Multifunctional anticancer platform for multimodal imaging and visible light driven photodynamic/photothermal therapy

Article abstract of DOI:10.1021/cm504566f

As a potential photosensitizer for photodynamic therapy (PDT), pure titanium dioxide has the drawbacks of low tissue penetration and possible damage to skin due to the triggered UV light. To realize near-infrared (NIR) laser-induced multimodal imaging guided therapy, we constructed a multifunctional core-shell structure (TiO₂@Y₂Ti₂O₇@YOF:Yb,Tm) by a facile coprecipitation route, followed by an annealing process. Under a single NIR laser irradiation, the highly cytotoxic reactive oxygen species (ROS) required for PDT can be generated due to the energy transfer from YOF:Yb,Tm to the Y₂Ti₂O₇ photocatalyst which is responsive to blue emission (visible light), and the thermal effect can be simultaneously produced due to the nonradiative transition and the recombination of electron-hole pairs. The NIR light induced PDT and photothermal therapy (PTT) can efficiently suppress tumor growth, which was evidenced by both in vitro and in vivo results. Moreover, the rare earth ions in the composite make the material have good up-conversion luminescence (UCL) imaging and CT imaging properties, thus achieving the target of synergistic PDT and PTT therapy under the multimodal imaging guidance.

Full text of DOI:10.1021/cm504566f

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Article
A Multifunctional Anticancer Platform for Multimodal Imaging
and Visible Light Driven Photodynamic/Photothermal Therapy
Ruichan Lv, Chongna Zhong, Rumin Li, Piaoping Yang,
Fei He, Shili Gai, Zhiyao Hou, Guixin Yang, and Jun Lin
Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/cm504566f • Publication Date (Web): 09 Feb 2015
Downloaded from http://pubs.acs.org on February 18, 2015
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Chemistry of Materials
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A Multifunctional Anticancer Platform for Multimodal Imaging
and Visible Light Driven Photodynamic/Photothermal Therapy
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Ruichan Lv,†,§ Chongna Zhong,†,§ Rumin Li,† Piaoping Yang,†,* Fei He,† Shili Gai,† Zhiyao Hou,‡
Guixin Yang,† and Jun Lin‡,*
† Key Laboratory of Superlight Materials and Surface Technology, Ministry of Education, College of Material Science and
Engineering, Harbin Engineering University, Harbin 150001, P. R. China
‡ State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of
Sciences, Changchun 130021, P. R. China
§ These authors contributed equally to this work.
KEYWORDS. Y2Ti2O7, Photodynamic, photothermal, bio-imaging, up-conversion
ABSTRACT: As a potential photosensitizer for photodynamic therapy (PDT), pure titanium dioxide has the drawbacks of low
tissue penetration and the possible damage to skin due to the triggered UV light. To realize nearꢀinfrared (NIR) laser induced mulꢀ
timodal imaging guided therapy, we constructed a multifunctional coreꢀshell structure (TiO2@Y2Ti2O7@YOF:Yb,Tm) by a facile
coꢀprecipitation route, followed by an annealing process. Under a single NIR laser irradiation, the highly cytotoxic reactive oxygen
species (ROS) required for PDT can be generated due to the energy transfer from YOF:Yb,Tm to the Y2Ti2O7 photocatalyst which
is responsive to blue emission (visible light), and the thermal effect can be simultaneously produced due to the nonꢀradiative transiꢀ
tion and the recombination of electronꢀhole pairs. The NIR light induced PDT and photothermal therapy (PTT) can efficiently supꢀ
press tumor growth, which was evidenced by both in vitro and in vivo results. Moreover, the rare earth ions in the composite make
the material have good upꢀconversion luminescence (UCL) imaging and CT imaging properties, thus achieving the target of PDT
and PTT synergistic therapy under the multimodal imaging guidance.
