A Near Infrared Light Triggered Hydrogenated Black TiO2 for Cancer Photothermal Therapy

Abstract

White TiO2 nanoparticles (NPs) have been widely used for cancer photodynamic therapy based on their ultraviolet light-triggered properties. To date, biomedical applications using white TiO2 NPs have been limited, since ultraviolet light is a well-known mutagen and shallow penetration. This work is the first report about hydrogenated black TiO2 (H-TiO2 ) NPs with near infrared absorption explored as photothermal agent for cancer photothermal therapy to circumvent the obstacle of ultraviolet light excitation. Here, it is shown that photothermal effect of H-TiO2 NPs can be attributed to their dramatically enhanced nonradiative recombination. After polyethylene glycol (PEG) coating, H-TiO2 -PEG NPs exhibit high photothermal conversion efficiency of 40.8%, and stable size distribution in serum solution. The toxicity and cancer therapy effect of H-TiO2 -PEG NPs are relative systemically evaluated in vitro and in vivo. The findings herein demonstrate that infrared-irradiated H-TiO2 -PEG NPs exhibit low toxicity, high efficiency as a photothermal agent for cancer therapy, and are promising for further biomedical applications.
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A Near Infrared Light Triggered Hydrogenated Black TiO
2
for Cancer Photothermal Therapy
Wenzhi Ren , Yong Yan , Leyong Zeng , Zhenzhi Shi , An Gong , Peter Schaaf , Dong Wang , *
Jinshun Zhao , Baobo Zou , Hongsheng Yu , Ge Chen , * Eric Michael Bratsolias Brown ,
and Aiguo Wu *
DOI: 10.1002/adhm.201500273
White TiO 2 nanoparticles (NPs) have been widely used for cancer photody-
namic therapy based on their ultraviolet light–triggered properties. To date,
biomedical applications using white TiO
2 NPs have been limited, since ultra-
violet light is a well-known mutagen and shallow penetration. This work is the
fi rst report about hydrogenated black TiO
2 (H-TiO 2 ) NPs with near infrared
absorption explored as photothermal agent for cancer photothermal therapy
to circumvent the obstacle of ultraviolet light excitation. Here, it is shown
that photothermal effect of H-TiO
2 NPs can be attributed to their dramati-
cally enhanced nonradiative recombination. After polyethylene glycol (PEG)
coating, H-TiO
2 -PEG NPs exhibit high photothermal conversion effi ciency of
40.8%, and stable size distribution in serum solution. The toxicity and cancer
therapy effect of H-TiO
2 -PEG NPs are relative systemically evaluated in vitro
and in vivo. The fi ndings herein demonstrate that infrared-irradiated H-TiO
2 -
PEG NPs exhibit low toxicity, high effi ciency as a photothermal agent for
cancer therapy, and are promising for further biomedical applications.
delivery, and even TiO
2 nanotube for
photothermal therapy (PTT).
[ 2 ] According
to the reports, as an inorganic photosen-
sitizer for cancer PDT is the most widely
and important application of TiO
2 nano-
particles (NPs) in biomedicine.
[ 3 ] The prin-
ciple of PDT is based on the accumulation
of a photosensitizer in a tumor; light of a
specifi ed wavelength is applied resulting
in the generation of reactive oxygen spe-
cies (ROS) and subsequent killing of
tumor cells. The band gap of TiO
2 is
3.23 eV for anatase, which is equal to the
energy of UV light at 385 nm. Therefore,
upon irradiating light with energies higher
than 385 nm (i.e., lower wavelengths),
the photoinduced pairs of electrons and
holes can induce ROS including O
2− and
OH
• radicals leading to the killing of
cancer cells.
[ 4 ] Based on the theory of UV
light–triggered radical production, cancer
PDT of TiO
2 NPs have attracted much attention over the last
two decades. However, due to very shallow penetration and
toxicity of UV light, cancer PDT with TiO
2 NPs met obstacles
that impeded further clinical applications. Many researchers
attempted to change the exciting light from UV to visible
light through doping or surface modifi cation, although these
advances solved the problem in some extent.
[ 5 ] Visible light
1. Introduction
TiO 2 nanomaterials have been widely used in many fi elds such
as energy, environment, cosmetics, food, and biomedicine.
[ 1 ] Its
low toxicity, good biocompatibility, stable structure, and unique
photocatalytic properties, make TiO
2 nanomaterials be applied
in cancer therapy, such as photodynamic therapy (PDT), drug
W. Ren, Dr. L. Zeng, Dr. Z. Shi, A. Gong, Prof. A. Wu
Key Laboratory of Magnetic Materials and Devices
& Division of Functional Materials and Nanodevices
Ningbo Institute of Materials Technology and Engineering
Chinese Academy of Sciences
1219 ZhongGuan West Road , Ningbo 315201 , China
E-mail: aiguo@nimte.ac.cn
Y. Yan, Prof. P. Schaaf, Dr. D. Wang
Chair Materials for Electrical Engineering and Electronics
Institute of Materials Engineering and Institute of
Micro- and Nanotechnologies MarcoNano, TU Ilmenau
Gustav-Kirchhoff-Str. 5 , Ilmenau 98693 , Germany
E-mail: dong.wang@tu-ilmenau.de
Prof. J. Zhao, Prof. B. Zou
Public Health Department
Ningbo University
818 Fenghua Road , Ningbo 315211 , China
Prof. H. Yu
Affi liated Hospital of Medical School
Ningbo University
247 People Road , Ningbo 315020 , China
Prof. G. Chen
College of Environmental & Energy Engineering
Beijing University of Technology
100 Pingleyuan , Beijing 100124 , China
E-mail: chenge@bjut.edu.cn
Prof. E. M. B. Brown
Department of Biological Sciences
University of Wisconsin-Whitewater
800 W. Main St. , Whitewater , WI 53190 , USA
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activation does circumvent the mutagenic problems of UV exci-
tation, but it still does not provide optimal penetration through
biological tissue compared with near infrared (NIR) light.
