Article

www.acsnano.org

Duality of Iron Oxide Nanoparticles in Cancer

Therapy: Amplification of Heating Efficiency
by Magnetic Hyperthermia and Photothermal
Bimodal Treatment
Ana Espinosa,† Riccardo Di Corato,† Jelena Kolosnjaj-Tabi,† Patrice Flaud,† Teresa Pellegrino,‡
and Claire Wilhelm*,†
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†
Laboratoire Matière et Systèmes Complexes, UMR 7057, CNRS and University Paris Diderot, 75205 Paris Cedex 13, France
‡
Istituto Italiano di Tecnologia, I-16163 Genoa, Italy
Downloaded via UNIV FED DE GOIAS on April 14, 2021 at 17:51:27 (UTC).

*
S Supporting Information

ABSTRACT: The pursuit of innovative, multifunctional, more efficient, and safer

treatments is a major challenge in preclinical nanoparticle-mediated thermotherapeutic
research. Here, we report that iron oxide nanoparticles have the dual capacity to act as
both magnetic and photothermal agents. We further explore every key aspect of this
magnetophotothermal approach, choosing iron oxide nanocubes for their high efficiency
for the magnetic hyperthermia modality itself. In aqueous suspension, the nanocubes’
exposure to both: an alternating magnetic field and near-infrared laser irradiation
(808 nm), defined as the DUAL-mode, amplifies the heating effect 2- to 5-fold by com-
parison with magnetic stimulation alone, yielding unprecedented heating powers (specific
loss powers) up to 5000 W/g. In cancer cells, the laser excitation restores the optimal effi-
ciency of magnetic hyperthermia, otherwise inhibited by intracellular confinement, resulting
in a remarkable heating efficiency in the DUAL-mode (up to 15-fold amplification), with
respect to the magnetophotothermal mode. As a consequence, the dual action yielded
complete apoptosis-mediated cell death. In solid tumors in vivo, single-mode treatments (magnetic or laser hyperthermia)
reduced tumor growth, while DUAL-mode treatment resulted in complete tumor regression, mediated by heat-induced tumoral
cell apoptosis and massive denaturation of the collagen fibers, and a long-lasting thermal efficiency over repeated treatments.
KEYWORDS: magnetic hyperthermia, photothermia, iron oxide nanoparticles, nanomedicine, combined tumor therapy

clinics, mediated by metallic nanoparticles (gold,9−11 silver,12

T he development of nanostructures with combined

therapeutic features represents a potential preclinical
approach to fight cancer.1−3 The earliest representatives
of the nanoscale-based antitumoral thermotherapeutic probes
metallic4 and magnetic nanoparticles5are among the promis-
copper,13,14) or even semiconducting carbon nanotubes15 or
graphene,16 which could be activated by near-infrared (NIR) light,
where the absorption of tissues is minimal. Current research led to
promising results in cancer cell laser ablation, both in vitro,17,18 and
ing agents. Hyperthermic tumor therapy has traditionally been in vivo in animal models.15,16,19,20 On the other hand, magnetic
applied to the whole body or to superficial tissues, with cancer iron oxide nanoparticles were even studied in humans after direct
cells being more heat-sensitive than normal tissues.6 Radiation injection of nanoparticles into solid tumors for magnetic hyper-
beams and remotely activated implanted probes have been thermia treatment of glioblastoma21 and prostate carcinoma.22
developed, but the experimental nanoparticles-mediated hyper-
However, both magnetic and photothermal approaches still
thermia seems to have some advantages because heat-generating
suffer from certain disadvantages. Some of these drawbacks
nanoparticles could be prepared in colloidal suspensions, which
could be injected and could avoid the need for macroscopic include the facts that high doses of laser irradiation can damage
implants. In addition, their biodistribution could potentially be bystander tissues and that some of the nanoparticles (namely,
controlled by different targeting strategies, and the particles could carbon nanotubes or gold nanoparticles) might be biopersistant
be made small enough to cross biological barriers and generate and potentially toxic.23 In contrast, iron oxide nanoparticles have
heat very close to the target, be it a tissue, cell, or organelle.7,8
Nanoheating agents can be activated by either light or Received: November 17, 2015
magnetism. Photothermal therapy (PTT) is an experimental Accepted: January 14, 2016
cancer treatment that might one day eventually find its way to Published: January 14, 2016

© 2016 American Chemical Society 2436 DOI: 10.1021/acsnano.5b07249

ACS Nano 2016, 10, 2436−2446
ACS Nano Article

already been approved for human use as magnetic resonance absorbance in the NIR region, iron oxide nanoparticles can still
imaging (MRI) contrast agents.24 They also have excellent compete with plasmonic nanoparticles in the same region. For
biodegradibility in vivo, and the iron ions they release upon instance, it was reported that gold nanoshells and magnetite
dissolution can be assimilated by the body through a tightly nanomaterials can exhibit a similar absorbance at 808 nm at the
regulated physiological process.25,26 same mass concentration.35 Here, the absorbance of iron oxide
Nevertheless, in the case of magnetic hyperthermia nanocubes at 808 nm is still effective and increases linearly with
applications, to date, the need for high nanoparticle concen- the concentration (below [Fe] = 25 mM) (Figure 1B), before
trations ([Fe] = 1−2 M, which is several orders of magnitude saturating at a higher concentration (with absorbance values over 3
higher than the concentrations used for MRI27) remains a major (99.99% absorbed) for 100 mM). These measurements were
disadvantage in their clinical use. Efforts are therefore being made performed in cuvettes of 2 mm optical path-length.
to optimize the heating efficiency of iron oxide nanoparticles. Nanocubes’ photothermal and magnetothermal effects were
Recently, iron oxide-based nanomaterials, such as nano- thus first tested in aqueous suspensions as a function of Fe
cubes,28,29 core−shell nanostructures,30 and multicore nano- concentrations, ranging from 0.75 to 250 mM. Three heating
particles,31 were reported to generate unprecedented magnetic protocols were selected: (i) photothermia alone with an 808 nm
heating, with specific loss powers (SLP, or heat dissipation per laser with powers ranging from 0.3 to 2.5 W/cm2; (ii) magnetic
unit mass of magnetic material) in the range of 1000 W/g, which hyperthermia alone (MHT), at frequencies of 110, 320, 520, 700,
is 5−10 times higher than all previous iron oxide nanoparticles and 900 kHz with a magnetic field amplitude in the range of
developed for magnetic hyperthermia. Unfortunately, with these 12−25 mT; and (iii) combined hyperthermia (DUAL:
high SLP values in the 1000 W/g range, heating performance simultaneous application of both MHT and LASER). The laser
seems to have reached its limit. In addition, high heating power in was adapted to the magnetic coil (Figure 1C), and reliable
aqueous suspensions may not translate into efficient heating in temperature measurements were obtained with a high-resolution
the cellular environment.32,33 infrared (IR) camera calibrated with a fluorescent temperature
Very recently, iron oxide nanoparticles were also tested for
probe (calibration shown in supplementary Figure S1). Samples
photothermal therapy in vitro and in vivo.34−39 However, in
were placed within the coil (maintained at 37 °C), and the
previous studies, intense irradiation was used (in the range
temperature increase was recorded at the bottom of the sample.
2−5 W/cm2), exceeding by far the safe limit for cutaneous tissues
Figure 1D shows IR images and temperature profiles obtained
(0.33 W/cm2 for a 808 nm laser).40
with each protocol, at [Fe] = 12 and 25 mM, with 25 mT
Here we demonstrate that simultaneous stimulation of iron
oxide nanocubes with both an alternating magnetic field and magnetic field at 520 kHz frequency and 0.3 W/cm2 laser power.
NIR laser irradiation provides unrivaled heating efficiency The temperature increase after 5 min obtained with the DUAL
at compatible clinical doses with a low iron concentration modality (17 and 26 °C at 12 and 25 mM, respectively) matched
(0.25 M) and with acceptable irradiation (0.3 W/cm2), thus the sum of the increases obtained with the two individual
overcoming the main drawbacks of stand-alone iron oxide modalities (9 and 10 °C for LASER only; 8 and 16 °C for MHT
nanoparticle-mediated magnetic hyperthermia or photothermia. only, at 12 and 25 mM, respectively). The heating induced by
This dual heating effect was tested in environments of increasing MHT increased linearly with the nanocube concentration
biological complexity, from aqueous suspensions to tumor cells (Figure 1E). In the experimental setup, the optical path-length
in vitro and solid tumors in vivo. In all these situations the two of the laser is equal to the height of the sample (∼1 cm; see
heating effects were cumulative, if not synergistic. In suspension Figure 1C). In this configuration, the temperature elevation
and in cancer cells in vitro, nanocube heating power reached induced by the LASER increased with Fe content at low
unprecedented levels up to 5000 W/g. While each heating concentrations ([Fe] = 0.75−25 mM, Figure 1F), in agreement
modality alone yielded a moderate cytotoxicity compromising with the optical absorbance at 808 nm shown in Figure 1B and
partially cancer cell viability, only the combination of magnetic also reported elsewhere,34−36 before initiating saturation over
hyperthermia and photothermia was sufficient to ensure 25 mM concentration (Figure S2). This effect with concen-
complete cancer cell death. Triggered apoptosis was confirmed tration is due to the rapid increase of the attenuation coefficient
by proteomic analysis of apoptotic effectors. Finally, the dual at high concentration: from 1.7 cm−1 (at 12 mM) to 27.7 cm−1
heating modality totally eradicated solid tumors in mice. (at 100 mM). Figure S3 illustrates this effect of different laser
penetrations as a function of Fe concentration.
RESULTS AND DISCUSSION Here, it is important to emphasize that the photothermal
Heat Generation by Iron Oxide Nanocubes in heating was totally specific to the nanocubes, as the controls in
Suspension: Dual Magnetophotothermal Modality Am- water or in a gallol-PEG solution (to mimic the surface coating)
plifies Heating Capacity (SLP) up to 5000 W/g. Iron oxide provided very low temperature increase under laser exposure
nanocubes with an average edge length of 20 nm (transmission (see Figure 1F and supplementary Figures S2 and S4). In the
electron microscopy (TEM) image, Figure 1A) were prepared by DUAL-mode, a cumulative effect was achieved for all con-
one-pot synthesis32,41 and homogeneously dispersed in water by a centrations: the heating measured when both LASER and MHT
shell of gallol-polyethylene glycol (gallol-PEG). Mössbauer were simultaneously applied equaled the sum of the heating for
characterization of this synthesis revealed a pure magnetite MHT or LASER alone (Figure 1G).
structure.41 Any NIR photothermal effect mediated by nano- The photothermal effect was also recorded at 680 and 1064 nm
particles is directly correlated to their NIR optical absorption. As irradiation (temperature elevation shown in supplementary
already widely demonstrated for iron oxide nanoparticles, the Figure S5), in agreement with the absorbance properties: similar
nanocubes exhibited an extended optical absorbance, which slowly heat effect at 680 and 808 nm and a slight increase in efficiency at
decreases from the near-UV to the NIR region42−44 (absorption 1064 nm due to the higher absorbance in the NIR second region
spectra shown in Figure 1B), mainly due to charge transfer and (over 1000 nm) resulting from Fe2+−Fe3+ transitions for
ligand transitions of iron. Despite the important decrease in magnetite iron oxides.44
2437 DOI: 10.1021/acsnano.5b07249
ACS Nano 2016, 10, 2436−2446
ACS Nano Article