under visible light irradiation.7 However, the required UV or
visible light still have the drawbacks of low tissue penetration
INTRODUCTION
Nowadays, the traditional antiꢀcancer therapies, such as chemꢀ
and possible damage to skin. Thus it is of special importance if
otherapy and the ionizing radiotherapy, usually bring about
the exciting laser locates at the longer wavelength, such as 980
serious side effects, drug resistance, and inevitably induce
lesions on irradiated tissues.1 As mild and noninvasive techꢀ
nm NIR, which has the high penetration to kill the inner tumor
cells to achieve improved therapy efficacy.8 It is well known
niques based on the photochemical reactions of photosensitizꢀ
that lanthanideꢀdoped nanocrystals possess a unique ability to
ers (PSs) and thermal effect, photodynamic therapy (PDT) and
convert excited NIR light to visible emissions, which can be
photodynamic therapy (PTT) have become booming powerful
used as the imaging agent on one side.9 On the other side, the
tools to apply in the biological and medical field.2 The convenꢀ
UCL emissions in visible regions can be utilized as the donors
tional organic PDT agents, such as ZnPc and Ce6 photosensiꢀ
for transferring energy to PDT agents.8 Meanwhile, the NIR
tizers, suffer from poor waterꢀsolubility, easy premature leakꢀ
ing, low bioꢀstability, and other unclear security problems.3
laser which locates within the optical transmission window of
biological specimens has the merits of high penetration depth
Alternatively, titanium dioxide (TiO2), which can absorb ulꢀ
and low tissue absorbance.10 Furthermore, if referred to imagꢀ
traviolet (UV) light to generate reactive oxygen species (ROS)
ingꢀguided PDT, two separate lights with different waveꢀ
to kill tumor cells, has motivated intensive experimental and
lengths were often adopted to realize diagnosis and therapy,
theoretical studies, due to its excellent biocompatibility, high
which makes it difficult for realꢀtime monitoring and assessing
photoꢀcatalytic properties, low cost, and high chemical stabilꢀ
efficacy.11 Therefore, developing a multifunctional platform
ity.4 However, in the field of titanium dioxide based PDT
combining the UCL and TiO2ꢀbased materials which can
agents, there are serious problems which inhibit the biomediꢀ
achieve the imagingꢀguided PDT therapy triggered by a single
cal application.
NIR light is highly desirable.
Firstly, most of the TiO2 crystals can only be irradiated by
UV light to generate ROS.5 To improve the separation probaꢀ
In the UCL field, lanthanide oxyfluorides (LnOF) have been
considered as one of the most excellent luminescent hosts for
bility and increase photoꢀinduced electronꢀhole pairs, it is necꢀ
achieving efficient UCL, which are even better than lanthanide
essary to transfer the absorption peaks of TiO2 from UV to
fluoride.12 Among all the commonly used coꢀdopants, only
visible regions. Some modification strategies have been emꢀ
ployed to achieve this target, such as decoration with metal
Yb3+/Tm3+ ions can emit the strong blue emissions used for the
complexes.6 In particular, doping is a powerful strategy to
bioꢀimaging and optical carrier. Meanwhile, Y2Ti2O7, as one
of the classical titanium dioxideꢀbased compound, has attractꢀ
modify electronic structure and construct heteroꢀatomic surꢀ
ed considerable attention recently as a possible candidate for
face structures, which allows high photoꢀcatalysis efficiency
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photoꢀcatalysis due to its unique structural characteristics of
for 2 h. Then, the asꢀsynthesized TYY UCNPs dispersed in 20
which the electron acceptor of Y3+ ions may attract the elecꢀ
tron to improve the photocatalysis efficiency.13 It is thus meanꢀ
ingful if the good UCL host and PSs (Y2Ti2O7) are integrated.
mL deionized water were added and stirred in dark overnight.
The product was collected by centrifugation and washed with
deionized water and ethanol several times to remove the free
FA. The asꢀprepared TYY UCNPs modified with FA was
stored for subsequently biological test.
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In this contribution, a core/shell/shell structured TiO2@
Y2Ti2O7@YOF:Yb,Tm (denoted as TYY) platform has been
synthesized through a facile and mild coꢀprecipitation method,
followed by a calcination process. The phase, composition,
and structure of the composites were tuned by adjusting the
molar ratio of TiO2/Ln3+. Core/shell/shell structured TYY
UCNPs have been optimized for the biomedical experiments.