In 2011, Chen et al. reported that hydrogenated black TiO
2
(H-TiO
2 ) NPs with enhanced visible and NIR light absorp-
tion exhibited substantial solar-driven photocatalytic activities,
including the photo-oxidation of organic molecules in water
and the generation of hydrogen through photocatalytic water
splitting.
[ 6 ] H-TiO 2 then attracted wide research attention in
applications of sustainable energy sources and cleaning of
the environment.
[ 7 ] Although it has been demonstrated that
the enhanced visible and NIR light absorption of H-TiO
2 NPs
was mainly due to the formation of oxygen vacancies in the
H-TiO
2 NPs, but the detailed mechanism for the enhanced
photocatalytic performance of H-TiO
2 NPs is still unknown and
under debate.
[ 8 ] However, it is accepted that H-TiO
2 NPs pos-
sess strong NIR absorption, which is a desired attribute of any
agents for cancer PTT.
As an effective treatment for cancer, the principle of PTT is
based on accumulation of photothermal agents in tumor which
absorbs and converts NIR light into heat to kill the cancer cells.
Compared with traditional therapeutic ways of cancer, such
as chemotherapy, radiotherapy, and surgery, PTT is targeted,
noninvasive, and consequently highly effective, but it does not
involve the side effects of traditional therapies. The type of
photothermal agent is a key factor for PTT and has attracted
numerous research attentions.
[ 9 ] Recently, a variety of inor-
ganic and organic nanomaterials with high photothermal per-
formance have been explored as effective photothermal agents
for cancer therapy.
[ 10 ] Based on the dramatic NIR absorption
of H-TiO
2 NPs, there is much promise to develop a new NIR
triggered photothermal agent; however, there is still no report
of H-TiO
2 applied in biomedicine, especially in diagnosis and
therapy of cancer.
[ 11 ]
Herein, we fi rst attempt to study in hydrogenated black
TiO
2 NPs even hydrogenated semiconducting nanomaterials
applied in the fi eld of cancer diagnosis and therapy. In this
work, H-TiO
2 NPs were coated with polyethylene glycol (PEG)
to improve the stability in physiological environment. Photo-
thermal effect of PEG-coated H-TiO
2 (H-TiO 2 -PEG) was meas-
ured. Finally, the toxicity and therapy effects of H-TiO
2 -PEG
NPs were relative systemically evaluated in vitro and in vivo.
Our results demonstrated that H-TiO
2 -PEG NPs possess 40.8%
of photothermal conversion effi ciency, low toxicity, and high
anticancer effect in vitro and in vivo. These fi ndings suggest
that the facile synthetic H-TiO
2 NPs are promising for further
applications in biomedicine.
2. Results and Discussion
2.1. Photothermal Principle of H-TiO 2 NPs
H-TiO 2 NPs were obtained by a high-power density H
2 plasma
treatment based on our previous studies.
[ 7b,c ] Figure 1 a shows
the UV–vis–NIR absorption spectra of pristine- and H-TiO
2
NPs. The highly increased absorption of H-TiO
2 NPs in the
region of visible and NIR light clarifi ed their color change
from white to black (as seen in the insets in Figure 1 a), which
might be correlated with a large amount of deep level defects
(Ti
3+ species) after H
2 plasma treatment.
[ 12 ] In addition, elec-
tron paramagnetic resonance (EPR) was measured to investi-
gate the concentration of defects in H-TiO
2 NPs. As indicated
in Figure 1 b, no obvious signal was observed for pristine-TiO
2
NPs, indicating their limited amount of defects; while the
H-TiO
2 NPs showed a much stronger signal at an average g
value of ≈1.957, implying the presence of a large amount of
Ti
3+ species in the bulk of the NPs.
[ 13 ] It has been demonstrated
that Ti
3+ species created by hydrogenation process could induce
the formation of additional electronic states below the conduc-
tion band of TiO
2 . In this case, H-TiO
2 NPs with the substan-
tial enhancement of visible and near infrared light absorption
might be attributed to the transitions from the TiO
2 valence
band to these additional electronic states or from these addi-
tional electronic states to the TiO
2 conduction band.
