Biomaterials 39 (2015) 67e74

Contents lists available at ScienceDirect

Biomaterials
journal homepage: www.elsevier.com/locate/biomaterials

Magnetic nanoparticle clusters for photothermal therapy with

near-infrared irradiation
Shun Shen a, 1, Sheng Wang a, c, 1, Rui Zheng b, Xiaoyan Zhu d, Xinguo Jiang a, Deliang Fu c,
Wuli Yang b, *
a

School of Pharmacy & Key Laboratory of Smart Drug Delivery, Fudan University, Shanghai 201203, China
State Key Laboratory of Molecular Engineering of Polymers & Department of Macromolecular Science, Fudan University, Shanghai 200433, China
Pancreatic Disease Institute, Department of Pancreatic Surgery, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai 200040, China
d
Department of Integrative Oncology, Shanghai Cancer Center, Department of Oncology, Shanghai Medical University, Fudan University,
Shanghai 200032, China
b
c

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 20 August 2014
Accepted 19 October 2014
Available online 15 November 2014

In this study, the photothermal effect of magnetic nanoparticle clusters was rstly reported for the
photothermal ablation of tumors both in vitro in cellular systems but also in vivo study. Compared with
individual magnetic Fe3O4 nanoparticles (NPs), clustered Fe3O4 NPs can result in a signicant increase in
the near-infrared (NIR) absorption. Upon NIR irradiation at 808 nm, clustered Fe3O4 NPs inducing higher
temperature were more cytotoxic against A549 cells than individual Fe3O4 NPs. We then performed
in vivo photothermal therapy (PTT) studies and observed a promising tumor treatment. Compared with
PBS and individual magnetic Fe3O4 NPs by NIR irradiation, the clustered Fe3O4 NPs treatment showed a
higher therapeutic efcacy. The treatment effects of clustered Fe3O4 NPs with different time of NIR
illumination were also evaluated. The result indicated that a sustained high temperature generated by
NIR laser with long irradiation time was more effective in killing tumor cells. Furthermore, histological
analysis of H&E staining and TUNEL immunohistological assay were further employed for antitumor
efcacy assessment of PTT against A549 tumors.
2014 Elsevier Ltd. All rights reserved.

Keywords:
Magnetic nanoparticle clusters
Fe3O4
Tumor
Photothermal therapy

1. Introduction
Nanomaterials have been extensively used in biomedical
research during the past twenty years [1e4]. Especially in recent
years, more and more studies focused on photo-absorbing nanoagents, and photothermal therapy (PTT) employing photoabsorbers
can convert optical energy into thermal energy to kill cancer cells
without affecting healthy tissues [5,6]. Compared with radiotherapy, chemotherapy and surgical management, PTT is less
invasive, controllable and highly efcient. Our and other research
groups have developed a large number of nanomaterials as PTT
agents, such as gold-based nanomaterials [5,7e9], carbon nanotubes and graphene [10,11], all of which show strong optical
absorbance in the near-infrared (NIR) tissue optical transparency
window. The previous results demonstrated that photoabsorberbased therapy might be a promising approach for cancer therapy,

* Corresponding author. Tel.: 86 21 65642385.

E-mail address: wlyang@fudan.edu.cn (W. Yang).
1
Both authors contributed equally to this work.
http://dx.doi.org/10.1016/j.biomaterials.2014.10.064
0142-9612/ 2014 Elsevier Ltd. All rights reserved.

which could effectively reduce tumor growth and enhance survival

[12e15]. Even so, the potential toxicity induced by photothermal
agents (especially for carbon nanotubes or graphene, and so on), is
still an unresolved debate [16,17], which will inevitably limit future
clinic applications of PTT. These non-degradable or slowly
degradable nanoparticles easily accumulate in bodily organs,
resulting in increased oxidative stress, inammatory cytokine
production and cell death [18]. Therefore, it is signicant to explore
a bio-safety and biodegradable photoabsorber for the photothermal
ablation of cancer with NIR irradiation.
Magnetic iron oxides nanoparticles (namely Fe3O4 NPs) have
received tremendous attention for their excellent magnetic,
biocompatible and potentially non-toxic properties [19,20]. Fe3O4
NPs, which can be manipulated and controlled by an external
magnetic eld, are additional important materials and have been
employed in many areas, including biology, pharmaceuticals and
diagnostics. Moreover, iron is a nutrient and readily metabolized by
cellular regulation using the transferrin pathway. Thus, Fe3O4 NPs
are easily degradable and passes in and out of cells across the
plasma membrane [21]. For the acknowledged advantages of Fe3O4
NPs, they are extremely suitable for in vivo applications. In addition,