The asꢀsynthesized TYY upꢀconversion nanoparticles
(UCNPs) were employed for MTT assay using HeLa cells and
hemolysis experiment using human red blood cells to detect
the in vitro and in vivo biocompatibility. Meanwhile, the cytoꢀ
toxicity against tumor cells was systematically studied by
MTT assay, trypan blue and AM/PI marked HeLa cells in
vitro. The photoꢀthermal imaging, the UCL imaging and CT
imaging properties were carried out on tumorꢀbearing mice in
vivo simultaneously. The animal experiment and the histologic
section assay were performed to demonstrate the potential
clinical application of this antiꢀcancer nanotheranostic.
Singlet Oxygen Detection of UCNPs. DPBF was emꢀ
ployed as a chemical probe to determine singlet oxygen by
measuring the absorption via UVꢀvis spectroscopy. Typically,
2 mL of ethanol solution containing DPBF (10 mmol/L) was
added to 2 mL of TYY UCNPs solution and then transferred
into a 5 mL cuvette. The solution was kept in dark and irradiꢀ
ated by a 980 nm NIR laser for 1 min, 2 min, 3 min, and 5
min, respectively. Then, the solution was centrifuged at 6000
rpm and the supernatant was collected for UVꢀvis detection.
The other two samples obtained with TiO2/Ln3+ ratio of 2:1
and 1:2 were also detected by the same process.
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In Vitro Cytotoxicity and Biocompatibility of TYY
UCPCs. Briefly, HeLa tumor cells were seeded in a 96ꢀwell
plate with the number of 7000 per well, then cultured at 37 °C
in 5% CO2 for 12 h. After that, pure culture media were added
into the first and the third group, and TYY UCNPs were added
to the second and the fifth group, pure DOX was added into
the fourth group, and then incubated with cells at 37 °C in 5%
CO2 for another 24 h. Among them, the first group was the
control group; the second group with only added UCNPs was
without NIR irradiation in order to detect the cytotoxicity of
the UCNPs; the third group with only culture was under NIR
irradiation, which is used to detect the effect of the NIR irradiꢀ
ation to the cells; the fourth group was with pure DOX added;
and the fifth group added with UCNPs was under NIR irradiaꢀ
tion which is to detect the antiꢀcancer efficiency with integratꢀ
ed PDT/PTT. The second and fifth group were compared to
evaluate the cytotoxicity of the photodynamic effect of the asꢀ
synthesized UCNPs, and the results of fifth group compared
with the conventional pure DOX (the third group) to evaluate
the therapeutic effect of the asꢀsynthesized UCNPs. The
UCNPs samples were diluted into respective concentration of
15.63, 31.25, 62.5, 125, 250, and 500 ꢁg mL–1, and DOX soluꢀ
tion was diluted into different concentration of 3.13, 6.25,
12.5, 25, 50, and 100 ꢁg mL–1. Then, 20 ꢁL of asꢀprepared
MTT solution (5 mg/mL) was added to each well. The plate
was then incubated at 37 °C for another 4 h. Finally, 150 ꢁL of
DMSO was added to each well and shaken for 10 min to blend
the DMSO solvent and the formazan completely. The absorbꢀ
ance was measured at 490 nm using a microꢀplate reader. Opꢀ
tical density which received no drugs was regarded as 100%
growth.
EXPERIMENTAL SECTION
Reagents and Materials. All the chemical reagents are of
analytical grade and used without any further purification,
including HNO3, urea, KF, Y2O3, Yb2O3, and Tm2O3
(99.99%), glutaraldehyde (from Sinopharm Chemical Reagent
Co., Ltd.), 1ꢀ(3ꢀdimethy laminopropyl)ꢀ3ꢀethylcarbodiimide
hydrochloride (EDC), Nꢀhydroxysuccinimide (NHS), folic
acid (FA), doxorubicin (DOX), hydrophilic titanium dioxide
(TiO2), 3ꢀ[4,5ꢀdimethylthiazolꢀ2ꢀyl]ꢀ2,5ꢀdiphenyl tetrazolium
bromide (MTT), 4’,6ꢀdiamidinoꢀ2ꢀphenylindole (DAPI), diꢀ
methyl sulfoxide (DMSO), trypan blue, calcein AM, propidiꢀ
um iodide (PI), 1,3ꢀdiphenylisobenzofuran (DPBF) (from sigꢀ
maꢀAldrich Co. LLC.).