[ 14 ]
To understand the photothermal effect of H-TiO
2 NPs
through studying the properties of photogenerated charges
of H-TiO
2 , light-induced EPR measurements were performed
under 405 nm light irradiation (Figure 1 c). For comparison,
these spectra of pristine-TiO
2 and H-TiO
2 NPs have been sub-
tracted by that of the spectra without light irradiation. The
pristine-TiO
2 NPs showed two well-separated sets of resonance
lines. The high-fi eld small peaks with an average g value of
≈1.960 can be assigned to electrons trapped on Ti
3+ centers;
the low-fi eld sharp features with an average g value of ≈2.013,
correspond to the holes trapped on O
− sites. [ 15 ] These results
indicate that the separation of photogenerated electrons and
holes was more effi cient in pristine-TiO
2 than in H-TiO
2 NPs.
On the other hand, the intensity of O
− signals was signifi cantly
decreased for H-TiO
2 NPs, and an inverted broad resonance
Adv. Healthcare Mater. 2015,
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Figure 1. a) UV–vis–NIR absorption spectra, b) EPR spectra, and c) light-induced EPR spectra of the pristine- and H-TiO
2 NPs. The inserts show the
pristine- and H-TiO
2 NPs samples.

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was observed at an average g value of ≈1.957. This implies that
decreasing the amount of Ti
3+ species in the light-irradiated
H-TiO
2 may derive from the combination of the photogenerated
holes and the localized bulk Ti
3+ species. These results suggest
that bulk Ti
3+ species tend to act as charge carrier traps where
most of the photogenerated holes were consumed through
recombination with electrons.
[ 8,16 ] According to the litera-
ture, nonradiative recombination releasing photons is a major
pathway for the annihilation of photogenerated charges in the
semiconductor with high concentration of deep level defects,
and the energy is exchanged in the form of lattice vibration and
the thermal energy in materials is increased in this process.
[ 17 ]
Hence, the photothermal effect of H-TiO
2 NPs can be attributed
to their dramatically enhanced nonradiative recombination by
deep level defects (Ti
3+ species) which have also been observed
in our previous work.
[ 7 ]
2.2. Photothermal Conversion Effi ciency of H-TiO
2 NPs
Based on the dramatic absorption of NIR and clarifi ed principle
of photothermal effect, H-TiO
2 NPs were used as photothermal
agent in the following research. To enhance the stability
of H-TiO
2 NPs in aqueous solutions, the NPs were coated
by PEG to form H-TiO
2 -PEG NPs. As shown in Figure S1
(Supporting Information), due to H-TiO
2 NPs were hydro-
genated from Degussa P25 (commercial TiO
2 NPs), size
of H-TiO
2 NPs is in accordance with P25, and is about
25 nm. Although H-TiO
2 -PEG NPs show a little aggregation
in some extent, but they still show better dispersion ability
than H-TiO
2 NPs under the same measurement concentra-
tion (10 µg mL
−1 ), which indicates PEG coating can increase
dispersion of H-TiO
2 NPs. Figure S2 (Supporting Informa-
tion) shows hydrodynamic size distributions of H-TiO
2 NPs
before and after PEG coating. Average sizes of H-TiO
2 and
H-TiO
2 -PEG NPs are about 3705 and 205 nm, respectively,
which also suggests PEG coating signifi cantly improves dis-
persion of the NPs in water. As shown in Figure S3 (Sup-
porting Information), zeta potentials of H-TiO
2 NPs and
H-TiO
2 -PEG NPs are −13.40 ± 3.88 and −15.00 ± 4.46 mV,
respectively, and are not signifi cantly different. For further
evaluation of the stability of H-TiO
2 -PEG NPs, the NPs were
dispersed in serum solution for 7 d at room temperature. As
shown in Figure S4 (Supporting Information), the size distri-
butions of the NPs do not show dramatic change during the
period, The PDI value is 0.17 ± 0.03, which suggests H-TiO
2 -
PEG NPs are stable in serum solution, and can be used as a
potential photothermal therapy (PTT) agent for further study.
Figure S5 (Supporting Information) shows UV-vis-NIR absorp-
tion of H-TiO
2 -PEG NPs in aqueous solution. The absorbance
is 0.49 at 808nm which is a basic datum in calculation of photo-
thermal conversion.
As a photothermal agent in PTT, photothermal conversion
is a very important attribute. Consequently, the photothermal
conversion performance of H-TiO
2 -PEG NPs was evaluated.
H-TiO
2 -PEG was dispersed in water, and then irradiated with an
808 nm NIR laser at 2 W cm
−2 . Pure water was used as a nega-
tive control. As shown in Figure 2 a, the temperature of H-TiO
2 -
PEG NPs increases rapidly under NIR irradiation. After irradia-
tion for 600 s, the temperature of H-TiO
2 -PEG NPs aqueous dis-
persions is 66 °C and there is a temperature increase of 44 °C.
By comparison, the temperature of pure water is 28.5 °C and
the increase is only 4.5 °C. It has been demonstrated that the
cancer cells can be easily killed by exposure to temperatures
over 50 °C for few minutes,
[ 10 ] and therefore H-TiO
2 -PEG NPs
may be considered as a viable agent for photothermal therapy
of cancer. After 600 s of irradiation, the NIR laser was shut
off, and the decreased temperature was recorded for another
1140 s. The temperature change (Δ T ) response to the NIR laser
over a period of 1740 s is shown in Figure 2 b. Linear time data
versus −ln(
θ
) obtained from the cooling period of the NIR laser
is shown in Figure S6 (Supporting Information). The photo-
thermal conversion effi ciency (
η
) of H-TiO
2 -PEG can be calcu-
lated according to the following equation
[ 18 ]
hA T T
IA
η
Δ−Δ
−−
λ
=()
(1 10 )
max,mix max,H O
2
(1)
where
h
is the heat transfer coeffi cient, A is the surface area of
the container, Δmax,mi
x
T and Δmax,H O
2
T are the temperature change
of the H-TiO
2 -PEG NPs dispersion and solvent (water) at the
maximum steady-state temperature, respectively, I is the laser
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Figure 2. a) Temperature evaluation of H-TiO
2 -PEG NPs (100 µg mL
−1 ) and pure water with 808 nm laser irradiation at 2 W cm
−2 for different times.
b) The temperature change (Δ T ) response to NIR laser on and off in period of 2100 s.

