% reflecting a
moderate to weak cytotoxicity to PEG-coated
SPIONs (Fig. 3F).
If hyperthermia was applied, both cell
types expressed a mild cytotoxicity,
independent of concentrations, with a
decreased mitochondrial activity holding
Fig.4. A - Normal keratinocytes and melanoma
exposed for 24 hours to 50 nm naked SPIONs; B –
Normal keratinocytes and melanoma exposed for 48
hours to 50 nm naked SPIONs; C - Normal
keratinocytes and
melanoma exposed for 24 hours to 50 nm naked
SPIONs, then treated by hyperthermia, LDH analysis
at 24 hours post-hyperthermia; D - Normal
keratinocytes and melanoma exposed for 24 hours to
50 nm naked SPIONs, then treated by hyperthermia,
LDH analysis at 48 hours post-hyperthermia; E –
Normal keratinocytes and melanoma exposed for 24
hours to 50 nm PEG-coated SPIONs; F - Normal
keratinocytes and melanoma exposed for 48 hours to
50 nm PEG-coated SPIONs; G - Normal keratinocytes
and melanoma exposed for 24 hours to 50 nm PEGcoated SPIONs, then treated by hyperthermia, LDH
analysis at 24 hours post-hyperthermia; H - Normal
keratinocytes and melanoma exposed for 24 hours to
50 nm PEG-coated
SPIONs, then treated by hyperthermia, LDH analysis
at 48 hours post-hyperthermia (*indicate statistical
significance, p≤0.05).
At 100 and 500 μg/ml keratinocytes present
almost no mitochondrial activity, which can
The Romanian Society for Cell Biology ©, Annals of R. S. C. B., Vol. XXI, Issue 1, 2016, Maria Suciu, pp. 11 – 21
16
Annals of R.S.C.B., Vol. XXI, Issue 1, 2016, pp. 11 – 21
Received 14 November 2016; accepted 23 November 2016.
doi: 10.ANN/RSCB-2016-0017:RSCB
around 70% at the 24 hours' time point after
hyperthermia (Fig. 3G), and no more
cytotoxic effects at 48 hours after the
treatment (Fig. 3H).
Membrane integrity assay by LDH
Lactate dehydrogenizes release in cell media
is an indicator of cells’ status and, depending
on the calculation formulae used, one can
determine the extent of growth inhibition state
or necrosis (Smith et al., 2011).
In our case, when normal keratinocytes and
melanoma were exposed for 24 hours to 50
nm naked SPIONs, melanoma cells released a
more LDH than keratinocytes at all tested
concentrations at 24 hours, in a dose
dependent manner, but all values remained
close to normal LDH release (Fig. 4A). After
48 hours exposure both keratinocytes and
melanoma cells had a dose response LDH
release in the cell media, which was
significantly higher from normal LDH
release. After 48 hours of contact with the
naked SPIONs, melanoma cells suffered from
more membrane rupture and released more
LDH than normal keratinocytes and entered
an inhibition state (Fig. 4B). The
hyperthermia treatment affected both
keratinocytes and melanoma at 24 hours' time
point, irrespective of the SPIONs dose, but
melanoma cells were more affected, releasing
20% of the LDH released by the Tween 20%
treated cells, suggesting cytotoxicity (Fig.
4C). At 48 hours after the hyperthermia
treatment, keratinocytes returned to normal
LDH release, showing an increased LDH
release only at the 500 μg/ml concentration.
Melanoma cells kept the LDH release above
20% at all tested concentrations, having an
increased LDH than at the 24 hours' time
point, suggesting an increased cytotoxicity
(Fig. 4D).
When normal keratinocytes and melanoma
where exposed for 24 hours to 50 nm PEGcoated SPIONs, keratinocytes gave a lower
than normal LDH release in the presence of
PEG-coated SPIONs. Melanoma cells have an
increased LDH release in the cell medium of
up to 10 - 20%, reaching 30% at 500 μg/ml
concentration, an indication of mild
cytotoxicity (Fig. 4E). At 48 hours' time
point, both keratinocytes and melanoma cells
had a dose-dependent response to PEG-coated
SPIONs. Keratinocytes had a 15% LDH
release at the 50 μg/ml concentration reaching
20% LDH release at the highest concentration
tested (500 μg/ml), indicating inhibition of
growth. Melanoma cells also reported a dosedependent response, but at higher levels,
starting from 25% at the lowest concentration
(0.1 μg/ml) to 40% at the highest
concentration (Fig. 4F). Hyperthermia
treatment with PEG-coated SPIONs, has lead
keratinocytes to display an inverse dosedependent reaction at the 24 hours' time point,
in the range of growth inhibition. Melanoma
cells had a higher than normal LDH release
(20-25%) but not much different from the
levels obtained at 48 hours of contact with the
PEG-coated SPIONs (Fig. 4G). At 48 hours
after the hyperthermia treatment both cell
types released 20-25% LDH in the cell media
independent
of
the
nanoparticles
concentrations, indicating mild cytotoxicity
(Fig. 4H).