Synthesis of TYY UCNPs. 1 M Ln(NO3)3 (Ln = Y, Yb,
Tm) was firstly prepared by dissolving corresponding Ln2O3
into dilute HNO3 with gradual heating. Meanwhile, hydroꢀ
philic TiO2 were dispersed in 20 mL deionized water by ultraꢀ
sonic wave. In a typical process for the synthesis of TYY
UCNPs (TiO2/Ln3+ molar ratio of 1:1), 1 mL of 1 M Ln(NO3)3,
3 g of urea, and 0.1 g of KF were dissolved in 15 mL deionꢀ
ized water mixed with 15 mL ethanol with continuous stirring,
and then the previously dispersed TiO2 nanoparticles were
added. Another 15 mL ethanol was added to the solution. Afꢀ
ter continuously stirring for 10 min, the mixture was heated to
90 °C and kept for 3 h. The resulting mixture was collected by
centrifugation at 6000 rpm for 4 min, and was repeated three
times. After dried at 60 °C for 12 h, the obtained powder was
calcinated at 600 °C for 3 h. The final product was
TiO2@Y2Ti2O7@YOF:Yb,Tm (simply labeled as TYY).
TiO2@YOF:Yb,Tm and Y2Ti2O7@YOFꢀYOF:Yb,Tm were
synthesized by the similar procedure except for adding
TiO2/Ln3+ with molar ratio of 1:2 and 2:1, respectively.
For the trypan blue measurement, three different plates were
used for each cell sample set, the first plate of HeLa cells was
incubated with culture only with and without NIR irradiation,
the second plate was incubated with TYY UCNPs with and
without NIR irradiation, and the third plate was incubated with
pure DOX. The cells on the bottom of the wells in one plate
were stained with 1 mL of 0.4% trypan blue dye to determine
the level of cell damage, and the dead cells were marked with
blue color while the live cells were not dyed. The three plates
could be divided to five groups (with and without NIR irradiaꢀ
tion) corresponding to the MTT cytotoxicity assay.
Surface Modification of TYY UCNPs with FA. FA moleꢀ
cules
were
conjugated
onto
the
surface
of
TiO2@Y2Ti2O7@YOF:Yb,Tm by coupling effect of NHS and
EDC. Briefly, 6 mg of EDC, 2 mg of NHS, and 1 mg of FA
were added into 20 mL of deionized water and stirred in dark
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Chemistry of Materials
The in vitro biocompatibility of TYY UCNPs was detected
cinoma cell lines) in the left axilla of each female Kunming
mouse (about 20 g). After grown for one week, the tumor sizes
reached about 5ꢀ8 mm in size with the body weight of 26ꢀ29 g.
The tumorꢀbearing mice were randomized into five groups (n
= 5, each group) and were treated by intravenous injection
with different treatment. Group 1: with none treatment, Group
2: with TYY UCNPs only without irradiation, Group 3: under
NIR irradiation only, Group 4: with pure DOX, and Group 5:
with TYY UCNPs under NIR irradiation. The first group was
made for blank control. The injected TYY UCNPs amount is
100 ꢁL (1 mg/mL) every two days, and the injected pure DOX
is 100 ꢁL (1 mg/mL) every two days. For the NIR irradiation
process, the tumor site was irradiated with 980 nm laser for 10
min after injecting different amount of materials for 2 h. The
body weights and tumor sizes were measured every two days
after treatment.
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similar to the cytotoxicity MTT assay by incubation of L929
fibroblast cells instead of HeLa cells. Hemolysis assay of TYY
UCNPs was used to determine the in vivo biocompatibility.