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power, and
λ
A is the absorbance of H-TiO
2 -PEG NPs at 808 nm.
Details of the calculation are given in the Supporting Informa-
tion. According to the equation,
η
value of H-TiO
2 -PEG was cal-
culated to be about 40.8%.
2.3. Cytotoxicity and Uptake of H-TiO 2 NPs In Vitro
It has been demonstrated that bare, white TiO
2 NPs are envi-
ronmentally friendly and low toxicity.
[ 1 ] However, there is still
no report about the toxicity of H-TiO
2 NPs. Thus, cytotox-
icity of H-TiO
2 -PEG NPs on both human and murine breast
cancer cells was carried out through the MTT method. As
shown in Figure 3 a, MCF-7 or 4T1 cells were incubated with
50–300 µg mL
−1 of H-TiO
2 -PEG NPs for 24 h. The relative via-
bility of the cancer cell is not decreased signifi cantly, which sug-
gests H-TiO
2 -PEG NPs are relatively low toxic in vitro.
Previous report showed that TiO
2 NPs can be stained by fl uo-
rescent dye (alizarin red S, ARS).
[ 19 ] To investigate the uptake
of H-TiO
2 -PEG in cancer cells, both MCF-7 and 4T1 cells were
incubated with ARS-stained H-TiO
2 -PEG (H-TiO 2 -PEG-ARS).
Free ARS incubated cells were used as negative control. The
cells were collected and 20 000 cells were analyzed by fl ow
cytometer in each group. As shown in Figure 3 b, X -axis is red
fl uorescence signal of ARS. The cells incubated with free ARS
show very weak fl uorescence, and mean signal intensities are
3.20 (MCF-7) and 2.37 (4T1). However, cells incubated with
H-TiO
2 -PEG-ARS show relative stronger signal, and mean
fl uorescence intensities are 13.80 (MCF-7) and 12.00 (4T1),
respectively. The results indicate that H-TiO
2 -PEG NPs can be
absorbed by both MCF-7 and 4T1 cells. These results further
suggest H-TiO
2 -PEG NPs are relatively low toxic in vitro.
2.4. MTT and Calcein Acetoxymethyl Ester (AM)/Propidium
Iodide (PI) Staining Assay of Photothermal Therapy In Vitro
Based on the high photothermal conversion effi ciency, sta-
bility in serum solution, and low toxicity, the photothermal
therapeutic effi cacy of H-TiO
2 -PEG NPs was evaluated on
cancer cells in vitro. MCF-7 and 4T1 cells were incubated with
H-TiO
2 -PEG NPs, and were then irradiated with an 808 nm
NIR laser for 0–5 min. Cells incubated without H-TiO
2 -PEG
NPs were also irradiated by laser as a negative control. As
shown in Figure 4 a,b, neither control group of MCF-7 or 4T1
cells receiving only laser treatment (without using H-TiO
2 -
PEG NPs), shows any signifi cant loss in viability after 5 min
of irradiation. However, the viability of the cells incubated with
H-TiO
2 -PEG NPs decreases signifi cantly with increasing irra-
diation time, and more than 80% of both MCF-7 and 4T1 cells
were killed upon 5 min of irradiation. These results demon-
strate that H-TiO
2 -PEG NPs are effective PTT agent for cancer
therapy in vitro.
In addition, to further verify the PTT performance of H-TiO
2 -
PEG in vitro, the cells were stained with calcein AM and PI
solutions, which can discern live or dead cells through emitted
green or red fl uorescence, respectively. As shown in Figure 4 c,
the majority of MCF-7 and 4T1 cells in control, NPs, and laser
groups are alive (green). However, most of the cells are dead
(red) in laser + NPs group, indicating that the H-TiO2-PEG NPs
can be applied as an effective PTT agent in vitro and may be
useful for in vivo applications as well.
2.5. Toxicity and Distribution In Vivo
As a potential in vivo PTT agent, the toxicity of H-TiO
2 -PEG
NPs must be evaluated in vivo. In this study, histological anal-
ysis and blood analysis were used to evaluate the toxicity of the
NPs in vivo according to previous report.
[ 20 ] Healthy Blab/c
mice were injected with different doses of H-TiO
2 -PEG NPs,
and saline injected mice were used as control. Over one month
period, behaviors of mice such as eating, drinking, excretion,
activity, and neurological status were observed. There is no sig-
nifi cant difference in the above behaviors between control and
H-TiO
2 -PEG-injected groups. After one month, the mice were
sacrifi ced; the main organs and blood were analyzed. Figure 5 a
shows histological analyses of the organs including heart, liver,
spleen, kidney, and lung. There is no detectable tissue damage
or other lesions such as necrosis, infl ammatory, or pulmonary
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Figure 3. a) Cell viability of MCF-7 and 4T1 cells after incubation with increased dose of H-TiO
2 -PEG NPs for 24 h. Data are expressed as the
mean ± standard ( n = 5). b) Uptake of red fl uorescence dye (ARS) stained TiO
2 -PEG NPs in MCF-7 and 4T1 cells. X -axis shows intensity of red fl uo-
rescence which indicates amount of the NPs absorbed by the cells. Each datum was obtained from 20 000 cells.