Fig. 5. A - Normal keratinocytes, control (bar = 1 um);
B - Normal keratinocytes exposed for 24 hours to 50
nm naked SPIONs (bar = 2 um); C - Normal
keratinocytes exposed for 24 hours to 50 nm naked
The Romanian Society for Cell Biology ©, Annals of R. S. C. B., Vol. XXI, Issue 1, 2016, Maria Suciu, pp. 11 – 21
17
Annals of R.S.C.B., Vol. XXI, Issue 1, 2016, pp. 11 – 21
Received 14 November 2016; accepted 23 November 2016.
doi: 10.ANN/RSCB-2016-0017:RSCB
SPIONs, then treated by hyperthermia, analysis at 24
hours post-hyperthermia (bar = 2 um); D – Normal
keratinocytes exposed for 24 hours to 50 nm PEGcoated SPIONs (bar = 500 nm); E - Normal
keratinocytes exposed for 24 hours to 50 nm PEGcoated SPIONs, then treated by hyperthermia, analysis
at 24 hours post-hyperthermia (bar = 2 um; N-nucleus,
m-mitochondrion, Ly-lysosome, black arrow indicate
nanoparticles’ presence).
size used (Kjellman et al., 2015), for our
study we chose the 100 μg/ml 50 nm SPIONs
in order to be sure that most cells will contain
SPIONs, and that their effect will be visible at
a morphological scale. It was shown that
keratinocytes can load up with iron oxides in
concentrations of up to 33 pg Fe/cell (Ito et
al., 2004).
50 nm naked SPIONs applied on cells
seem to have lost their round cluster form and
keratinocytes have engulfed large quantities
of nanoparticles. Naked SPIONs were found
inside lysosomes, apparently with the
crystalline form lost. Many singular
nanoparticles could be seen attached to the
cell membrane, decorating it along its surface
(Fig. 5B). Hyperthermia affected the
keratinocytes by inducing a massive cell lysis:
the cytoplasm has lacked electron dense
material, and mitochondria had rarefied
content, and the nucleus seem to have lost
content as well (Fig. 5C).
In keratinocytes PEG-coated SPIONs
could be found in clusters in the cytoplasm
without any membrane enclosure, near-by the
nucleus but not inside it. Cells appeared to
have no morphological changes after 24 hours
of exposure to nanoparticles (Fig. 5D). After
the hyperthermia treatment with the PEGcoated SPIONs, nanoparticles can be found in
lysosomes, with the iron oxides degraded to
hemosiderin deposits (Oiu, Wang and Mao,
2014; Jendelova et al., 2003) or outside the
cells, in the membrane proximity. Still, cells
have normal morphology with large nuclei,
with thin heterochromatin disposed at the
nucleus periphery. Long shaped mitochondria
can be seen in the cytoplasm, but few
endoplasmic reticulum and ribosomes,
suggesting a loss of protein synthesis (Fig.
5E).
In the case of melanoma cells, 50 nm naked
SPIONs induced a foam-like appearance of
the cells full of lysosomal vesicles. SPIONs
were found in lysosomes and also membrane
free in the cytoplasm, but the cells cytoplasm
was very electron-dense, suggesting high
protein content (Fig. 6B). At the 24 hours'
Prodan et al. (2013) using 10 nm iron
oxides found similar responses to HeLa cells:
the toxic effect was enhanced with increased
concentration and time. In another
experiment, using 9 nm iron oxide
nanoparticles covered in Pluronic, a highly
toxicity response was obtained in macrophage
RAW 264.7 cell line (Gonzales et al., 2010).