Briefly, red cells were obtained by removing the serum from
human blood after washing with 0.9% saline, and centrifugatꢀ
ed at 4000 rpm for five times to make the supernatant clear.
Subsequently, blood cells were diluted with PBS solution. 0.3
mL of diluted cells suspension was then mixed with 1.2 mL of
PBS (as a negative control), 1.2 mL of deionized water (as a
positive control), and 1.2 mL of UCNPs suspensions with
varying concentration of 15.63, 31.25, 62.5, 125, 250, and 500
ꢁg mL–1. The eight samples were shaken for several minutes,
and then kept steady for 2 h. Finally, the mixtures were centriꢀ
fuged at 4000 rpm and the absorbance of the upper supernaꢀ
tants was measured by UVꢀvis spectroscopy. The hemolysis
percentage was calculated as follows: Hemolysis (%) = (Asample
ꢀ Acontrol(ꢀ)) / (A control(+) ꢀ Acontrol(ꢀ)), where A is the absorbance.
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Histology Examination of TYY UCNPs. Histology analyꢀ
sis was studied at the 14th day after treatment. The typical
heart, liver, spleen, lung, kidney, and tumor tissues of the mice
in the five different treatment group were isolated. After that,
the organs were dehydrated by buffered formalin, ethanol with
different concentrations, and xylene, then embedded in liquid
paraffin. After that, the organs and tumor tissues were sliced to
3ꢀ5 mm and stained with Hematoxylin and Eosin (H&E) and
examined by CLSM (Leica TCS SP8).
Cellular Uptake of TYY UCNPs. The cellular uptake was
examined by confocal laser scanning microscope (CLSM)
incubated with HeLa cells. Typically, HeLa cancer cells were
cultured in a 6ꢀwell plate and grew overnight to obtain a monꢀ
olayer. Then, they were incubated with the asꢀsynthesized
TYY UCNPs at 37 °C for 0.5 h, 1 h, and 3 h, respectively.
After that, the cells were rinsed with PBS solution several
times, fixed for 10 min with 2.5% glutaraldehyde (1 mL well–
1), and further rinsed with PBS three times. Subsequently, the
nuclei were stained for 10 min with DAPI solution (20 ꢁg mL–
Characterization. Powder Xꢀray diffraction (XRD) patꢀ
terns were measured on a Rigaku D/max TTRꢀIII diffractomeꢀ
ter at a scanning rate of 15°/min in the 2θ range from 20° to
80°, using graphite monochromatized Cu Kα radiation (λ =
0.15405 nm). The morphology and structure were studied on
scanning electron microscope (SEM, JSMꢀ6480A) and transꢀ
mission electron microscopy (TEM, FEI Tecnai G2 SꢀTwin).
UCL emission spectra were acquired using 980 nm laser diode
(LD) Module (K98D08Mꢀ30W, China) as the irradiation
source and detected by R955 (HAMAMATSU) from 400–800
nm. DOX concentration was detected by UVꢀ1601 UVꢀvis
spectrophotometer at the wavelength of 480 nm. Inductively
coupled plasma (ICP) was carried out on a Thermo Electron X
Series II ICP instrument.
in PBS, 1 mL well–1) in order to perform nucleus labeling.
Finally, the cells were rinsed with PBS three times. The coꢀ
verslips were placed on a glass microscope slide, and the samꢀ
ples were measured by CLSM (Leica TCS SP8).
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UC Luminescence Microscopy (UCLM) Measurement.
To obtain a monolayer, HeLa cells (5 × 104 well–1) were firstly
seeded in 6ꢀwell culture plates. Then, TYY UCNPs were inꢀ
troduced into the 6ꢀwell culture. After that, these cells with
UCNPs were incubated at 37 °C for different times (0.5 h, 1 h,
and 3 h), respectively. At each incubated time, the cells in
each well were washed with PBS solution and fixed with 2.5%
formaldehyde (1 mL) at 37 °C for 10 min. Subsequently, the
asꢀprepared cells were washed with PBS several times to reꢀ
move the attached UCNPs.
RESULTS AND DISCUSSION
Phase, Structure, Morphology and UCL Properties.