Figure 1. Nanocube-mediated heating in suspension. (A) Transmission electron microscopy (TEM) of iron oxide nanocubes (20 nm) at low and
high magnifications (100 and 20 nm scale bars, respectively). (B) (Left) UV−vis−NIR absorbance spectra of the iron oxide nanocubes at
different iron concentrations [Fe] = 0.75−25 mM and (right) absorbance at 808 nm as a function of iron concentration. (C) Scheme of the
experimental device for combined hyperthermia experiments, consisting of a magnetic coil in which the sample is placed so that it can be
illuminated by the near-infrared (NIR) laser (808 nm). The temperature increase was recorded with an infrared thermal imaging (IR) camera
located at the end of the coil cavity. (D) (Left) Panel of thermal images acquired by the IR camera on samples at the same iron concentration
([Fe] = 12 and 25 mM) measured inside the coil setup (MHT at 520 kHz, 25 mT; LASER at 0.3 W/cm2). The images correspond to cross-
sectional views of the bottom of the samples. (Right) Temporal response curves for [Fe] = 12 and 25 mM. Temperature increase for different
concentrations ([Fe] = 0−25 mM) after 5 min of each treatment: (E) MHT at 520 kHz, (F) LASER at 0.3 W/cm2, and (G) DUAL-mode. Heating
capacity of iron oxide nanocubes (SLP (W/g)) as a function (H) of the frequency (320, 520, 700, and 900 kHz, with a magnetic field of
H = 25 mT) and (I) of NIR-laser powers (0.3, 0.8, 1.5, and 2.5 W/cm2) in the DUAL-mode (MHT at 900 kHz, 25 mT). (J) Heating capacity of
iron oxide nanoparticles (9 nm) (SLP (W/g)) in the DUAL-mode (MHT at 900 kHz, 25 mT, and LASER at 0.3 and 0.8 W/cm2).

Both the magnetic and the photoinduced heating can be tuned specific loss power, SLP (also called the specific absorption rate,
by varying the magnetic field and laser parameters. The efficiency SAR), as defined in the Methods section. As expected, in
of magnetic hyperthermia is classically expressed in terms of the MHT-mode, this heating power SLP increased with the
2438 DOI: 10.1021/acsnano.5b07249
ACS Nano 2016, 10, 2436−2446
ACS Nano Article

amplitude and frequency of the magnetic field (best efficiency However, nanocubes were chosen here for their excellent heating
recorded at 900 kHz), as summarized in Figure 1H and performance for magnetic hyperthermia (2−3 times higher
supplementary Figure S6. To date, the best efficiency reported than for the two other ones), making the DUAL modality more
for iron oxide nanoparticles is in the range of 1000 W/g. On the appealing.
other hand, photothermic efficiency increased linearly with laser Dual Heating of Cultured Cancer Cells: Cumulative
power, expressed in W/cm2 (see Figure S7). Figure 1I shows the Efficiency and Cell Death. In vitro, dual heating was tested on
SLP obtained here with MHT alone (25 mT, 900 kHz) and with three malignant cell lines, namely, SKOV3 (ovarian cancer), PC3
MHT combined with laser powers ranging from 0.3 to 2.5 W/cm2 (prostate cancer), and A431 (epidermoid cancer). The cells were
(DUAL-mode). The SLP was easily doubled at low laser power first incubated for 2 h with nanocubes at [Fe] = 0.2 mM. This
(0.8 W/cm2), equaling up to 4850 W/g at 2.5 W/cm2, a power extracellular concentration condition was chosen after establish-
used in previous studies for stand-alone photothermia. ing the cell capture profile of nanocubes as a function of the iron
Finally, we emphasize that this amplified heating in the DUAL- concentration (see Figure S9). The nanocubes were taken up via
mode is not specific to iron oxide nanocubes. The same effect was the endocytotic pathway and concentrated within late endosomes
achieved with spherical 9 nm iron oxide nanoparticles (Figure 1J) and lysosomes, as evidenced by electron microscopy (Figure 2A).32
or 9 nm cobalt ferrite nanoparticles (supplementary Figure S8). Cellular iron uptake, quantified by magnetophoresis, was in the