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fi brosis when comparing the H-TiO
2 -PEG-injected groups with
control group. Figure 5 b shows hematological analysis of the
mice including white blood cell (WBC), red blood cell (RBC),
platelet (PLT), and their relevant data such as neutrophil (NE),
lymphocyte (LY), monocyte (MO), eosinophil (EO), basophil
(BASO), and hemoglobin (HGB), hematocrit (HCT), mean
corpuscular volume (MCV), mean corpuscular hemoglobin
(MCHC), red blood cell distribution width (RDW-CV), platelet
distribution width (PDW-CV), and mean platelet volume (MPV).
Number and distribution changes of blood cell are an impor-
tant indicator of disease. As shown in Figure 5 b, there is no
signifi cant difference between control and H-TiO
2 -PEG-injected
groups, suggesting the mice are healthy. Furthermore, blood
biochemical analysis was carried out by blood autoanalyzer. Six
important hepatic indicators for liver functions (direct bilirubin,
DBIL; albumin, ALB; globin, GLOB; alkaline phosphatase, ALP;
gamma glutamyl transpeptidase, GGT), three indicators for
kidney functions (urea nitrogen, UREA; creatinine, CREA; uric
acid, URCA), total cholesterol (CHOL), triglyceride (TG), and
glucose (GLU) were evaluated. As shown in Figure 5 c, H-TiO
2 -
PEG NPs injection does not cause signifi cant change of these
indicators compared with the control group. Our relative sys-
temic results demonstrate that H-TiO
2 -PEG NPs are not toxic
to the mice at the injected doses for one month. However, more
efforts are still required to systematically evaluate the potential
long-term toxicity of H-TiO
2 -PEG NPs at higher doses in vivo.
In order to evaluate the distribution of H-TiO
2 -PEG NPS in
tumor-bearing mice, contents of Ti in main organs were meas-
ured. As shown in Figure S7 (Supporting Information), after
intravenously injected with H-TiO
2 -PEG for 24 h, the NPs accu-
mulation in tumor is 9.43 ± 0.03 µg g
−1 . The distribution of
H-TiO
2 -PEG in tumor-bearing mice is similar to other reported
nanomaterials.
[ 18 ]
2.6. Photothermal Therapy In Vivo
Because H-TiO 2 -PEG NPs possess low toxicity and good bio-
compatibility in vitro and in vivo, and also show effective
photothermal therapy in vitro, their application for cancer
photothermal therapy in vivo was carried out in tumor-bearing
mice. The mice injected with H-TiO
2 -PEG NPs and irradiated
with NIR laser are named as laser + NPs group. Mice neither
injected nor irradiated are control group. Mice injected with
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Figure 4. Viability of a) MCF-7 and b) 4T1 cells treated with or without 100 µg mL
−1 H-TiO 2 -PEG NPs and 808 nm laser irradiation at 2 W cm
−2 for
≈5 min. Data are expressed as the mean ± standard ( n = 5). Statistically signifi cant differences were evaluated using the Student’s t -test (* p < 0.05, ** p
< 0.01, ns > 0.05). c) Microscope images of calcein AM (green, live cells) and propidium iodide (red, dead cells) costained MCF-7 or 4T1 cells treated
with or without 100 µg mL
−1 H-TiO 2 -PEG NPs and laser irradiation for 5 min. (Scale bar = 50 µm.)

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saline and irradiated with NIR laser are called laser group. Mice
only injected with H-TiO
2 -PEG NPs are NPs group. In order
to record temperature change of NIR irradiated tumor, pho-
tothermal imaging of tumor-bearing mice was measured. As
shown in Figure 6 a, temperatures of tumor site in laser and
laser + NPs groups are imaged in each minute during NIR
irradiation. In laser group, temperature of tumor site increases
very slowly and limits after 5 min irradiation of 2 W cm
−2 NIR
and temperature is about 36.5 °C which is accepted and toler-
ated by tissue. However, the temperature increases quickly,
and reaches about 52.6 °C under the same NIR irradiation in
laser + NPs group. Whole body temperature imaging is shown
in Figure 6 b. Figure 6 c shows the temperature change (Δ T ) of
tumor sites during NIR irradiation. Δ T is about 11 °C in laser
group; however, the value is about 27.1 °C in laser + NPs group
which is 2.5 times more than the laser group. It has been
considered that cancer cells can be easily killed in few minutes
when their temperature is over 50 °C.
[ 9 ] Therefore, the tumor
could be ablated in laser + NPs group.
In order to justify our supposition of tumor ablation, four
mice were sacrifi ced immediately after NIR irradiation, and
tumors were analyzed by hematoxylin and eosin (H&E) stain.