Schematic diagram of the antiꢀcancer therapy mechanism of
TiO2@Y2Ti2O7@YOF:Yb,Tm under a single NIR irradiation
is presented in Scheme 1, which indicates the asꢀprepared
UCNPs may have synergistic PDT/PTT effect to cancer cell.
As shown, the blue emissions could be obtained due to the upꢀ
conversion energy transfer process of YOF:Yb,Tm (the outer
shell of the TYY UCNPs) under the 980 nm laser irradiation.
After that, the Y2Ti2O7 (the middle of the TYY UCNPs) as the
photocatalyst responsive to the blue light plays an important
role in generating the ROS with high level due to the deꢀ
creased distance between the valence band and conduction
band. During the energy transfer process, the thermal effect
could be also acquired due to the nonꢀradiative process and the
recombination of electron and hole pairs. The generated ROS
and thermal effect may result in the nuclei apoptosis and kill
the cancer cells. Figure S1 shows TEM images of hydrophilic
TiO2 precursor. The precursor is wellꢀdispersed with the size
of 20ꢀ25 nm. In Figure S2, when the TiO2/Ln3+ molar ratio is
In Vivo Biodistribution and Circulation of TYY UCNPs.
Female Kunming mice (20ꢀ25 g) were purchased from Harbin
Veterinary Research Institute, Chinese Academy of Agriculꢀ
tural Sciences (Harbin, China), and all the mouse experiments
were performed in compliance with the criterions of The Naꢀ
tional Regulation of China for Care and Use of Laboratory
Animals. Balb/c mice were intravenously injected with 100 ꢁL
of TYY UCNPs (20 mg/mL). The mice were euthanized at
different time (10 min, 1 h, 4 h, 12 h, 1 day, 3 days, 7 days,
and 14 days). The tumor, blood, and major organs of kidney,
lung, spleen, liver, heart were collected and dissolved in 5 mL
of HNO3 and HCl (v/v = 1:3), and then heated at 70 °C for
several minutes to obtain clear solutions. Then, the solutions
were centrifuged and the supernatant were collected for ICPꢀ
OES detection. For blood circulation assay, more dense time
points (10 min, 30 min, 1 h, 2 h, and 4 h) were acquired.
In Vivo Toxicity of TYY UCNPs. The tumors were foundꢀ
ed by subcutaneous injection of H22 cells (murine hepatocarꢀ
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2:1, highꢀdispersed microspheres with an average size of 140
particles uniformly, which further prove the coreꢀshell strucꢀ
ture.
It is well known the luminescence intensity and energy
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nm are obtained which can be due to the uniform coating of
Y(OH)CO3F:Yb,Tm shell on the surface of TiO2 cores. When
the molar ratio is altered to 1:1, uniform nanospheres with an
average size of 70 nm are generated. During this coꢀ
precipitation process, the extra Y3+ ions react with electronꢀ
negative TiO2, to form a layer of Y2Ti2O7 shell between TiO2
inner core and Y(OH)CO3F:Yb,Tm outer shell, leading to a
core/shell/shell structure.14 When the molar ratio is adjusted to
1:2, because more Y3+ ions exist in the solution, the inner surꢀ
vived TiO2 crystals completely transfer to Y2Ti2O7 (100 nm).
Meanwhile, the excessive Y3+ ions further react with F– ions to
form YOF particles (200 nm).15 After calcination,
Y(OH)CO3F transform to YOF phase, while the size and disꢀ
persibility (Figure S3) are kept similar to those of the precurꢀ
sors.