Figure 2. Nanocube-mediated heating inside cells (in vitro). (A) TEM of a cancer cell (PC3) after incubation with iron oxide nanocubes. The
nanocubes are totally internalized and concentrated within endosomes (see zoom on the left). (B) Mass of iron internalized per cell for the three
cancer cell types tested (SKOV3, A431, and PC3). (C) Temperature curves of SKOV3 cells labeled with nanocubes, at a final iron concentration
[Fe] = 12 mM, and subjected to magnetic hyperthermia (MHT, 520 kHz, 25 mT, black curve), NIR-laser irradiation (LASER, 808 nm at
0.3 W/cm2, green), or both treatments simultaneously (DUAL, red). (D) Corresponding panels of thermal images acquired inside the coil setup
by the IR camera are shown below for MHT, LASER, and DUAL. (E) Temperature increase for all three labeled cancer cell types after 5 min of
each treatment (MHT, LASER at 0.3 and 0.8 W/cm2, and DUAL). Temperature contribution of unlabeled cells is also shown as controls (C0).
The iron concentration was [Fe] = 12 mM in all samples. (F) Heating power for all three cell samples, expressed as the SLP (in W/g), for the
MHT (0 W/cm2) and DUAL conditions (0.3 and 0.8 W/cm2). (G) Magnetothermal temperature contribution in cells (ΔTMHT) when only MHT
is applied or when MHT is combined with LASER application (DUAL) for 20 nm iron oxide nanocubes and 9 nm nanoparticles. (H) Cell viability
(SKOV3 cells) assessed with the Alamar Blue assay (assessing metabolic activity) after each heating treatment applied during 600 s. Nanocubes
subjected to dual stimulation induced a significant decrease in cell viability compared to magnetic hyperthermia or laser irradiation alone and to
untreated control cells (unlabeled (C0) and nanocube-labeled (C1) cells). (I) Expression of apoptosis-related proteins in SKOV3 cells after the
different treatments (MHT, LASER, and DUAL for 100 s and DUAL for 600 s). Cell lysates were analyzed in duplicate with the Proteome Profiler
Array (R&D Systems, Inc.) according to the supplier’s instructions. The error bars represent the standard deviation. p-Values were calculated
from the t test: *** indicates p < 0.01; ** indicates p < 0.05; * indicates p < 0.1.

2439 DOI: 10.1021/acsnano.5b07249

ACS Nano 2016, 10, 2436−2446
ACS Nano Article

range 6−9 pg of iron per cell for all three cell types (Figure 2B). By contrast, at high doses, cell death was not compensated by
PC3 cells contained slightly more iron (9 pg) than SKOV3 and activation of survival pathways, and the expression of apoptosis
A431 cells (6 pg), which is due to their larger diameter (20 μm inhibitors decreased.
for PC3, 17 μm for the other two cell lines). Cell viability was not Dual Heating in Vivo: Complete Tumor Regression.
affected by nanocube internalization (97 ± 4% of control Finally, the heating effect was tested in vivo on mice bearing solid
viability). tumors. Mice were injected intratumorally with 50 μL of
To mimic the tumor mass, about 15 million labeled cells were suspension containing 14 mg of nanocubes per mL of saline
concentrated in a small volume (150 μL) at a final iron ([Fe] = 0.25 M) and subsequently exposed to MHT, LASER, or
concentration adjusted with media to reach [Fe] = 12 mM. The DUAL treatment, repeated three times at 24 h intervals. In vivo,
tumor cells loaded with magnetic nanocubes were then subjected MHT must fall within the limits of magnetic field frequency ( f)
to three heating protocols. Nanocube heating remained efficient and amplitude (H): the H × f product should be lower than
in these cellular conditions (see Figure 2C,D for typical tem- 5 × 109 A m−1 s−1, in order to avoid nonspecific inductive tissue
perature curves and IR images), and the same time, temperature heating and cardiac stimulation.45 This restriction implies the use
elevations and heating powers (SLP) were recorded for the three of lower frequencies, and we thus selected 110 kHz at 12 mT.
cell lines (Figure 2E,F). However, magnetic hyperthermia was The surface temperature of the mice and tumors was
far less efficient than in suspension, the temperature increase monitored with an IR camera (Figure 3A). On day 0, the
after 5 min (ΔT) falling from 5−7 °C (Figure 1D, 12 mM) to temperature of the tumor reached 40 °C (7 °C increase) with the
1−3 °C (Figure 2C,E, same 12 mM iron concentration). As a
result, the heating power (SLP) fell dramatically, from 700 to
900 W/g to 200−250 W/g (at 900 kHz). This loss is due to the
inhibition of magnetic dynamics when the nanocubes are densely
sequestrated in endosomes.32,33 By contrast, cellular confine-
ment did not hinder photothermal efficiency (same 7−9 °C
temperature increase in cultured cells as in suspension at 12 mM
iron; compare Figure 1D and Figure 2C). As a consequence, SLP
values were far higher with DUAL treatment than with magnetic
hyperthermia alone (4-fold at 0.3 W/cm2, 15-fold at 0.8 W/cm2,
Figure 2F). Remarkably, heating in the DUAL-mode was
synergistic, with an additional temperature elevation from
LASER-mode alone to the DUAL-mode (which is due to the
MHT contribution, ΔTMHT) of 8−9 °C, while the MHT-mode
alone provided only a 2−3 °C temperature increase (see Figure 2G).
This 8−9 °C increase matches the magnetic heating capacity of
the nanocubes in suspension, with competent Brownian motion
(about 7−8 °C at 520 kHz and 10−11 °C at 900 kHz). It thus
appears that the thermal energy provided by laser stimulation
frees the nanocubes and restores their Brownian motion (and
thus the Brownian relaxation heating mode) in the cellular
environment. On the contrary, this effect is not observed
for 9 nm maghemite nanoparticles when internalized in cells
(Figure 2G): for these maghemite nanoparticles, the magnetic
relaxation dynamics is governed by Néel relaxation.32
The next step consisted in the assessment of the induced
tumor cell death. The three protocols were tested for durations
of 100 and 600 s. Significant cytotoxicity was observed in
DUAL-mode during 600 s, where cell viability fell to 14 ± 7%,
compared to only 74 ± 15% with MHT and 36 ± 3% with the
laser (Figure 2H).
The expression profiles of apoptosis-related proteins (Figure 2I
and Figure S10) showed that pro-caspase 3 was activated by all
three modalities, reflecting the onset of apoptosis. Both intrinsic Figure 3. In vivo heat therapy. (A) Thermal images obtained with the
and extrinsic pathways were involved in this caspase activation. IR camera in mice, after intratumoral injection of nanocubes (50 μL
DNA damage and changes in the mitochondrial membrane at [Fe] = 250 mM), in the left-hand tumor, and after 10 min
enhanced the release of pro-apoptotic proteins such as application of magnetic hyperthermia (MHT, 110 kHz, 12 mT),
cytochrome c and Smac/Diablo. Death receptors (TNF RI, NIR-laser irradiation (LASER, 808 nm at 0.3 W/cm2), or DUAL
Fas, DR3, TRAIL R1/DR4, TRAIL R2/DR5) were also over- (both effects). (B) Corresponding thermal elevation curves for all
expressed. Interestingly, inhibitors of apoptosis (Survivin, XIAP, treatments and for the noninjected tumor in the DUAL condition.
(C) Average final temperature increase obtained after 10 min (MHT,
Livin, cIAP-1, cIAP-2) were overexpressed after low-dose
LASER, and DUAL) on day 0 (1 h after injection) and 1 and 2 days
treatment (MHT, LASER, or DUAL, for 100 s), but underex- after injection and for noninjected tumors. (D) Average tumor
pressed at higher doses (DUAL, for 10 min). At low doses, growth (groups of six tumors each in noninjected mice submitted to
activation of both pro-apoptotic and anti-apoptotic events no treatment (Control) and in nanocube-injected mice exposed to
reflected a balance between apoptotic and survival pathways, MHT, LASER, and DUAL during the 8 days following the 3 days of
allowing partial recovery from damage (reversible apoptosis). treatment.