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S. Shen et al. / Biomaterials 39 (2015) 67e74

Fe3O4 NPs are exceptional materials for hyperthermia treatment of

tumors [22,23]. Under an alternating magnetic eld (AMF), magnetic hyperthermia of Fe3O4 NPs is produced via dipole relaxation,
which can be employed to destroy tumor cells because these cells
are more sensitive to temperatures in excess of ca. 41  C than their
normal counterparts [24]. Although magnetic hyperthermia has
been clinically used [25e28], the technique requires high current
and voltage due to large air volume within the applied eld in
which energy cannot be easily focused [29].
Very recently, NIR light induced photothermal effect for Fe3O4
NPs has been studied and it exhibits good photothermal converting
efciency. For example, Chu et al. applied individual Fe3O4 NPs with
high concentration for the photothermal ablation of cancer by NIR
laser irradiation [29]. Considering extra-high-dose magnetite
might generate potential toxicity to the body, the number of usable
elements should also be severely limited. Thus, Fe3O4 NPs used for
PTT must be improved, mainly involving two routes. One is to
modify NIR light-absorbing materials onto the surface of magnetic
NPs, another is to concentrate the magnetic NPs. Newly, it has been
shown that the clustering of magnetic NPs induces a signicant
increase in the magnetic moment, and consequently, the clustered
magnetic NPs have a much higher relatively high saturation
magnetization and specic absorption rate (SAR) than individual
magnetic NPs [30e33]. Hayashi et al. have utilized the unique merit
of magnetite clusters for the magnetic hyperthermia therapy of
tumors by AMF [32,33]. More signicantly, previous studies have
also indicated that metallic NPs clusters can induce a red-shift in
the light absorption spectra [34,35], which enhances the light
absorbance in the NIR region, expanding new application elds for
metallic NPs clusters to be utilized as photosensitizers in the NIR
PTT. Therefore, numerous papers have been reported the photothermal ablation of tumors by utilizing aggregation-induced
enhanced photothermo (AIEP) of metallic NPs clusters [36,37],
such as gold NPs aggregation. Until now, to the best of our
knowledge, the papers on the study of photothermal effect of
magnetite clusters were rarely reported, let alone for the use of
magnetite clusters with NIR irradiation to destroy tumor in vivo.
Herein, we present the synthesis of magnetic colloidal nanocrystal clusters that effectively produce heat in response to harmless NIR irradiation. The biomedical parameters were not only
assessed in vitro in cellular systems but also in vivo study. The results proved that the photothermal effects of clustered magnetic
NPs for the treatment of A549 (Human lung adenocarcinoma
epithelial cell line) tumor were better than those obtained by single
crystal magnetic NPs.

temperature in nitrogen atmosphere, and NaOH solution (10 M, 50 mL) was then
slowly dropped into the ask from the dropping funnel in 1 h. After that, the mixture
was continuously stirred at room temperature for 1 h, which was then stirred at
90  C for another 2 h and cooled to room temperature under continuous stirring. The
whole process was in the nitrogen atmosphere. The products were washed with
deionized water three times, collected with the help of a magnet. After that, nitric
acid solution (2 M, 100 mL) was added into the above products, which were then
washed with deionized water several times and collected with the magnet, until the
pH value of the supernatant is neutral. Then 100 mL (0.3 M) of trisodium citrate was
added into the resulting products, the mixture was stirred for 30 min at 90  C in the
nitrogen atmosphere. Then it was cooled to room temperature, transferred into a
beaker and collected with the magnet, and 20 mL of deionized water and a lot of
acetone were added into the beaker to wash the products twice. Finally, the obtained
products were dispersed in 50 mL of deionized water, and stirred at 80  C for a long
time to remove acetone.