Figure 4. A - Normal keratinocytes and
melanoma exposed for 24 hours to 50 nm
naked SPIONs; B - Normal keratinocytes and
melanoma exposed for 48 hours to 50 nm
naked SPIONs; C - Normal keratinocytes and
melanoma exposed for 24 hours to 50 nm
naked SPIONs, then treated by hyperthermia,
LDH analysis at 24 hours post-hyperthermia;
D - Normal keratinocytes and melanoma
exposed for 24 hours to 50 nm naked
SPIONs, then treated by hyperthermia, LDH
analysis at 48 hours post-hyperthermia; E Normal keratinocytes and melanoma exposed
for 24 hours to 50 nm PEG-coated SPIONs; F
- Normal keratinocytes and melanoma
exposed for 48 hours to 50 nm PEG-coated
SPIONs; G - Normal keratinocytes and
melanoma exposed for 24 hours to 50 nm
PEG-coated SPIONs, then treated by
hyperthermia, LDH analysis at 24 hours posthyperthermia; H - Normal keratinocytes and
melanoma exposed for 24 hours to 50 nm
PEG-coated SPIONs, then treated by
hyperthermia, LDH analysis at 48 hours posthyperthermia
(*indicate
statistical
significance, p≤0.05).
Nanoparticle uptake analysis by TEM.
Cells can phagocytose nanoparticles starting
from the smallest possibly synthesized to
micron sized nanoparticles (Shang, Nienhaus
and Nienhaus, 2014). A good SPIONs
endocytosis depends on concentrations and
The Romanian Society for Cell Biology ©, Annals of R. S. C. B., Vol. XXI, Issue 1, 2016, Maria Suciu, pp. 11 – 21
18
Annals of R.S.C.B., Vol. XXI, Issue 1, 2016, pp. 11 – 21
Received 14 November 2016; accepted 23 November 2016.
doi: 10.ANN/RSCB-2016-0017:RSCB
time point after hyperthermia, naked SPIONs
were found membrane free in the cytoplasm
or in lysosomes, and also degraded in
hemosiderin-like lysosomes. The cytoplasm
has lost the electron-dense appearance,
becoming very electron-transparent, with
many lysosomes. Also, the nuclei had very
thin heterochromatin on the margins of the
nucleus membrane (Fig. 6C).
cytoplasm, but the entire cytoplasm was
loaded with late endosomes, lysosomes, and
dyeing mitochondria, and was electrontransparent (Fig. 6E). According to Hsieh et
al.
(2015)
PEG-coated
iron
oxide
nanoparticles tend to escape lysosomes and
enter mitochondria where they produce
reactive oxygen species, inducing apoptosis.
It seems that iron oxide nanoparticles are
considered inert by the mitochondria and so,
are no longer regulated, and accumulate in the
mitochondrial lumen. This happens until the
loading impairs the normal activity of the
mitochondria and reactive oxygen species are
produced (Dancis and Lindhal, 2013).
Conclusions
The naked SPIONs treatment on melanoma and
keratinocyte cells affected the mitochondrial
activity of both cell types, but differently:
keratinocytes were not affected by the naked
SPIONs except at 24 hours after the
hyperthermia treatment; melanoma cells had a
mild cytotoxic response at all concentrations
and most times points. Melanoma cells were
most affected at 48 hours after the hyperthermia
treatment. Morphologically, SPIONs induce a
general lysis in melanoma cells mitochondria,
probably developing into slow progression
apoptosis.
Keratinocytes seem to react more rapidly to
nanoparticles and hyperthermia, but afterwards
they recover faster. Melanoma cells have a
delayed reaction which can only be seen at a
larger time frame, but they are affected at a
higher extent than the normal cells. It is
possible that the PEG coating affects the hyperthermic properties of the nanoparticles,
absorbing most of the local heat energy, that
would be otherwise released to the cell
components by the naked SPIONs, or that the
frequency or time of treatment used for PEGcoated SPIONs must be adapted for enhanced
responses
Fig. 6. A - Melanoma cells, control (bar = 1 um); B –
Melanoma cells exposed for 24 hours to 50 nm naked
SPIONs (bar = 2 um); C - Melanoma cells exposed for
24 hours to 50 nm naked SPIONs, then treated by
hyperthermia, analysis at 24 hours post-hyperthermia
(bar = 2 um); D - Melanoma cells exposed for 24 hours
to 50 nm PEG-coated SPIONs (bar = 1 um); E Melanoma cells exposed for 24 hours to 50 nm
PEGcoated SPIONs, then treated by hyperthermia,
analysis at 24 hours post-hyperthermia (bar = 1 um,
Nnucleus, m-mitochondrion, Ly-lysosome, black arrow
indicate nanoparticles’ presence).