2440 DOI: 10.1021/acsnano.5b07249

ACS Nano 2016, 10, 2436−2446
ACS Nano Article

two individual treatment modalities and 50 °C (15 °C increase) the near-infrared region of the spectra and hence could penetrate
with DUAL treatment (Figure 3B). On days 1 and 2, MHT biological tissues. Although these plasmonic nanomaterials may
yielded a smaller temperature increase (3−4 °C), while LASER have a bright therapeutic future,46 the long biopersistence
efficiency almost doubled. The net result of the DUAL heating of gold remains a matter of concern.47 By contrast, iron oxide
was a remarkably consistent increase of 15 °C (Figure 3C). nanoparticles are biodegradable, and free iron can be handled by
These in vivo results also show that combined therapy amplifies physiological metabolic pathways. Iron oxide nanoparticles thus
the heating. The latter clearly impacts tumor therapy (Figure 3D). have a net advantage over other metallic or carbon nanoparticles.
While tumor growth is slightly inhibited by single-mode It was very recently reported that iron oxide nanoparticles can
therapies, a complete tumor regression is observed in all mice also be used for photothermal imaging and photothermal
after the DUAL treatment. In comparison, the tumors that were therapy.35,36,38,39,48 These studies used different nanoformula-
not injected with the nanocube suspension but exposed to tions and illumination doses (Table 1): 13 nm iron/iron oxide
DUAL treatment had a growth that was comparable to core−shells illuminated at 0.3−0.4 W/cm2;38 Fe3O4 chitosan-
nontreated and noninjected tumors (Figure S11). modified spherical particles of about 180 nm diameter at 2 W/cm2;35
Representative sections of tumors excised at day 8 after highly crystallized 15 nm iron oxide nanoparticles at 2.5 W/cm2;36
injection are shown in Figure 4A,B. Control tumors, as well as and nanoparticle clusters 225 nm in diameter at 2−5 W/cm2.39
LASER- or MHT-treated tumors, exhibited a relatively viable and Here, we used 20 nm iron oxide nanocubes and illumination doses
homogeneous tumor tissue. The nanocubes (appearing as brown between 0.3 and 2.5 W/cm2, yielding promising photoheating
dots mainly situated on the left side of the panels of the control efficiency (see Table 1 for comparison with the previously tested
mouse) were mainly restrained to the tumor capsula and did not nanoparticles).
affect the viability of adjacent cells when hyperthermia was not Magnetic Hyperthermia. The other prospective heat-
applied, as neither zones of necrosis nor immune cell infiltrates mediated therapeutic modality is magnetic hyperthermia. Its
were observed in the vicinity of the nanocubes. In contrast, the advantages include unlimited tissue penetration and the
thermal treatments led to diffusion and subsequent dilution of possibility of multiple hyperthermia cycles. Its efficacy and safety
the nanocubes throughout the tumors, triggering in turn their explain why magnetic hyperthermia has already reached the
internalization within cancer cells (Figure 4C). The three heating clinic (MagForce, Berlin) for the treatment of solid tumors. In
cycles, provided by the DUAL treatment, resulted in complete addition, iron oxide nanoparticles’ intrinsic properties to act as
tumor eradication. As exemplified in the micrographs (Figure 4B), MRI contrast agents would allow following their fate inside the
viable tumor tissues (Figure 4B, left) were replaced by scar tissue body in a noninvasive manner. One limitation of magnetic
occurring under scabs and exhibited second intention healing hyperthermia is the low heat yield of iron oxide nanoparticles: it
(Figure 4B, right) with inflammatory cell infiltrates and involves a large dose of nanoparticles to achieve a therapeutic
granulation tissue among scattered aggregates of iron oxide temperature rise, and in vivo protocols required the injection of
nanocubes (brown aggregates). TEM revealed intracellular about 30 mg of iron per cm3 of tumoral tissue. Recently, this limit
(endolysosomal) confinement of nanocubes and extracellular was raised by tuning nanoparticles’ architecture, yielding
localization of nanocubes within denatured collagen fibers unprecedented heating capacities in the range of 1000 W/g
(Figure 4C,D, right panel). The magnetophotothermal approach with nanocubes28,29 or multicore31 or core−shell nanoparticles30
thus affects two tumoral constituents, tumor cells and collagen, (compared to tens of W/g for other iron oxide nanoparticles).
which is the most important stromal structural constituent Until now, yields over 1000−2000 W/g seemed beyond reach.
(Figure 4C left panel). While control tumors were characterized The other inconvenience with magnetic hyperthermia is
by tumor cells tightly adjacent to a rich collagen network, DUAL- that heating efficiency declines markedly when nanoparticles
treatment-exposed tumor leftovers exhibited nanocube-loaded are taken up by cancer cells, because their endocytosis and
phagocytic cells and nanocube-loaded denatured collagen fibers aggregation inhibit their Brownian motion. Similarly, the
(Figure 4C right panel and Figure S12). In contrast to organized diffusion (thereby local dilution) or enhanced distribution of
collagen fibers, which were present in control tumors (Figure 4D nanoparticles (also known as the “thermal bystander effect”49)
left panel), the collagen in DUAL-treatment-exposed tumors also decreases nanoparticle heating, which is concentration-
was defibrillated in the close vicinity of the nanocubes, as well as dependent.
rare, scattered, and chaotic in the proximity of electron-dense, Unprecedented Heating with Iron Oxide Nanocubes and
denatured collagen zones, shown on the TEM micrographs Combined Magnetic Hyperthermia−Photothermia. The
(Figure 4D right panel; see also Figure S12). drawbacks of magnetic hyperthermia could be overcome by
Magnetophotothermia as an Efficient Therapeutic combination with photothermia (and vice versa). Surprisingly, no
Modality Using Iron Oxide Nanoparticles. Cancer can be one so far has studied the possibility of coupling magnetic
fought with early detection and efficient treatment. Among novel hyperthermia with photothermia when employing iron oxide
experimental approaches in the old combat, magnetic hyper- nanomaterials as a bimodal therapy. The most obvious way of
thermia and photothermal therapy might provide potentially implementing a magnetophotothermal strategy would be to
efficient means for tumor heating at therapeutic levels. Sur- use a magnetic (for the magnetic hyperthermia modality) and
prisingly, magnetic hyperthermia, based on magnetic nano- plasmonic (for the photothermal modality) hybrid. This
particles, and photothermal therapy, applying mostly plasmonic approach has been very recently proposed using nanohybrids
nanoparticles, have been developed and researched independ- composed of gold and iron oxide (e.g., a magnetic core and a gold
ently. shell50,51 and in the form of a nanocomposite)52 or nano-
Photothermal Treatment. Currently, plasmonic nanopar- compounds based on cobalt/manganese ferrite nanoparticles.53
ticles are the prime candidates for photothermia. Optical (laser) Using nanoparticles made with iron oxide only displays however
excitation causes local plasmonic heating that could destroy a net advantage over other composite materials (nanohybrids)
surrounding cancer cells. Nanorods, nanoshells, or nanostars are by reducing the risk of adverse reactions. Indeed, iron oxide
the designs of choice, which exhibit plasmonic resonance toward nanoparticles have an excellent biocompatible status, in that iron
2441 DOI: 10.1021/acsnano.5b07249
ACS Nano 2016, 10, 2436−2446
ACS Nano Article

Figure 4. Optical and transmission electron micrographs of tumor tissue. (A) Tumor sections after hematoxylin and eosin staining. (Note that
tumor capsula is located on the left of each panel.) Control tumors exhibit a viable, cohesive tissue. LASER- and MHT-treated tumors show
diffuse regions of nanocube-rich zones (brown color) and immune cell infiltrates. DUAL treatment resulted in tumor regression. Nanocube
aggregates were disseminated throughout the scar tissue (×10). (B) Viable control tumor core (left) and core of the scar that remained after
DUAL treatment (right), showing second intention scarring with inflammation and granulation tissue. (C) Ultrastructural characteristics of the
control tumor (left) and scar tissue (right). The control tissue was characterized by numerous tumor cells (N = nucleus) surrounded by organized
collagen fibers (single asterisk). The scar tissue was characterized by immune cell infiltrates (N = nucleus). The remaining nanocubes were
localized either within endolysosomes (L on the panel) or within denatured collagen fibers (double asterisk). (D) Magnification of collagen fibers
in control tumors and a remnant scar. Left: Tumors that were not exposed to hyperthermia exhibited typical tightly bound threadlike axially (left
side) or longitudinally (right side) sectioned dense and organized collagen fibers. Right: Treated tumors exhibited scattered, poorly organized
threadlike collagen fibers that do not contain nanocubes (axially (bottom) and longitudinally (top) sectioned fibers) that were disseminated
around electron-dense zones of denatured collagen in close vicinity to nanocubes (the black appearing dots).