2. Materials and methods

2.7. Cell viability assay

2.1. Materials

A549 cells were seeded in 96-well plates with a density of 104 cells/well, and
allowed to adhere for 24 h prior to assay. The cells were exposed to the clustered
magnetic NPs or individual magnetic NPs with the same concentration of 50 mg/mL,
respectively. The cells were or were not irradiated by NIR laser light at a power
density of 5 W/cm2 with different illumination time from 60 to 180 s. After the cells
were incubated at 37  C for another 24 h, they were incubated with 0.5 mg/mL MTT
in DMEM for 4 h in dark and then mixed with dimethyl sulfoxide after the supernatant was removed. The OD value at 570 nm was read using the microplate reader
(Synergy TM2, BIO-TEK Instruments Inc. USA). Cell viability was determined by the
percentage of OD value of the study group over the control group. Afterward, cells
were co-stained by a mixture of Calcein AM and PI solution for 20 min. The samples
were subjected to observe using a ZEISS LSM710 live cell confocal laser imaging
System (Carl Zeiss, German).

Iron (III) chloride (FeCl3$6H2O), iron (II) chloride (FeCl2$4H2O), trisodium citrate
dehydrate (C8H5Na3O7$2H2O), sodium acetate anhydrous (NaOAc), ethylene glycol
(EG), acetone, sodium hydroxide (NaOH), and nitric acid (HNO3) were purchased
from Shanghai Chemical Reagents Company (China). MTT (3-(4, 5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) assay, and other biological reagents were
purchased from Invitrogen Corp. Dulbecco's modied Eagle medium (DMEM), Fetal
bovine serum (FBS), Penicillin-Streptomycin solution and Trypsin-EDTA solution
were purchased from Gibco (Tulsa, OK, USA). All the other chemicals were of
analytical grade, and puried water was produced by a Millipore water purication
system.

2.3. Synthesis of clustered magnetic NPs

The clustered magnetic NPs (magnetic particles) were prepared by a modied
solvothermal reaction [39]. Briey, FeCl3$6H2O (1.028 g), C8H5Na3O7$2H2O
(0.24 g), and NaOAc (1.2 g) were rst dissolved in 20 mL of EG under vigorous
stirring for 0.5 h. The resulting solution was then transferred into a Teon-lined
stainless-steel autoclave with a capacity of 50 mL. The autoclave was sealed and
heated at 200  C for 10 h. Then it was cooled to room temperature. The asprepared black products were washed with ethanol and deionized water several
times, and collected with a magnet. The nal products were dispersed in 10 mL of
ethanol for the further use.
2.4. Characterization
Ultravioletevisible (UVevis) spectra were performed using a PerkineElmer
Lambda 750 spectrophotometer. Transmission electron microscopy (TEM) images
were obtained on a Tecnai G2 20 TWIN transmission electron microscope. Magnetic
characterization was carried out with a vibrating sample magnetometer (VSM) on a
Model 6000 physical property measurement system (Quantum, USA) at 300 K. X-ray
diffraction (XRD) measurements were recorded on a X'pert PRO diffractometer to
determine the composition of Fe3O4 particles. All the diffraction peaks in the XRD
patterns were indexed and assigned to the typical cubic structure of Fe3O4 (JCPDS
75-1609).
2.5. Cell lines and culture conditions
A549 cells obtained from Chinese Academy of Sciences Cells Bank, Shanghai,
China, were routinely cultured in RPMI-1640 cell medium supplemented with 10%
FBS, 100 U/mL penicillin and 100 mg/mL streptomycin, at 37  C in 5% CO2 and 95% air
atmosphere with >95% humidity. All experiments were performed on cells in the
logarithmic phase of growth.
2.6. NIR-heating effect of magnetite NPs in solution
The individual magnetic NPs or clustering of magnetic NPs stock solution at
1 mg/mL was diluted to the different concentrations (10e80 mg/mL) and 200 mL of
aliquots were deposited into wells of a 48-well cell culture plate. Wells were illuminated by an 808-nm continuous-wave NIR laser (Changchun New Industries
Optoelectronics Technology, Changchun, China; uence: 5 W/cm2, spot size: 5 mm)
with the different exposure time from 60 to 180 s. Pre- and postillumination temperatures were taken by thermocouple.