When PEG-coated SPIONs were applied
to melanoma cell culture, they kept their
cluster form, and could be found inside the
cells freely in the cytoplasm, or outside the
cells, but drawn in clusters. The cytoplasm
was electron-dense and contained many
vesicles and lysosomes (Fig. 6D). After
hyperthermia PEG-coated SPION clusters
were located in lysosomes or free in the
Acknowledgments
This work was supported by the Romanian
National Authority for Scientific Research and
The Romanian Society for Cell Biology ©, Annals of R. S. C. B., Vol. XXI, Issue 1, 2016, Maria Suciu, pp. 11 – 21
19
Annals of R.S.C.B., Vol. XXI, Issue 1, 2016, pp. 11 – 21
Received 14 November 2016; accepted 23 November 2016.
doi: 10.ANN/RSCB-2016-0017:RSCB
cancer/treatment/types/surgery/hyperthermia-factsheet
ISO 10993-5:2009 Biological Evaluation of Medical
Devices. Part 5: Tests for In Vitro Cytotoxicity.
International Organization for Standardization;
Geneva, Switzerland, 2009.
Ito, A., Hayashida, M., Honda, H., Hata, K., Kagami,
H., Ueda, M., Kobayashi, T.: Construction and
harvest of multilayered keratinocyte sheets using
magnetite nanoparticles and magnetic force. Tissue
Eng. 10(5-6):873-80, 2004
Jendelová, P., Herynek, V., DeCroos, J., Glogarová,
K., Andersson, B., Hájek, M., Syková, E.: Imaging
the fate of implanted bone marrow stromal cells
labeled with superparamagnetic nanoparticles,
Magn. Res.Med. 50(4):767–776, 2003
Kjellman, P., Fredriksson, S., Kjellman, C., Strand,
S.E., Zandt, R.: Size-dependent lymphatic uptake of
nanoscale-tailored particles as tumor mass
increases, Future Science OA, 1(4) FSO60, 2015
López-García, J., Lehocký, M., Humpolíček, P., Sáha,
P.:
HaCaT
Keratinocytes
Response
on
Antimicrobial Atelocollagen Substrates: Extent of
Cytotoxicity, Cell Viability and Proliferation.
Journal of Functional Biomaterials., 5(2):43-57,
2014
Osman, O., Zanini, L.F., Frénéa-Robin, M., DumasBouchiat, F., Dempsey, N.M., Reyne, G., Buret, F.,
Haddour, N.: Monitoring the endocytosis of
magnetic nanoparticles by cells using permanent
micro-flux
sources,
Biomed
Microdevices.
14(5):947-54, 2012
Prodan, A.M., Iconaru, S.L., Ciobanu, C.S., Chifiriuc,
M.C., Stoicea, M., Predoi, D.: Iron Oxide Magnetic
Nanoparticles: Characterization and Toxicity
Evaluation by In Vitro and In Vivo Assays, J
Nanomat. vol. 2013, ID 587021, 10 pgs., 2013
Qiu, P., Wang, L., Mao, C.B.: TEM characterization of
biological and inorganic nanocomposites in
Transmission
Electron
Microscopy
Characterization of Nanomaterials, Eds. Kumar
CSSR, Springer-Verlag, p: 1-41 2014,
Riker, A.I., Enkemann, S.A., Fodstad, O., Liu, S., Ren,
S., Morris, C., Xi, Y., Howell, P., Metge, B.,
Samant, R.S., Shevde, L.A., Li, W., Eschrich, S.,
Daud, A., Ju, J., Matta, J.: The gene
expression profiles of primary and metastatic
melanoma yields a transition point of tumor
progression and metastasis, BMC Med Genomics.
28;1:13, 2008 Riss, T.L., Moravec, R.A., Niles,
A.L., Duellman, S., Benink, H.B., Worzella, T.J.,
Minor, L.: Cell Viability Assays, in Assay
Guidance Manual [Internet], Sittampalam GS,
Coussens NP, Nelson H, et al., editors. Bethesda:
Eli Lilly & Company and the National Center for
Advancing Translational Sciences, 2016
Rosenweig, R.E.: Heating magnetic fluid with
alternating magnetic field, J.Magnetism Magnetic
Mat, 252:370-374, 2002
Shang, L., Nienhaus, K., Nienhaus, G.U.: Engineered
nanoparticles interacting with cells: size matters,
J.Nanobiotech.
12:5,
2014
Innovations,
CNCSIS-UEFISCDI
project
number PN2-RU-TE-2014-4-0608. We thank
dr. A. Sesarman for the HaCaT cells.