2442 DOI: 10.1021/acsnano.5b07249

ACS Nano 2016, 10, 2436−2446
ACS Nano Article

Table 1. Temperature Increases Reported in All Published Studies of Photothermia with Iron Oxide Nanomaterialsa
In Aqueous Suspensions
nanoparticles (iron oxide) size (nm) [Fe] (mg/mL) irradiation (W/cm2) time (s) wavelength (nm) heating ΔT (°C) ref

Fe@Fe3O4 core−shell NPs 13 0.05 0.3−0.4 500 808 12 38

Fe3O4 NPs 15 0.5 2.5 600 885 33 36
Fe3O4 clusters of NPs 225 0.05 5 180 808 25 39
CMCTS@Fe3O4 NPs 177 0.15 2 300 808 50 35
Fe3O4 NCs 20 0.7 0.3 300 808 7 present work
20 0.7 0.8 300 808 22
In Vivo
nanoparticles (iron oxide) size (nm) mFe injected (mg/kg) irradiation (W/cm2) time (s) wavelength (nm) heating ΔT (°C) ref
Fe@Fe3O4 core−shell NPs 13 1460 0.3 600 808 6, 12 (magnet) 38
Fe3O4 NPs 15 20 2.5 600 885 25 36
Fe3O4 clusters of NPs 225 3 5 180 808 34 39
CMCTS@Fe3O4 NPs 177 70−100 1.5 300 808 22 35
Fe3O4 NCs 20 35 0.3 600 808 15 present work
a
Size (in nm) ranges between 13 and 225 nm, the iron concentration in suspension between 0.05 and 0.7 mg/mL, the iron dose injected
in vivo between 3 and 1500 mg/kg, laser power between 0.3 and 5 W/cm2, time exposure between 1 and 10 min, and the temperature increase from
10 to 50 °C.

is assimilated by the body through a tightly regulated DUAL-mode, at injected iron doses 4−8 times lower than those
physiological process.26 used for existing methods of magnetic hyperthermia alone. The
However, it is admitted that the practical limitation for thermal main targets of this DUAL treatment are indeed the tumoral
therapies with iron oxide nanoparticles (mainly magnetic cells, but also the extracellular matrix. The tumoral environment,
hyperthermia) is the poor heating efficiency. The simultaneous particularly the collagen matrix, is directly affected by the local
application of both magnetic hyperthermia and photothermia temperature increase. Immediately after administration, nano-
could subsequently increase the delivered heating (and cubes mainly perfuse between collagen fibers and are, at the
consequently help to decrease the dose of iron oxide nano- beginning, rarely internalized by cells. As a result, when MHT or
particles needed for magnetic hyperthermia). The aim of the LASER only are applied, the collagen fibers slacken, as we
magnetophotothermal approach is thus to increase the heating evidenced previously for MHT.54 Herein we observed that, when
power of iron oxide nanomaterials. This should be considered as the DUAL-mode is applied, the collagen fibers completely
an additional strategy to supplement the efforts devoted to denature. These structural modifications are due to phase
optimizing nanoparticle heating efficiency, due to innovative transitions of collagen fibers, which are in close vicinity to heating
designs (cubes, multicores, core−shells). iron oxide nanocubes. A mild hyperthermia (typically up to
Here, we compare the two thermal effects both individually 37 °C) induces the depolymerization of the smallest collagen
and simultaneously, mediated by the same iron oxide nanoma- fibrils and is reversible.55 In contrast, the thermal transition that
terial, under three different settingsin aqueous dispersion, in occurs between 37 and 55 °C is irreversible and accounts for an
different tumor cell lines, and in solid tumorswhich is also irreversible destructuration, which arises from a total defibril-
the first implementation of the magnetophotothermal strategy
lation and unfolding of the triple-helical collagen structure.55 The
in vivo.
first hyperthermia treatment thus locally modifies the fibrillar
The heating power (SLP) in suspension markedly increased
stroma and allows the penetration of the nanoparticles across the
when laser and magnetic stimulation were combined, boosting
tumor and their subsequent internalization within cancer cells.
the SLP from 700 to 900 W/g with magnetic hyperthermia alone
to 4850 W/g at the maximum laser power (2.5 W/cm2). This explains the significant diminution of temperature increase
However, this laser condition exceeds biologically safe limits. At in the following cycles of MHT, since MHT efficiency depends
0.3 W/cm2, which avoids nonspecific tissue heating, the magnetic on local concentration and is strongly inhibited in the intra-
and photoinduced effects were similar, and heating efficiency cellular environment due to nanocube immobilization. The
was doubled in the DUAL-mode. The heating effect after contrasting phenomenon occurring in LASER-treated mice
intracellular uptake was particularly interesting. As expected, (temperature increase is higher in the second and third
magnetic heating efficiency fell dramatically when nano- hyperthermia cycle) can be explained as well by the nanocube
cubes were confined inside cancer cells (the SLP fell from redistribution across the tumoral mass. While during the first
700−900 W/g to 200−250 W/g); photothermia then became hyperthermia cycle the nanocube-rich zone is smaller and more
predominant, because its efficiency was not impacted concentrated, the particles infiltrate a larger zone during the two
by intracellular confinement. This resulted in 4-fold and following cycles. Because the LASER heating is concentration-
15-fold increases in SLP at 0.3 and 0.8 W/cm2, respectively. independent (above a limit concentration found at [Fe] = 12 mM
Another noteworthy finding is the synergistic heating effect in suspension), a larger heating zone then yielded a higher
in the DUAL-mode, where LASER stimulation restored temperature increase, regardless of the decrease in concentration.
Brownian relaxation that had been inhibited by endosomal As a final consequence, DUAL-mode heating efficiency remained
confinement. remarkably consistent. This long-lasting heating for magneto-
The treatment of solid subcutaneous tumors in mice gave photothermal therapy, together with the overall tumor temper-
very encouraging results, leading to tumor eradication in the ature increase, explains why complete tumor regression was
2443 DOI: 10.1021/acsnano.5b07249
ACS Nano 2016, 10, 2436−2446
ACS Nano Article