2.2. Synthesis of individual magnetic NPs

The individual magnetic NPs were prepared by chemical coprecipitation method
[38]. In a typical recipe, FeCl3$6H2O (10.81 g, 0.04 mol) was dissolved in 50 mL
deionized water. The resulting solution was transferred into a three-necked ask
(250 mL), which was equipped with a mechanical stirrer, a dropping funnel, and a
nitrogen inlet. Then 3.98 g of FeCl2$4H2O (0.02 mol) was also dissolved in another
50 mL deionized water, and the resulting solution was transferred into the abovementioned three-necked ask. The as-prepared mixture was then stirred at room

2.8. Flow cytometry analysis

To investigate the photothermal ablation of A549 cells by clustered Fe3O4 NPs
without or with the 808 nm laser irradiation using ow cytometry, clustered
Fe3O4 NPs dispersion (100 mg/mL in PBS solution) was added to a 12-well cell
culture plate containing A549 cells with a density of 5  105 cells/well in RPMI1640 medium supplemented with 10% FBS and 1% penicillinestreptomycin, then
the A549 cells were incubated for 4 h at 37  C in 5% CO2 and 95% air atmosphere

S. Shen et al. / Biomaterials 39 (2015) 67e74

with >95% humidity. Then, a group of cell solution was exposed to an 808 nm
laser at a power intensity of 5 W/cm2 for 180 s. After the laser irradiation treated,
A549 cells were stained with Annexin-V-FITC and PI (Becton Dickinson, Mountain View, CA, USA), and analyzed by BD FACSAria I ow cytometry with excitation at 488 nm. Fluorescent emission of FITC was measured at 515e545 nm and
that of DNA-PI complexes at 564e606 nm. Compensation was used wherever
necessary.
2.9. In vivo photothermal treatment
Male Balb/c mice, aging 4e5 weeks and weighing 18.8 1.9 g, were supplied by
the Department of Experimental Animals, Fudan University (Shanghai, China), and
maintained under standard housing conditions. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of Fudan University. Through a subcutaneous injection of 2  106 cells
suspended in 100 mL PBS into the ank region, the mice bearing A549 tumor were
successfully established. The longest dimension and the shortest dimension of tumors were monitored by digital calipers. Once tumors were measured ~5.0 mm in
longest dimension, mice were randomized into 4 treatment groups (n 5 per
group): PBS with laser treated (Group I), individual Fe3O4 NPs with laser treated
(Group II), clustered Fe3O4 NPs with laser treated (Group III and IV for different NIR
illumination time). Each type of dispersion (2 mg/mL, injection volume of 25 mL) was
intratumorally injected into mice. Two hours after injection, the 808-nm
continuous-wave NIR laser was applied to irradiate tumors under a power intensity of 5 W/cm2 (Group I, II and IV for 180 s, Group III for 120 s). The temperature
was recorded using an infrared camera thermographic system (InfraTec, VarioCAMhr research, German). After treatment, all mice were returned to animal
housing and tumor volumes were tracked every 2 days by digital caliper measurements. The tumor volume was calculated by the formula V 1/2(L$W2), where L is
length (longest dimension) and W is width (shortest dimension). At 19th day, all the
animals were euthanized, and the tumors were dissected and weighed. According to
our previous reports [40e43], some histological examinations included hematoxylin
and eosin (H&E) stains and TUNEL assays were carried out for the assessment of
therapy efcacy.

69

2.10. The retention of the magnetic NPs in the tumor sites

According to the previous literature [29], the retention of the Fe3O4 NPs in the
tumor sites was also studied. Once tumors were measured ~5.0 mm in longest
dimension, mice were randomized into 2 groups (n 9 per group): individual Fe3O4
NPs with laser treated (Group I), clustered Fe3O4 NPs with laser treated (Group II).
Each type of dispersion (2 mg/mL, injection volume of 25 mL) was intratumorally
injected into mice. Two hours after injection, the 808-nm continuous-wave NIR laser
was applied to irradiate tumors under a power intensity of 5 W/cm2 for 180 s. They
were sacriced on day 1, 5 and 19 after injection, respectively. Three mice for one
group were randomly selected and sacriced at each time point and the tumors
were extracted immediately after. The tumors were weighed and washed with
distilled water. Afterward, the tissue was mixed with 0.5 mL of aqua regia and
incubated at room temperature. Two days later, the samples were centrifuged and
the upper clear solutions were collected and adjusted with distilled water. The
concentrations of iron atoms in the solutions were performed by ame atomic absorption spectroscopy (Hitachi, Japan). An iron hollow cathode lamp was used as a
light source. The excitation current, slit width and absorption wavelength of iron
were set at 15 mA, 0.2 nm and 248.3 nm, respectively.
2.11. Statistical analysis
Unpaired student's t test was used for between two-group comparison and oneway ANOVA with Fisher's LSD for multiple-group analysis. A probability (P) less than
0.05 was considered statistically signicant. Results were expressed as
mean standard deviation (SD) unless otherwise indicated.