References
Atkinson, W.J., Brezovich,,I.A., Chakraborty, D.P.:
Usable frequencies in hyperthermia with thermal
seeds, IEEE Trans. Biomed. Eng. 31:70–75, 1984
Boukamp, P., Breitkreutz, D., Hornung, J., Markham,
A.: Normal keratinizationin a spontaneously
immortalized aneuploid keratinocyte cell line. J.
Cell Biol., 106:761–771, 1988,
Calero, M., Chiappi, M., Lazaro-Carrillo, A.,
Rodríguez, M.J., Chichón, F.J., Crosbie-Staunton,
K., Prina-Mello, A., Volkov, Y., Carrascosa, J.L.:
Characterization of interaction of magnetic
nanoparticles
with
breast
cancer
cells,
J.Nanobiotech. 13:16. 2015
Cancer
Facts
and
Figures,
2016,
http://www.cancer.org/acs/groups/content/@researc
h/documents/document/acspc-047079.pdf
Chan, F.K.-M., Moriwaki, K., De Rosa, M.J.,
Detection of Necrosis by Release of Lactate
Dehydrogenase (LDH) Activity. Methods in
molecular biology (Clifton, NJ). 979:65-70, 2013.
Craciunescu, I., Petran, A., Liebscher, J., Vekas, L.,
Turcu, R.: Synthesis and characterization of sizecontrolled magnetic clusters functionalized with
polymer layer for wastewater depollution, Materials
Chemistry
and
Physics,
in
press:http://dx.doi.org/10.1016/j.matchemphys.201
6.10.009, 2016
Dancis, A., Lindhal, P.A.: Mitochondrial iron
metabolism and the synthesis of iron-sulfur clusters
in Metals in Cells, Eds: Cullota V, Scott RA, John
Wiley and Sons, p: 473-487, 2013
Fannin, P.C.: Magnetic spectroscopy as an aide in
understanding magnetic fluids, J.Magnetism
Magnetic Mat., 252:59-64, 2002
Gonzales, M., Mitsumori, L.M., Kushleika, J.V.,
Rosenfeld, M.E., Krishnan, K.M.: Cytotoxicity of
iron oxide nanoparticles made from the thermal
decomposition of organometallics and aqueous
phase transfer with Pluronic F127. Contrast media
& molecular imaging. 5(5):286-293, 2010.
Herget. R., Dutz, S., Muller, R,. Zeisberger, M.:
Magnetic particle hyperthermia: nanoparticle
magnetism and materials development for cancer
therapy, Journal of Physics Condensed Matter,
8:S2919-S2934, 2006
Hsieh, H.C., Chen, C.M., Hsieh, W.Y., Chen, C.Y.,
Liu, C.C., Lin, F.H.: ROS-induced toxicity:
exposure of 3T3, RAW264.7, and MCF7 cells to
superparamagnetic iron oxide nanoparticles results
in cell death by mitochondria-dependent apoptosis,
Nanopart Res 17: 71, 2015
Hyperthermia in Cancer Treatment, 2016, National
Cancer Institute, National Institute of Health,
https://www.cancer.gov/about-
The Romanian Society for Cell Biology ©, Annals of R. S. C. B., Vol. XXI, Issue 1, 2016, Maria Suciu, pp. 11 – 21
20
Annals of R.S.C.B., Vol. XXI, Issue 1, 2016, pp. 11 – 21
Received 14 November 2016; accepted 23 November 2016.
doi: 10.ANN/RSCB-2016-0017:RSCB
Smith, S.M., Wunder, M.B., Norris, D.A., Shellman,
Y.G.: A Simple Protocol for Using a LDH-Based
Cytotoxicity Assay to Assess the Effects of Death
and Growth Inhibition at the Same Time. Roemer
K, ed. PLoS ONE. 6(11):e26908, 2011 Turcu, R.,
Socoliuc, V., Craciunescu, I., Petran, A., Paulus,
A., Franzreb, M., Vasile, E., Vekas, L.: Magnetic
microgels, a promising candidate for enhanced
magnetic adsorbent particles in bioseparation:
synthesis, physicochemical characterization, and
separation performance, Soft Matter 11:1008-1018,
2015
Yamada, S., Chomsuwan, K., Mukhopadhyay, S.C.,
Iwahara, M., Kakikawa, M., Nagano, I.: Detection
of Magnetic Fluid Volume Density with a GRM
Sensor, Journal of Magnetic Soc. Jpn. 31:44-47,
2007
The Romanian Society for Cell Biology ©, Annals of R. S. C. B., Vol. XXI, Issue 1, 2016, Maria Suciu, pp. 11 – 21
21