achieved in the DUAL condition but not with the individual the coil cavity, providing a cross-sectional view of the bottom of the
modalities. sample.
The temperature inside the coil was maintained at 37 °C (initial
temperature) with a nonane cooling circuit in order to avoid thermal
CONCLUSIONS fluctuations.
In conclusion, this work offers a dual magnetophotothermal The temperature elevation was measured as a function of time
therapeutic approach with iron oxide nanoparticles as the sole (dT/dt) at the initial linear slope (t ≈ 30 s) in order to evaluate the
heating effect in terms of specific loss power. SLP is defined as the power
heat mediators. We demonstrate that iron oxide nanocubes can dissipation per unit mass of iron (W/g).
be remotely activated with an alternating magnetic field and SLP was calculated as follows:
a near-infrared laser, achieving a very efficient heat conversion
for both modalities. Remarkably, the dual magnetic and photo- CVs dT
SLP =
thermal action resulted in complete cell death in vitro and in total m dt
solid tumor ablation in vivo at low iron doses, tolerable magnetic where m is the total mass of iron in the sample, C is the specific heat
field and frequency conditions, and acceptable laser power doses. capacity of the sample (Cwater = 4185 J/L/K, Ccell = 4125 J/L/K), and
This cumulative, if not synergistic, heat therapy is thus promising V is the sample volume.
for tumor treatment with minimal collateral tissue damage. Photothermal and Dual Magnetophotothermal Measure-
ments in Aqueous Suspension and in Cells in Vitro. Laser
hyperthermia was induced by a near-infrared continuous laser at 808 nm
METHODS (Laser Components S.A.S. (France)) with external adjustable power
Iron Oxide Nanocube Synthesis. Magnetite iron oxide nanocubes (0−5 W). The sample (aqueous nanoparticle suspension or suspension
were synthesized by thermal decomposition, as described by Guardia of nanoparticle-loaded cells) contained in a 0.5 mL tube was illuminated
et al.41 For the preparation of nanocubes with an edge length of about by the laser placed on the same holder as for magnetic hyperthermia.
20 nm, 1 mmol of iron(III) acetylacetonate and 4 mmol of decanoic acid The path-length is around 1 cm. This configuration has two goals: (i)
were dispersed in 25 mL of dibenzyl ether. The suspension was heated to use of the same cooling circuit to place the sample at 37 °C initially and
200 °C at 5 °C/min and maintained at this temperature for 2.5 h. The (ii) application of combined hyperthermia (with simultaneous magnetic
mixture was then raised to reflux temperature (at 10 °C/min) and induction). The distance between the sample and the laser was 2−3 cm,
reacted for 1 h. Finally, the nanoparticles were washed three times, and the laser spot was 1 cm2. The laser power range was between 0.3 and
dispersed in 15 mL of chloroform, and transferred in water, a ligand 2.5 W/cm2. The temperature elevation was recorded as described above,
exchange protocol that allows replacing the decanoic acid with a and SLP was calculated.
PEG(3000)-gallol molecule polymer coating with poly(maleic anhy- Proteomic Analysis. An antibody-based apoptosis detection kit
dride alt-1-octadecene), following a well-established procedure (Human Apoptosis Array, R&D System, #ARY009) was used to assess
developed by us. PEG(3000)-gallol molecules were synthesized in the impact of the different treatments on the cellular proteome. After
order to coat the as-synthesized nanocubes (PEG-gallol-coated nano- each treatment, 107 cells were processed following the manufacturer’s
cubes). protocol. Antibody spot intensities were analyzed with an LAS-4000
Elemental Analysis. The iron concentration in nanocube chemiluminescence imaging system (FujiFilm) and processed with
suspensions and cells was analyzed by inductively coupled plasma ImageJ open-source software.
atomic emission spectrometry (ICP-AES). The samples were digested Magnetic Hyperthermia and Photothermal Measurements in
in concentrated HNO3 for 2 h at 100 °C, then recovered in purified Vivo. A total of 22 pathogen-free 8-week-old female immunodeficient
water for analysis. athymic nude NMRI mice with a mean weight of 20 ± 2 g were used
Cell Culture and Other in Vitro Experiments. Three malignant (Janvier Laboratories, France). The animals were allowed to acclimatize
cell lines (SKOV3, PC3, and A431) were cultured in Dulbecco’s (Animalerie Buffon, Institute Jacques Monod, Paris 7 University) and
modified Eagle’s medium (DMEM) for PC3 cells and McCoy’s medium were treated according to European standards of animal care and well-
for SKOV3 and A431 cells, both supplemented with 5% fetal bovine being. Solid tumors were induced by subcutaneous injection of 1.5 × 106
serum (FBS) and 1% penicillin at 37 °C with 5% CO2 until confluence. A431 human epidermoid carcinoma cells in 100 μL of physiological
Cells were incubated with PEG nanocubes at [Fe] = 0.2 mM for 2 h in saline in the left and right flanks. The animals were treated after 2 to
5% citrated RPMI medium at 37 °C. The medium was then removed, 3 weeks, when the tumors reached approximately 125 mm3. The animals
and the cells were washed with culture medium for a further 2 h (chase were then injected in one tumor with 50 μL of nanocube suspension
period). Labeled cells were detached, centrifuged, and resuspended at a (250 mM or 14 mg/mL Fe, corresponding to 35 mg/kg). The animals
concentration of about 15 million cells in 150 μL of culture medium, were subsequently divided into three groups: MHT (n = 6), LASER
exactly adjusted to match [Fe] = 12 mM. (n = 6), and DUAL (n = 6). Some collateral tumors (a total of 6) were
Cytotoxicity Assay. Cytotoxicity was measured with the Alamar used as controls (noninjected and nontreated). A last group of animals
Blue assay according to the supplier’s instructions (Life Technologies). (n = 4) with noninjected tumors were treated with DUAL treatment (as
Briefly, the culture medium was replaced with an Alamar Blue DUAL control). Magnetic hyperthermia experiments were conducted
suspension (10% in DMEM without red phenol). After 2 h incubation, on a 5 cm diameter inductive coil (Nanotherics, Corp). During the
it was transferred to a 96-well plate, which was analyzed with a hyperthermia treatment the animals were anesthetized with a ketamine/
microplate reader (BMG FluoStar Galaxy), at an excitation wavelength xylazine anesthesia and placed inside the coil with an ac magnetic field of
of 550 nm and fluorescence detection at 590 nm. Viability was 12 mT and a frequency of 110 kHz (within the safe limit for tissue
determined by comparison with control cells. All reported experimental exposure) and/or laser irradiation of 808 nm at 0.3 W/cm2. An infrared
points were done in triplicate. thermal camera (FLIR SC7000, FLIR Systems, Inc.) was used to
Magnetic Hyperthermia Measurement in Aqueous Suspen- monitor the suface temperature of the tumors. All the acquisitions were
sions and Cells in Vitro. The alternating magnetic field was generated processed with Altair software (FLIR Systems, Inc.). The animals were
with a homemade magnetothermal setup consisting of a resonant RLC sacrificed when collateral tumors reached 1 cm3.
circuit and a 16 mm copper coil. The field amplitude can be tuned from Histology. Tumors were excised and fixed with phosphate-buffered
5 to 24 kA/m and the magnetic field frequency from 320 kHz to 10% formalin at pH 7.4, dehydrated in graded ethanol solutions, and
1.1 MHz. The suspension of magnetic nanocubes (150 μL) or embedded in paraffin. Six-micrometer-thick sections were stained with
nanocube-loaded cells was placed inside the coil in 0.5 mL Eppendorf hematoxylin and eosin for optical microscopy observation.
tubes. The temperature increase was recorded with an infrared thermal Transmission Electron Microscopy. Parts of excised tumors were
imaging camera (FLIR SC7000) in real time, every second, in a cut into 1 mm3 pieces and fixed with 2% glutaraldehyde in 0.1 M sodium
temperature range of 25 to 70 °C. The camera was located at the end of cacodylate buffer, then postfixed with 1% osmium tetroxide containing