3. Results and discussion

3.1. Characterization of magnetic NPs
A TEM image shows the dimension of our obtained individual
Fe3O4 NPs is about 15 nm (Fig. 1a), while that of the clustered Fe3O4

Fig. 1. TEM images of (a) individual magnetic Fe3O4 NPs and (b) clustered magnetic Fe3O4 NPs. (c) The XRD pattern of individual and clustered magnetic Fe3O4 NPs. (d) Magnetization loops of individual and clustered magnetic Fe3O4 NPs.

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S. Shen et al. / Biomaterials 39 (2015) 67e74

NPs has a nearly uniform size of ca. 225 nm with spherical shape
(Fig. 1b). A TEM image of the clustered Fe3O4 NPs at higher
magnication (Fig. S1) illustrates that the magnetite conglobulations are loose clusters, and the particles are composed of
nanocrystals with the size of about 5e10 nm. According to the
previously reported literature [44], these nanocrystals are connected with each other by amorphous matrix as a bridge. The
samples of individual and clustered Fe3O4 NPs both show similar
typical XRD patterns of magnetite (JCPDS no. 75-1609; Fig. 1c),
which indicates that the nanocrystalline structure of the clustered
Fe3O4 NPs is the same as individual Fe3O4 NPs. The magnetic
property characterization (Fig. 1d) shows that both individual and
clustered Fe3O4 NPs have superparamagnetic property and high
magnetization with saturation value of 58.69 emu/g for the individual Fe3O4 NPs, and 63.70 emu/g for the clustered Fe3O4 NPs,
respectively.
3.2. Photothermal effect of magnetic NPs
Fig. 2a illustrates the UVevis spectra of the individual and
clustered Fe3O4 NPs dispersed in deionized water. The individual
Fe3O4 NPs have a very weak absorption in the NIR spectroscopy
from 600 to 1000 nm. Surprisingly, the clustered Fe3O4 NPs show an
obvious absorption band containing a maximum at ca. 420 nm.
Moreover, compared with individual Fe3O4 NPs, the clustered Fe3O4
NPs also resulted in a signicant ~3.6 fold increase in the NIR absorption at 808 nm for the aggregation of nanoparticles. Thus, the
clustered Fe3O4 NPs that have a stronger absorption in the NIR
spectroscopy may be conducive to destroying tumors in vivo as an
effective thermal generator due to its in-depth tissue penetration.
The photothermal effects of the PBS solutions of the individual and
clustered Fe3O4 NPs were rstly examined and compared in vitro by
NIR laser irradiation (l 808 nm, 5 W/cm2). As seen from Fig. 2b,
the clustered Fe3O4 NPs were more efcient than the individual
Fe3O4 NPs in inducing a temperature increase with the same concentration and exposure time. For example, the temperature raised
by the clustered Fe3O4 NPs solution could reach 51.4 1.2  C with
the NPs concentration of 50 mg/mL and NIR exposure time of 180 s,
while that of the individual Fe3O4 NPs only reached 42.0 1.5  C.
Compared with the previous report using individual Fe3O4 NPs
with high concentration [29], the higher temperature was easily
acquirable by clustered Fe3O4 NPs with lower concentration and