As shown in Figure 7 , there is no obvious pathological change
in control, laser, and NPs groups. However, there are signifi cant
necrosis features in laser + NPs group, such as pyknosis, kary-
orrhexis, and karyolysis happening in nucleus region; cell mem-
brane is destroyed and fused with intercellular substance to
form a fuzzy red-stained substance without any granular struc-
ture. Moreover, red blood cells are observed (shown by green
arrows) which indicates tumor vessels are also destroyed by
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Figure 5. a) Histological analyses of mice main organs injected with saline or various doses of H-TiO
2 -PEG NPs. (Scale bar = 20 µm.) b) Hematological
analysis and c) blood biochemical analysis of the mice. Data are expressed as the mean ± standard ( n = 3). Statistically signifi cant differences were
evaluated using the Student’s t -test (* p < 0.05, ** p < 0.01, ns > 0.05).

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heat. These results justify our supposition that tumor cells can
be ablated after H-TiO
2 -PEG NPs injection plus NIR irradiation.
The changes of tumor volume were recorded in the fol-
lowing two weeks. As shown in Figure 8 a,d, the tumors grow
consistently in control, laser, and NPs groups. The relative
tumor volumes ( V / V O ) on 14th day are 12.30 ± 2.38 in the
control group, 14.20 ± 3.45 in the laser group, and 13.71 ±
2.82 in the NPs group. These results demonstrate that laser
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Figure 6. a) Photothermal images of tumor site in Laser group (injected with saline) and Laser + NPs group (injected with NPs) during 5 min irradia-
tion of 2 W cm
−2 NIR. b) Whole body temperature images of the mice at the fi fth min of NIR irradiation. c) Temperature change (Δ T ) of tumor sites
during NIR irradiation.
Figure 7. Histological HE stain analysis of tumor injury after the tumor-bearing mice injected with or without H-TiO
2 -PEG NPs, and irradiated with
or without 808 nm NIR for 5 min.

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irradiation alone or H-TiO
2 -PEG NPs injection alone does not
affect tumor development. However, upon NIR irradiation of
the NPs injected tumor, edemas appear on the tumors within 3
d due to thermal damage. On the fi fth day, the tumors shrink,
and black scars are left on the tumor sites. On the 14th day,
the tumors disappear, leaving only smooth scars on the original
tumor sites. These results clarify that H-TiO
2 -PEG NPs are an
effective PTT agent for in vivo cancer therapy.
Change of body weight is an important parameter for the
evaluation of toxicity or damage during treatment. Conse-
quently, the body weights of mice were recorded during the
therapy period. As shown in Figure 8 b, in the fi rst 3 d, body
weights of mice decrease slightly in laser group, NPs group, and
laser + NPs group, but increase slightly in control group. This
is likely due to the fact that the mice were anesthetized during
this period of time, which negatively affects food consumed
and thus losses body weight slightly. However, mice in control
group were not anesthetized so their body weights increase.
From 4th to 14th day, body weights of all mice increase, which
demonstrates that the PTT agent and treatment used in this
study are nontoxic and safe to tumor-bearing mice. However,
body weight in laser + NPs group is signifi cantly less than the
other three groups after the 4th day, due to tumor shrinkage as
measured by reduction in tumor volume.
To further evaluate the PTT performance of H-TiO
2 -PEG,
survivals of the mice after treatment were also recorded. As
shown in Figure 8 c, mice live healthily for more than 50 d in
laser + NPs group. However, some mice in the other three
groups die from days 25 to 34, and all of the mice in these
groups die after day 47. These results demonstrate that H-TiO
2 -
PEG NPs, as high-performance PTT agents, are promising for
further biomedical application.
3. Conclusion
In summary, H-TiO
2 NPs were explored as a new near infrared
triggered photothermal agent for cancer therapy. We have dem-
onstrated the photothermal effect of H-TiO
2 NPs can be attrib-
uted to its dramatically enhanced nonradiative recombination,
which lead to an excellent performance of photothermal con-
version in the NIR range. Our results also demonstrate that
Figure 8. a) The relative tumor volume, b) body weight, and c) survival rate of the mice after different treatments described above. Data are expressed
as the mean ± standard ( n = 5). Statistically signifi cant differences were evaluated using the Student's t -test (* p < 0.05, ** p < 0.01, ns > 0.05). d) Photos
of 4T1 tumor-bearing mice at the 1st, 5th, and 14th day after the treatments.

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H-TiO
2 NPs coated by PEG possess several features: 1) high
NIR photothermal effect, 2) low toxicity and good biocom-
patibility, and 3) low cost and facile synthetic method. More
importantly, H-TiO
2 -PEG NPs are successfully used as photo-
thermal agent for NIR-triggered cancer therapy both in vitro
and in vivo. This work also provides an experimental basis for
the promising application of H-TiO
2 -PEG NPs as an effective
photothermal agent for cancer therapy. It is demonstrated that
NIR-triggered cancer photothermal therapy of H-TiO
2 shows
better applicability than UV light–induced photodynamic
therapy of TiO
2 and thus can advance the applications of TiO
2
in biomedicine in future.
4. Experimental Section
Preparation of H-TiO
2 Nanoparticles : Commercial TiO
2 nanoparticles
(Degussa P25) were purchased from Sigma-Aldrich. 0.10 g TiO
2 NPs
were dispersed in 50 mL ethanol, and then drop-casted onto a 6 inch-Si
wafer. The drop-casting process was repeated several times to achieve a
TiO
2 mass loading of 0.5–0.6 mg cm
−2 (100–150 mg on a whole wafer).
This wafer was then transferred into a chamber for the hydrogenation
treatment with H
2 plasma, and there an instrument of inductively
coupled plasma (ICP) (Plasmalab 100 ICP-CVD, Oxford Instruments)
was used. The H
2 plasma treatment was performed at 150 °C for 20 min.