2444 DOI: 10.1021/acsnano.5b07249

ACS Nano 2016, 10, 2436−2446
ACS Nano Article

1.5% potassium cyanoferrate, gradually dehydrated in ethanol, and (11) Yavuz, M. S.; Cheng, Y.; Chen, J.; Cobley, C. M.; Zhang, Q.;
embedded in Epon. Thin sections (70 nm) were observed with a Zeiss Rycenga, M.; Xie, J.; Kim, C.; Song, K. H.; Schwartz, A. G. Gold
EM902 electron microscope operating at 80 keV. Nanocages Covered by Smart Polymers for Controlled Release with
near-Infrared Light. Nat. Mater. 2009, 8, 935−939.
ASSOCIATED CONTENT (12) Boca, S. C.; Potara, M.; Gabudean, A.-M.; Juhem, A.; Baldeck, P.
*
S Supporting Information
L.; Astilean, S. Chitosan-Coated Triangular Silver Nanoparticles as a
Novel Class of Biocompatible, Highly Effective Photothermal Trans-
The Supporting Information is available free of charge on the ducers for in Vitro Cancer Cell Therapy. Cancer Lett. 2011, 311, 131−
ACS Publications website at DOI: 10.1021/acsnano.5b07249. 140.
Additional figures (PDF) (13) He, R.; Wang, Y.-C.; Wang, X.; Wang, Z.; Liu, G.; Zhou, W.; Wen,
L.; Li, Q.; Wang, X.; Chen, X. Facile Synthesis of Pentacle Gold−
Copper Alloy Nanocrystals and Their Plasmonic and Catalytic
AUTHOR INFORMATION Properties. Nat. Commun. 2014, 5.10.1038/ncomms5327
Corresponding Author (14) Zhou, M.; Zhang, R.; Huang, M.; Lu, W.; Song, S.; Melancon, M.
*E-mail: [email protected]. P.; Tian, M.; Liang, D.; Li, C. A Chelator-Free Multifunctional [64cu]
Notes Cus Nanoparticle Platform for Simultaneous Micro-Pet/Ct Imaging and
The authors declare no competing financial interest. Photothermal Ablation Therapy. J. Am. Chem. Soc. 2010, 132, 15351−
15358.
(15) Liu, Z.; Cai, W.; He, L.; Nakayama, N.; Chen, K.; Sun, X.; Chen,
ACKNOWLEDGMENTS X.; Dai, H. In Vivo Biodistribution and Highly Efficient Tumour
This work was supported by the Marie Curie Intra-European Targeting of Carbon Nanotubes in Mice. Nat. Nanotechnol. 2007, 2, 47−
Project FP7-PEOPLE-2013-740 IEF-62647, by the European 52.
project Magnifyco (Contract NMP4-SL-2009-228622), and by (16) Yang, K.; Zhang, S.; Zhang, G.; Sun, X.; Lee, S. T.; Liu, Z.
the AIRC project (contract no. 14527 to T.P.). Additionally, we Graphene in Mice: Ultrahigh in Vivo Tumor Uptake and Efficient
thank the European COST action TD1402 RADIOMAG. We Photothermal Therapy. Nano Lett. 2010, 10, 3318−3323.
are grateful to A. Djemat from Animalerie Buffon for animal care, (17) Huang, X.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. Cancer Cell
Imaging and Photothermal Therapy in the near-Infrared Region by
C. Longin for help with TEM analysis, and P. Guardia, S. Nitti, Using Gold Nanorods. J. Am. Chem. Soc. 2006, 128, 2115−2120.
and G. Pugliese for help with the nanoparticle preparation. (18) Loo, C.; Lowery, A.; Halas, N.; West, J.; Drezek, R.
SKOV3 (ovarian cancer) and A431 (epidermoid cancer) were a Immunotargeted Nanoshells for Integrated Cancer Imaging and
gift from the S. Canevari group at the Istituto Nazionale dei Therapy. Nano Lett. 2005, 5, 709−711.
Tunori, Milan (Italy). (19) Moon, H. K.; Lee, S. H.; Choi, H. C. In Vivo near-Infrared
Mediated Tumor Destruction by Photothermal Effect of Carbon
REFERENCES Nanotubes. ACS Nano 2009, 3, 3707−3713.
(1) Li, Y.; Lin, T.-y.; Luo, Y.; Liu, Q.; Xiao, W.; Guo, W.; Lac, D.; (20) Wang, Y.-H.; Chen, S.-P.; Liao, A.-H.; Yang, Y.-C.; Lee, C.-R.; Wu,
Zhang, H.; Feng, C.; Wachsmann-Hogiu, S. A Smart and Versatile C.-H.; Wu, P.-C.; Liu, T.-M.; Wang, C.-R. C.; Li, P.-C. Synergistic
Theranostic Nanomedicine Platform Based on Nanoporphyrin. Nat. Delivery of Gold Nanorods Using Multifunctional Microbubbles for
Commun. 2014, 5.471210.1038/ncomms5712 Enhanced Plasmonic Photothermal Therapy. Sci. Rep. 2014, 4.10.1038/
(2) Di Corato, R.; Béalle, G.; Kolosnjaj-Tabi, J.; Espinosa, A.; Clément, srep05685
O.; Silva, A. K.; Menager, C.; Wilhelm, C. Combining Magnetic (21) Maier-Hauff, K.; Ulrich, F.; Nestler, D.; Niehoff, H.; Wust, P.;
Hyperthermia and Photodynamic Therapy for Tumor Ablation with Thiesen, B.; Orawa, H.; Budach, V.; Jordan, A. Efficacy and Safety of
Photoresponsive Magnetic Liposomes. ACS Nano 2015, 9, 2904−2916. Intratumoral Thermotherapy Using Magnetic Iron-Oxide Nano-
(3) Pasparakis, G.; Manouras, T.; Vamvakaki, M.; Argitis, P. particles Combined with External Beam Radiotherapy on Patients
Harnessing Photochemical Internalization with Dual Degradable with Recurrent Glioblastoma Multiforme. J. Neuro-Oncol. 2011, 103,
Nanoparticles for Combinatorial Photo−Chemotherapy. Nat. Commun. 317−324.
2014, 5.10.1038/ncomms4623 (22) Johannsen, M.; Thiesen, B.; Wust, P.; Jordan, A. Magnetic
(4) Jain, P. K.; Huang, X.; El-Sayed, I. H.; El-Sayed, M. A. Noble Metals Nanoparticle Hyperthermia for Prostate Cancer. Int. J. Hyperthermia
on the Nanoscale: Optical and Photothermal Properties and Some 2010, 26, 790−795.
Applications in Imaging, Sensing, Biology, and Medicine. Acc. Chem. Res. (23) Soenen, S. J.; Parak, W. J.; Rejman, J.; Manshian, B. Intra) Cellular
2008, 41, 1578−1586. Stability of Inorganic Nanoparticles: Effects on Cytotoxicity, Particle
(5) Reddy, L. H.; Arias, J. L.; Nicolas, J.; Couvreur, P. Magnetic Functionality, and Biomedical Applications. Chem. Rev. 2015, 115,
Nanoparticles: Design and Characterization, Toxicity and Biocompat- 2109−2135.
ibility, Pharmaceutical and Biomedical Applications. Chem. Rev. 2012, (24) Anselmo, A. C.; Mitragotri, S. A Review of Clinical Translation of
112, 5818−5878. Inorganic Nanoparticles. AAPS J. 2015, 17, 1−14.
(6) Crile, G. The Effects of Heat and Radiation on Cancers Implanted (25) Levy, M.; Luciani, N.; Alloyeau, D.; Elgrabli, D.; Deveaux, V.;
on the Feet of Mice. Cancer Res. 1963, 23, 372−380. Pechoux, C.; Chat, S.; Wang, G.; Vats, N.; Gendron, F. Long Term in
(7) Yu, M. K.; Park, J.; Jon, S. Targeting Strategies for Multifunctional Vivo Biotransformation of Iron Oxide Nanoparticles. Biomaterials 2011,
Nanoparticles in Cancer Imaging and Therapy. Theranostics 2012, 2, 3. 32, 3988−3999.
(8) Quarta, A.; Bernareggi, D.; Benigni, F.; Luison, E.; Nano, G.; Nitti, (26) Lartigue, L.; Alloyeau, D.; Kolosnjaj-Tabi, J.; Javed, Y.; Guardia,
S.; Cesta, M. C.; Di Ciccio, L.; Canevari, S.; Pellegrino, T. Targeting Fr- P.; Riedinger, A.; Péchoux, C.; Pellegrino, T.; Wilhelm, C.; Gazeau, F.
Expressing Cells in Ovarian Cancer with Fab-Functionalized Nano- Biodegradation of Iron Oxide Nanocubes: High-Resolution in Situ
particles: A Full Study to Provide the Proof of Principle from in Vitro to Monitoring. ACS Nano 2013, 7, 3939−3952.
in Vivo. Nanoscale 2015, 7, 2336−2351. (27) Johannsen, M.; Gneveckow, U.; Thiesen, B.; Taymoorian, K.;
(9) Nikoobakht, B.; El-Sayed, M. A. Preparation and Growth Cho, C. H.; Waldöfner, N.; Scholz, R.; Jordan, A.; Loening, S. A.; Wust,
Mechanism of Gold Nanorods (Nrs) Using Seed-Mediated Growth P. Thermotherapy of Prostate Cancer Using Magnetic Nanoparticles:
Method. Chem. Mater. 2003, 15, 1957−1962. Feasibility, Imaging, and Three-Dimensional Temperature Distribution.
(10) Bardhan, R.; Lal, S.; Joshi, A.; Halas, N. J. Theranostic Nanoshells: Eur. Urol. 2007, 52, 1653−1662.
From Probe Design to Imaging and Treatment of Cancer. Acc. Chem. (28) Guardia, P.; Di Corato, R.; Lartigue, L.; Wilhelm, C.; Espinosa, A.;
Res. 2011, 44, 936−946. Garcia-Hernandez, M.; Gazeau, F.; Manna, L.; Pellegrino, T.