shorter NIR irradiation time in our study, which was benecial for
the safe application in vivo. In addition, the NIR-heating effects of
the individual and clustered Fe3O4 NPs were both time and
concentration-dependent. The NPs with higher concentration
performed markedly better than that of low concentration. Likewise, the longer the laser irradiation time, the higher the solution
temperature is. The results indicate that optoelectronic excitation
of clustered Fe3O4 NPs by NIR irradiation can occur rapidly, and the
extra energies are efciently transferred into molecular vibration
modes to generate signicant amounts of thermal energy, which
could be useful as photothermal mediators.
In order to validate the feasibility of the Fe3O4 NPs in biomedicine, the nanomaterials of individual or clustered Fe3O4 NPs were
administered to A549 cells in 96-well plates, respectively, and each
group was subdivided into groups of with and without NIR laser
exposure. The cell counting Kit-8 (CCK-8) assay was employed for
the quantitative evaluation of cell viability [45]. The viability of
untreated cells was assumed to be 100%. The cell viability remained
about 92.63% when they were incubated with individual or clustered Fe3O4 NPs at higher concentration of 500 mg/mL for 24 h
(Fig. S2, Supporting information), which indicated our magnetic
nanomaterials had no inherent toxicity. Subsequently, the magnetic
nanomaterials were applied for the photothermal ablation of cancer cells. The cytotoxic effects by NIR irradiation were shown in
Fig. 3a. About 8.9%, 33.5% and 72.8% of cells were killed by clustered
Fe3O4 NPs at the concentration of 50 mg/mL with a power density of
5 W/cm2 for different illumination time of 60, 120 and 180 s,
respectively. And only 0.8%, 3.5% and 14.5% of cells were killed by
individual Fe3O4 NPs. In addition, direct irradiation of the cells
remained close to 100%, exhibiting no effect on cell viability. This is
mainly because the low light absorption by natural endogenous
cytochromes of these cells caused minimal temperature elevation
accounts for their high survival [5]. The CCK-8 experimental result
indicated that clustered Fe3O4 NPs inducing higher temperature
were more cytotoxic against A549 cells than individual Fe3O4 NPs
by NIR irradiation. These results also proved that cancer cells could
be effectively killed by clustered Fe3O4 NPs with lower concentration and shorter NIR irradiation time compared with previous
report [29]. Fluorescence images of calcein AM (green, live cells)
and PI (red, dead cells) co-stained cells after PTT were further
conrmed the effectiveness of PTT using clustered Fe3O4 NPs
(Fig. 3b). A large number of killed A549 cells were observed by

Fig. 2. (a) UVevis spectra of aqueous dispersion of individual and clustered magnetic Fe3O4 NPs. (b) The photothermal effects of the PBS solutions of individual and clustered
magnetic Fe3O4 NPs with the concentration of 0e80 mg/mL were examined ex vitro by irradiating NIR (l 808 nm, 5 W/cm2) for 60e180 s. Temperature was measured by
thermocouple.

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71

Fig. 3. (a) Relative viabilities of PBS, individual and clustered magnetic Fe3O4 NPs with the concentration of 50 mg/mL treated A549 cells without or with NIR laser irradiation
(l 808 nm, 5 W/cm2) for 60e180 s. (b) Confocal uorescence images of calcein AM (green, live cells) and propidium iodide (red, dead cells) co-stained A549 cells treated by
clustered magnetic Fe3O4 NPs at the concentration of 50 mg/mL with NIR laser irradiation (l 808 nm, 5 W/cm2) for (1) 0 s, (2) 60 s, (3) 120 s, (4) 180 s. (c) Flow cytometry graphs of
A549 cells treated by clustered magnetic Fe3O4 NPs at the concentration of 50 mg/mL without or with NIR laser irradiation (l 808 nm, 5 W/cm2) for 180 s. The treated A549 cells
were double stained by Annexin-V-FITC/PI and analyzed by ow cytometry. Quadrant I doesn't representatively correspond to live or damaged cells; Quadrant II representatively
corresponds to the population of dying cells. The membranes of these cells had been compromised so PI could penetrate and then hybridize with their DNAs; Quadrant III represents
the population of live cells. Since the live cells have intact cell membranes, PI could not stain their DNAs; The population of dead cells, or stained DNAs, exhibits a high PI and small
forward scattering signal as shown in quadrant IV. These are free DNAs, no longer enclosed within a membrane. The analysis data reveal that cells targeted by the clustered
magnetic Fe3O4 NPs combined with NIR lighting display irreversible damage, as shown by the larger population in quadrant IV, where the cellular membrane is broken to such an
extent that the cell can no longer function nor recover from the damage [46].