The ICP power was 3000 W, the chamber pressure was 25.8–27.1 mTorr,
and the H
2 fl ow rate was 50 sccm. After this treatment, hydrogenated
TiO
2 (H-TiO 2 ) NPs was obtained and scratched from the Si wafer.
IR Absorption and Photothermal Principle of H-TiO
2 NPs : X-ray
diffraction (XRD) pattern of the samples was recorded on a
diffractometer (SIEMENS D5000) with Cu-K radiation. The optical
absorption in the range from UV to the NIR was measured by a diffuse
refl ectance accessory of a UV–vis–NIR spectrometer (Cary 5000). EPR
spectra were recorded at the temperature of 77 K using a Bruker BioSpin
CW X-band (9.5 GHz) spectrometer (ELEXYS E500). PL spectroscopy
was performed by using a Czerny–Turner spectrograph (Jobin Yvon SPEX
1000M) with a focal length of 1000 mm. The excitation wavelength of
266 nm was generated by a femtosecond laser (Coherent MIRA 900-F)
followed by a pulse picker (Coherent Pulse Picker) and a third harmonic
generator (APE HarmoniXX THG).
PEG Coating and Photothermal Conversion Effi ciency of H-TiO
2 -PEG
NPs : For biomedical applications, the H-TiO
2 should be disperse and
stable in serum. In this study, PEG (molecular weight 1500) was used
to enwrap the H-TiO
2 NPs to improve their stability. 20 mg of H-TiO
2
powder was dispersed in 75 mL ethanol by an ultrasound treatment of
30 min. The H-TiO
2 contained ethanol was dropped into a 25 mL PEG-
ethanol solution (20 mg mL
−1 of PEG), and stirred for 24 h. H-TiO
2 -PEG
NPs were separated by centrifugation, and were washed with ultrapure
water. The as-prepared H-TiO
2 -PEG NPs were dispersed in the ultrapure
water and stored at 4 °C. Micromorphologies of H-TiO
2 before and
after PEG coating were investigated at the same concentration through
a transmission electron microscope (FEI Tecnai F20). Size distribution
and zeta potential of H-TiO
2 and H-TiO
2 -PEG were measured by a
particle size zeta potential analyzer (Nano ZS, Malvern Instruments
Ltd, England). The UV–visible spectra of H-TiO
2 and H-TiO
2 -PEG were
determined by using an UV–visible spectrophotometer (T10CS, Beijing
Purkinje General Instrument, China). For evaluating the photothermal
conversion effi ciency of H-TiO
2 -PEG, 2 mL aqueous dispersion of
H-TiO
2 -PEG (100 µg mL
−1 ) were moved into a well of 24-well culture
plate, and irradiated under an 808 nm NIR laser at a power density of
2 W cm
−2 for 600 s. The temperature of the dispersion was measured
every 60 s after the start of irradiation. After the laser irradiation was shut
off, the temperature was further measured for another 1500 s with the
same intervals. Ultrapure water as control group was treated under the
same conditions. Photothermal conversion effi ciency (
η
) of H-TiO
2 -PEG
was then calculated according to the methods reported previously.
[ 18 ]
In order to investigate the stability, H-TiO
2 -PEG NPs were dispersed in
sterile fetal bovine serum (FBS) solution at room temperature for 7 d,
and size distributions of the nanoparticles were measured every day by
the particle size zeta potential analyzer.
Cell Culture and Cytotoxicity of H-TiO
2
-PEG NPs In Vitro : MCF-7 cell
line of human breast cancer and 4T1 cell line of murine breast cancer
were cultured in RPMI1640 medium and supplemented with 10% FBS.
The cells were maintained at 37 °C incubator with 5% CO
2 . To evaluate
the cytotoxicity of H-TiO
2 -PEG, MCF-7 or 4T1 cells were plated in
96-well plates (1 × 10
4 cells per well) and cultured for 24 h. The cells
were incubated with different doses of H-TiO
2 -PEG (50–300 µg mL
−1 )
for 24 h. The viability of cells was assayed by the MTT assay. Briefl y, 10
µL of MTT (5 mg mL
−1 in PBS) was added into every well, and incubated
for 4 h. Next, DMSO was used to dissolve the formazan crystals. The
absorbance was measured by a microplate absorbance reader (Biorad
iMARKTM, USA), and the cell viability was calculated.
Uptake of H-TiO
2 -PEG NPs by Cancer Cells : To investigate the uptake
of H-TiO
2 -PEG in the cancer cells, fl uorescent dye ARS was used to stain
TiO
2 as previously reported.
[ 19 ] MCF-7 and 4T1 cells (2 × 10
5 cells) were
seeded into 35 mm culture dishes and cultured for 24 h, respectively.
The culture media was then replaced by fresh medium contained
ARS (10 µg mL
−1 ) or ARS-H-TiO
2 -PEG (100 µg mL
−1 ). The cells were
incubated for 2 h, then washed with PBS, and moved to tubes after
incubation with trypsin-EDTA. After centrifugation and resuspension
with PBS, red fl uorescence of cells was analyzed by fl ow cytometer
(FACSCalibur, BD, USA).