2445 DOI: 10.1021/acsnano.5b07249

ACS Nano 2016, 10, 2436−2446
ACS Nano Article

Water-Soluble Iron Oxide Nanocubes with High Values of Specific Treatment: Gold-Nanoparticle-Mediated Thermal Therapies. Small
Absorption Rate for Cancer Cell Hyperthermia Treatment. ACS Nano 2011, 7, 169−183.
2012, 6, 3080−3091. (47) Donaldson, K.; Murphy, F. A.; Duffin, R.; Poland, C. A. Asbestos,
(29) Martinez-Boubeta, C.; Simeonidis, K.; Makridis, A.; Angelakeris, Carbon Nanotubes and the Pleural Mesothelium: A Review of the
M.; Iglesias, O.; Guardia, P.; Cabot, A.; Yedra, L.; Estradé, S.; Peiró, F. Hypothesis Regarding the Role of Long Fibre Retention in the Parietal
Learning from Nature to Improve the Heat Generation of Iron-Oxide Pleura, Inflammation and Mesothelioma. Part. Fibre Toxicol. 2010, 7, 5.
Nanoparticles for Magnetic Hyperthermia Applications. Sci. Rep. 2013, (48) Bogart, L. K.; Taylor, A.; Cesbron, Y.; Murray, P.; Lévy, R. l.
3.10.1038/srep01652 Photothermal Microscopy of the Core of Dextran-Coated Iron Oxide
(30) Lee, J.-H.; Jang, J.-t.; Choi, J.-s.; Moon, S. H.; Noh, S.-h.; Kim, Nanoparticles During Cell Uptake. ACS Nano 2012, 6, 5961−5971.
J.-w.; Kim, J.-G.; Kim, I.-S.; Park, K. I.; Cheon, J. Exchange-Coupled (49) Jordan, A.; Scholz, R.; Wust, P.; Fähling, H.; Felix, R. Magnetic
Magnetic Nanoparticles for Efficient Heat Induction. Nat. Nanotechnol. Fluid Hyperthermia (Mfh): Cancer Treatment with Ac Magnetic Field
2011, 6, 418−422. Induced Excitation of Biocompatible Superparamagnetic Nanoparticles.
(31) Lartigue, L. n.; Hugounenq, P.; Alloyeau, D.; Clarke, S. P.; Lévy, J. Magn. Magn. Mater. 1999, 201, 413−419.
M.; Bacri, J.-C.; Bazzi, R.; Brougham, D. F.; Wilhelm, C.; Gazeau, F. (50) Abdulla-Al-Mamun, M.; Kusumoto, Y.; Zannat, T.; Horie, Y.;
Cooperative Organization in Iron Oxide Multi-Core Nanoparticles Manaka, H. Au-Ultrathin Functionalized Core−Shell (Fe3O4@Au)
Potentiates Their Efficiency as Heating Mediators and Mri Contrast Monodispersed Nanocubes for a Combination of Magnetic/Plasmonic
Agents. ACS Nano 2012, 6, 10935−10949. Photothermal Cancer Cell Killing. RSC Adv. 2013, 3, 7816−7827.
(32) Di Corato, R.; Espinosa, A.; Lartigue, L.; Tharaud, M.; Chat, S.; (51) Espinosa, A.; Bugnet, M.; Radke, G.; Neveu, S.; Botton, G.;
Wilhelm, C.; Abou-Hassan, A. Can Magneto-Plasmonic Nanohybrids
Pellegrino, T.; Ménager, C.; Gazeau, F.; Wilhelm, C. Magnetic
Efficiently Combine Photothermia with Magnetic Hyperthermia?
Hyperthermia Efficiency in the Cellular Environment for Different
Nanoscale 2015, 7, 18872−18877.
Nanoparticle Designs. Biomaterials 2014, 35, 6400−6411.
(52) Balasubramanian, S.; Girija, A. R.; Nagaoka, Y.; Fukuda, T.; Iwai,
(33) Soukup, D.; Moise, S.; Céspedes, E.; Dobson, J.; Telling, N. D.
S.; Kizhikkilot, V.; Kato, K.; Maekawa, T.; Nair, S. D. An ‘All in One’
In Situ Measurement of Magnetization Relaxation of Internalized Approach for Simultaneous Chemotherapeutic, Photothermal and
Nanoparticles in Live Cells. ACS Nano 2015, 9, 231−240. Magnetic Hyperthermia Mediated by Hybrid Magnetic Nanoparticles.
(34) Chu, M.; Shao, Y.; Peng, J.; Dai, X.; Li, H.; Wu, Q.; Shi, D. Near- RSC Adv. 2015, 5, 25066−25078.
Infrared Laser Light Mediated Cancer Therapy by Photothermal Effect (53) Wang, J.; Zhou, Z.; Wang, L.; Wei, J.; Yang, H.; Yang, S.; Zhao, J.
of Fe3o4Magnetic Nanoparticles. Biomaterials 2013, 34, 4078−4088. CoFe2O4@MnFe2O4/Polypyrrole Nanocomposites for in Vitro Photo-
(35) Shen, S.; Kong, F.; Guo, X.; Wu, L.; Shen, H.; Xie, M.; Wang, X.; thermal/Magnetothermal Combined Therapy. RSC Adv. 2015, 5,
Jin, Y.; Ge, Y. Cmcts Stabilized Fe3o4 Particles with Extremely Low 7349−7355.
Toxicity as Highly Efficient near-Infrared Photothermal Agents for (54) Kolosnjaj-Tabi, J.; Di Corato, R.; Lartigue, L.; Marangon, I.;
in Vivo Tumor Ablation. Nanoscale 2013, 5, 8056−8066. Guardia, P.; Silva, A. K.; Luciani, N.; Clément, O.; Flaud, P.; Singh, J. V.
(36) Chen, H.; Burnett, J.; Zhang, F.; Zhang, J.; Paholak, H.; Sun, D. Heat-Generating Iron Oxide Nanocubes: Subtle “Destructurators” of
Highly Crystallized Iron Oxide Nanoparticles as Effective and the Tumoral Microenvironment. ACS Nano 2014, 8, 4268−4283.
Biodegradable Mediators for Photothermal Cancer Therapy. J. Mater. (55) Liu, Y.; Liu, L.; Chen, M.; Zhang, Q. Double Thermal Transitions
Chem. B 2014, 2, 757−765. of Type I Collagen in Acidic Solution. J. Biomol. Struct. Dyn. 2013, 31,
(37) Chen, C.-L.; Kuo, L.-R.; Lee, S.-Y.; Hwu, Y.-K.; Chou, S.-W.; 862−873.
Chen, C.-C.; Chang, F.-H.; Lin, K.-H.; Tsai, D.-H.; Chen, Y.-Y.
Photothermal Cancer Therapy Via Femtosecond-Laser-Excited Fept
Nanoparticles. Biomaterials 2013, 34, 1128−1134.
(38) Zhou, Z.; Sun, Y.; Shen, J.; Wei, J.; Yu, C.; Kong, B.; Liu, W.; Yang,
H.; Yang, S.; Wang, W. Iron/Iron Oxide Core/Shell Nanoparticles for
Magnetic Targeting Mri and near-Infrared Photothermal Therapy.
Biomaterials 2014, 35, 7470−7478.
(39) Shen, S.; Wang, S.; Zheng, R.; Zhu, X.; Jiang, X.; Fu, D.; Yang, W.
Magnetic Nanoparticle Clusters for Photothermal Therapy with near-
Infrared Irradiation. Biomaterials 2015, 39, 67−74.
(40) Institute, A. N. S. American National Standard for the Safe Use of
Lasers; Inc., Laser Institute of America: New York, 2000.
(41) Guardia, P.; Riedinger, A.; Nitti, S.; Pugliese, G.; Marras, S.;
Genovese, A.; Materia, M. E.; Lefevre, C.; Manna, L.; Pellegrino, T. One
Pot Synthesis of Monodisperse Water Soluble Iron Oxide Nanocrystals
with High Values of the Specific Absorption Rate. J. Mater. Chem. B
2014, 2, 4426−4434.
(42) He, Y.; Miao, Y.; Li, C.; Wang, S.; Cao, L.; Xie, S.; Yang, G.; Zou,
B.; Burda, C. Size and Structure Effect on Optical Transitions of Iron
Oxide Nanocrystals. Phys. Rev. B: Condens. Matter Mater. Phys. 2005, 71,
125411.
(43) Cornell, R.; Schwertmann, U. Introduction to the Iron Oxides. In
Iron Oxides: Structure, Properties, Reactions, Occurences and Uses, Second
Edition; Wiley: Weinheim, 2004; pp 1−7.
(44) Tang, J.; Myers, M.; Bosnick, K. A.; Brus, L. E. Magnetite Fe3O4
Nanocrystals: Spectroscopic Observation of Aqueous Oxidation
Kinetics. J. Phys. Chem. B 2003, 107, 7501−7506.
(45) Hergt, R.; Dutz, S. Magnetic Particle HyperthermiaBiophysical
Limitations of a Visionary Tumour Therapy. J. Magn. Magn. Mater.
2007, 311, 187−192.
(46) Kennedy, L. C.; Bickford, L. R.; Lewinski, N. A.; Coughlin, A. J.;
Hu, Y.; Day, E. S.; West, J. L.; Drezek, R. A. A New Era for Cancer

2446 DOI: 10.1021/acsnano.5b07249

ACS Nano 2016, 10, 2436−2446