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S. Shen et al. / Biomaterials 39 (2015) 67e74

clustered Fe3O4 NPs exposed to laser illumination with a power

density of 5 W/cm2 for 180 s. Once the irradiation time was reduced
to 120 s, most of cells remained survival. In addition, cells incubated
with clustered Fe3O4 NPs for irradiation time of 60 s or without NIR
laser treatment both showed limited damage, which could be
attributable to insufciently lethal temperature rise. The uorescence imaging result was consistent with the CCK-8 result.
To make clear the cell death mode after photothermal treatment, an Annexin-V-FITC/PI method was conducted by ow
cytometry. Fig. 3c showed the ow cytometry graphs of the cells by
clustered Fe3O4 NPs with and without laser irradiation. Annexin-VFITC emission signal was plotted on the x-axis, while PI emission
signal was plotted on the y-axis. The graphs quadrants were
divided into four quadrants. The quantities of living cells, apoptotic
cells and necrotic cells were determined by the percentage of
Annexin V/PI, Annexin V/PI and Annexin V/PI. Almost no
apoptosis or necrosis cells were observed in the group of no NIR
treatment. After laser irradiation, the apoptosis rate of cells reached
67.7%, while the necrosis rate was only 6.4%. The ow cytometry
data revealed that cells treated by clustered Fe3O4 NPs displayed
irreversible damage upon photothermal treatment, and their
cellular membranes were broken to such an extent that the cells
could no longer function nor recover from the damage. Therefore,
the mechanism of in vitro PTT is mainly triggered by cell apoptosis,
not necrosis.
3.3. Photothermal ablation of tumor in vivo
Encouraged by the effective photothermal outcome in vitro, we
next performed a pilot in vivo photothermal treatment study. In
this work, A549 tumor-bearing mice were established by subcutaneous injection of 2  106 A549 cells suspended in RMPI-1640
medium into the ank region. Once the tumors were measured
ca. 5.0 mm in longest dimension, mice were randomized into four
treatment groups (n 5 per group) mentioned in the experimental
section. There was no statistical difference among group mean
tumor volumes at the onset of treatment (P > 0.05). Twenty-four
hours before irradiation, mice were then injected intratumorally
with 25 mL of treatment solution containing 50 mg of individual or
clustered Fe3O4 NPs in Group II, III and IV, leaving light gray scars on
the original tumor sites. The tumors were illuminated with an 808nm NIR laser (5 W/cm2; spot size, 5 mm) for 120 s in Group III and
for 180 s in Group II, IV. No mice died during the course of therapy.

Fig. 4 showed the infrared thermal images of tumor surface in

Group I, II and IV mice. In Group IV, the maximum temperature of
the tumor surface rapidly increased 21.6  C to reach 55.9  C
attributing to the clustered Fe3O4 NPs with a good NIR-heating
property. In addition, as can be seen from the gure, the increase
in NIR irradiation time from 120 s to 180 s could not signicantly
increase the temperature of tumor surface, showing the temperature reached a balance after two minutes of NIR irradiation. At that
time, the NIR laser didn't stop working, and kept irradiating for one
minute to maintain a high temperature in the tumor site to effectively kill A549 cells. In Group II, the maximum temperature of the
tumor surface reached 50.3  C, which was inferior to Group IV due
to the weak photothermal effects. In contrast, less temperature
changed in Group I by PBS with NIR treated, and increased only
4.2  C. Furthermore, in order to investigate the effect of PTT using
clustered Fe3O4 NPs with different NIR irradiation time, the 808nm continuous-wave NIR laser was employed to irradiate tumors
under a power intensity of 5 W/cm2 for 120 s in Group III.
Compared with Group IV, the duration time of high temperature in
Group III reduced one minute. As we know, high temperature can
lead to cell apoptosis, even instantaneous coagulative necrosis and
irreversible cell death [47]. Tumor sizes after diverse treatments
were measured every two days (Fig. 5a). The tumors of Group I, II
and III grew rapidly. There was no statistically signicant difference
in nal tumor size (P > 0.05), indicating that tumor growth was not
affected by PBS or individual Fe3O4 NPs with NIR irradiation for
180 s, and clustered Fe3O4 NPs with NIR irradiation time of 120 s.
On the contrary, a statistically signicant of tumor growth was
observed in mice treated with clustered Fe3O4 NPs plus NIR laser
for illumination time of 180 s (P < 0.05). Effects were most pronounced in the Group IV, where mean tumor volume at 19 days
was reduced from 955.3 mm3 in controls to 222.8 mm3
(P 0.0139). At 19th days, mice were euthanized, tumors were
excised and weighted (Fig. 5). The weights of tumors for Group I, II,
III, IV and V were 0.5311 0.0599 g, 0.4369 0.0452 g,
0.4072 0.0479 g and 0.2297 0.0333 g, respectively (Fig. 5d).
Obviously, the antitumor efciency of Group IV was particularly
prominent and was superior to all the other groups (P < 0.01),
showing an inhibition rate of 56.75%, 47.43% and 43.59% compared
with Group I, II and III. However, there was no statistically signicant difference in Group III compared with Group I, II, indicating
that a lack of sustained high temperature would hardly effectively
kill the tumor cells.