Photothermal Therapy of H-TiO
2 -PEG NPs In Vitro : To quantitatively
evaluate the photothermal therapy effi ciency of H-TiO
2 -PEG on cancer
cells, MCF-7 and 4T1 cells were seeded into 96-well plates (1 × 10
4 cells
per well), respectively. The cells were then incubated with fresh DMEM
and 100 µg mL
−1 of H-TiO
2 -PEG containing DMEM for 2 h. After 2 h
incubation, all of the media were replaced by fresh DMEM. The cells
were then irradiated by an 808 nm NIR laser (2 W cm
−2 ) for 0–5 min
and were cultured for another 24 h. 10 µL of MTT was added into
each well and incubated for 4 h. The MTT solution was removed and
100 µL DMSO was added to dissolve the formazan crystals. Finally, the
absorbance was measured and the cell viability was calculated. In order
to further evaluate the photothermal therapy of H-TiO
2 -PEG on cancer
cells, MCF-7 or 4T1 cells were cultured in 35 mm dishes. The cells were
then incubated with fresh DMEM containing 100 µg mL
−1 of H-TiO
2 -PEG
for 2 h. The culture media were then replaced by fresh DMEM, and the
cells were irradiated by 808 nm NIR (2 W cm
−2 ) for 5 min. The cells
were stained with both calcein AM and PI. The live and dead cells were
observed by confocal microscopy as previously described.
Toxicity Evaluation of H-TiO
2 -PEG NPs In Vivo : In the animal
experiments, the animal care and handing procedures were in agreement
with the guidelines of the Regional Ethics Committee for Animal
Experiments at Ningbo University (Permit No. SYXK Zhe 2013-0191). For
assessing toxicity of H-TiO
2 -PEG in vivo, 24 healthy Blab/C mice were
divided into four groups randomly. The mice were intravenously injected
with different doses (1, 5, 25 mg kg
−1 ) of H-TiO
2 -PEG, respectively. Mice
injected with saline were used as the control. Mice were observed for
behavioral changes over one month period. After one month, all mice
were sacrifi ced. Mice's blood were collected by a cardiac puncture
method for hematological and were analyzed by blood analyzer (Sysmex
XT-1800i, Japan) and Hitachi 7600-110 autoanalyzer (Hitachi, Tokyo,
Japan). The main organs including heart, liver, spleen, kidney, and lung
were preserved in a 10% formalin solution and stained with H&E for
histological analysis to assess the toxicity of H-TiO
2 -PEG.
Biodistribution of H-TiO
2 -PEG NPs In Vivo : For evaluating the
distribution of H-TiO
2 -PEG in tumor-bearing mice, tumor model was
established. 4T1 cells (1 × 10
6 cells for one mouse) suspended in 100 µL
of serum free medium were inoculated subcutaneously in several female
Balb/C mice (fi ve weeks old). A digital caliper was used to measure the
size of tumor. When the tumor grew to 3–4 mm, mice were intravenously
injected with 100 µL of H-TiO
2 -PEG aqueous dispersion (2000 µg mL
−1 ).
The mice were sacrifi ced after 24 h, and the concentration of Ti in main

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organs were measured by ICP-MS (NexION 300, Perkin-Elmer, USA) as
described previously.
[ 18 ]
Photothermal Imaging and Therapy of H-TiO
2 -PEG NPs In Vivo : Tumor
models were established in 24 female Balb/C mice (fi ve weeks old) as
described above. A digital caliper was used to measure the size of tumor.
Tumor volume = (tumor length) × (tumor width)
2 /2. When the tumors
grew to 3–4 mm in diameter, the mice were randomly divided into four
groups, and each group contained six mice. The mice were anesthetized
by intraperitoneal injection of chloral hydrate solution (8 wt%), and
were given an intratumor injection with 100 µL of H-TiO
2 -PEG aqueous
dispersion (100 µg mL
−1 ) or with 100 µL of saline. The tumor sites were
irradiated with or without an 808 nm NIR laser at 2 W cm
−2 for 5 min.
The temperature change of tumor site was measured by a photothermal
imaging system (Ti400, Fluke, USA) during NIR irradiation. Four mice
were sacrifi ced after NIR irradiation; their tumors were collected for
histological analysis to evaluate the nanoparticles triggered photothermal
injury of tumor. In order to kill the residual tumor cells, the tumor sites
were irradiated each of the following 2 d under the same conditions. The
tumor sizes of mice were measured by a digital caliper for 14 d and the
tumor volume was calculated according to the formula of tumor volume
mentioned above. Relative tumor volumes were calculated as V / V O
( V O was the tumor volume when the treatment was initiated). Body
weight and survival of the mice were also recorded.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
W.R. and Y.Y. contributed equally to this work. This work was supported
by the National Natural Science Foundation of China (31170964,
U1432114, U1332117, and 11475012), the Hundred Talents Program
of Chinese Academy of Sciences (2010-735), the Natural Science
Foundation of Zhejiang province (LY15C100002), and by the Natural
Science Foundation of Ningbo city (2014A610166). Prof. Xiaoyuan
Chen is appreciated for evaluating scientifi c sense of this study. The
authors thank Prof. Qiang Yao for supplying of a photothermal imaging
system. The authors also thank Mr. Fei Zhou in the Experimental Animal
Center of Ningbo University for conducting animal feeding. Yong Yan
is supported by means of a doctoral scholarship from the Carl Zeiss
Stiftung (Germany).
Received: April 14, 2015
Revised: April 30, 2015
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