Fig. 4. Infrared thermal images of PBS, individual and clustered magnetic Fe3O4 NPs with the concentration of 100 mg/mL injected A549 tumor sample under NIR laser irradiation
(l 808 nm, 5 W/cm2) for 0e180 s.

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73

Fig. 5. (a) Tumor growth curves of different groups after treatment. Tumor sizes were measured every 2 days. Means and standard errors are shown. The groups of PBS, individual
magnetic Fe3O4 NPs with NIR irradiation for 180 s and clustered magnetic Fe3O4 NPs with NIR irradiation for 120 s were statistically identical. The antitumor efciency of clustered
magnetic Fe3O4 NPs with NIR irradiation for 180 s was particularly prominent and was superior to all the other groups (P < 0.05). (b) Body weight changes were recorded after
therapy every 2 days. Means and standard errors are shown. (c) Photograph of tumors after excision from PBS, individual and clustered magnetic Fe3O4 NPs under NIR irradiation.
(d) Tumor weights of each group. n.s. no signicant difference, *P < 0.05, **P < 0.01.

Accordingly, histological analysis of H&E staining illustrated

that the tumors in test groups exhibited a scarlike structure containing numerous collagen bundles (Fig. S3), especially for Group
IV. However, a lot of tumor cells were observed in Group I, II and III.
The antitumor efcacy of PTT against A549 tumor was also evaluated by the TUNEL immunohistological assay (Fig. S4), which
exclusively detected apoptosis in tumor tissues. TUNEL signals
(brown uorescence) were observed in Group II, III and IV. Tumor
tissues treated with clustered Fe3O4 NPs plus laser irradiation for
180 s showed more TUNEL signals than did tumors treated with PBS
and individual Fe3O4 NPs plus laser irradiation for 180 s, and
clustered Fe3O4 NPs plus laser irradiation for 120 s, indicating that
photothermal ablation of clustered Fe3O4 NPs with long time NIR
treatment was more efcient for tumor therapy. Last but not the
least, to investigate the metabolism of the Fe3O4 NPs after therapy,
the iron atoms captured in the tumors were determined by a ame
atomic absorption spectroscopy. The analytical results exhibited
that 98.7%, 77.8%, 53.5% of individual Fe3O4 NPs or 99.1%, 72.3%,
46.7% of clustered Fe3O4 NPs in the tumors for 1, 5 and 19 days postinjection with NIR irradiation with 180 s (Fig. S5). The analytical
result showed that our Fe3O4 NPs could be slowly cleared from the
tumors after therapy. Besides, the high Fe3O4 NPs uptake in the rst
few days is particularly important for photothermal cancer treatment if repeated NIR irradiation therapy was needed.
4. Conclusions
The individual and clustered Fe3O4 NPs with the same nanocrystalline structure were successfully synthesized. The clustered
Fe3O4 NPs were more efcient than the individual Fe3O4 NPs in
inducing a temperature increase. Signicantly greater cell killing
was detected when A549 cells incubated with clustered Fe3O4 NPs

were irradiated with NIR illumination. The ow cytometry analysis

demonstrated that the mechanism of in vitro PTT was mainly
triggered by cell apoptosis, not necrosis. Using an A549 tumor
model, a higher therapeutic efcacy was obtained by the NIRinduced hyperthermia of the clustered Fe3O4 NPs. We believe
that our study has the potential in the future clinical application of
cancer therapies for the clustered Fe3O4 NPs with good photothermal effect and excellent biological safety.
Acknowledgment
This work was supported by the National Natural Science
Foundation of China (grant, No. 51273047, 81301974 and
81102847). W. Yang thanks Prof. Hao Zhang (Jilin University) for the
discussion about the properties of Fe3O4.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.biomaterials.2014.10.064.
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