transfer efficiency between donor and accepter are influenced
by the crystal sizes, the morphologies, the distances between
donor and accepter. Usually, in the UVꢀvis diffuse reflection
spectra of TiO2, there are two major peaks which locate at
around 370ꢀ470 nm. The wide peak at lower than 388 nm is
attributed to the band gap recombination of TiO2, and the
peaks at 450ꢀ480 nm are related with transition from localized
surface states to the valence band of TiO2.13a Researches show
that the absorbance peaks of Y2Ti2O7 at around 470 nm are
well crossed with the max luminescence peaks of Tm3+
ions.13a,16 In order to utilize the cross to generate ROS under
NIR irradiation, the influence factors (phase, size, and strucꢀ
ture) which play prominent roles in the energy transfer effiꢀ
ciency should be considered. Figure 2aꢀc shows the SEM
images with schematic morphology (insets) of the samples
synthesized with different TiO2/Ln3+ molar ratios. The respecꢀ
tive average size of TiO2@YOF:Yb,Tm and TYY is 140 nm
and 70 nm, while Y2Ti2O7@YOFꢀYOF:Yb,Tm has two size
distributions (100 nm and 200 nm). Figure 2dꢀf shows the
emission spectra of the three samples excited at 980 nm for 0.5
min and 3 min, respectively. The same emission peaks at 476
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The phases and compositions of the samples synthesized
with different TiO2/Ln3+ molar ratios can also be confirmed by
the XRD results, as shown in Figure S4. For the sample synꢀ
thesized with molar ratio of 2:1 (TiO2@YOF:Yb,Tm), two sets
of diffraction peaks can be found. One is assigned to the teꢀ
tragonal TiO2 phase (JCPDS No. 21ꢀ1272), and the other to
tetragonal YOF phase (JCPDS No. 06ꢀ0347). For
TiO2@Y2Ti2O7@YOF:Yb,Tm, three sets of diffraction peaks
can be well indexed to tetragonal TiO2, tetragonal YOF and
cubic Y2Ti2O7 phase (JCPDS No. 42ꢀ0413). In the XRD patꢀ
tern of the sample synthesized with the TiO2/Ln3+ molar ratios
of 1:2, only tetragonal YOF and cubic Y2Ti2O7 are found,
while no TiO2 phase is detected.
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nm and 803 nm are assigned to G4 → H6 and H4 → H6
transitions of Tm3+ ions.17
Figure S6A shows the UCL emission spectra of different
samples at 980 nm laser excitation. The TYY UCNPs almost
have the same intensity as the pure YOF:Yb,Tm synthesized
by coꢀprecipitation method, while the other two samples have
much weaker emission intensities. The different UCL intensiꢀ
ties should be attributed to the diverse compositions, surface
defects, sizes, and shapes. Figure S6B shows that the intensity
of TYY UCNPs decreases in the whole regions (blue and NIR
emissions) with the prolonged time due to the nonradiative
transitions. The two main peaks (476 nm and 803 nm) ratio
can indicate the energy transfer efficiency because only the
blue emission regions have the cross with the Y2Ti2O7 photoꢀ
catalyst. As shown in Figure 2dꢀf and Figure S6B, the deꢀ
creased blue emissions may transfer the energy to the photoꢀ
catalyst which can generate ROS and thus the two weak ratio
is decreased. For TYY with the strongest UC emission intensiꢀ
ty, the ratio decreases from 4.38 to 1.42 when the irradiation
time is adjusted from 0.5 min to 3 min, and the emission colꢀ
our changes from blue to purple. This emission transformation
is also proved by the CIE chromaticity (Figure S7) and the in
vitro and in vivo experiment (Figure S8), while there is a
slight change for TiO2@YOF:Yb,Tm (3.68 to 3.3) and
Y2Ti2O7@YOFꢀYOF:Yb,Tm (2.94 to 2.68). The low peak
ratio of Y2Ti2O7@YOFꢀYOF:Yb,Tm may be due to the fast
energy transfer process between YOF and Y2Ti2O7 within 0.5
min. Figure 2gꢀi presents the UC emission stability at 476 nm
(1G4 → 3H6 of Tm3+ ions) of the three samples with irradiation
time interval of 5 min. We can see the intensity of
TiO2@YOF:Yb,Tm doesn’t change with the prolonged irraꢀ
diation time, while the intensity of TYY and Y2Ti2O7@YOFꢀ
YOF:Yb,Tm both decreases obviously. The lifetime at 476 nm
of TYY UCNPs (Figure S9B) was detected compared with
pure YOF:Yb,Tm (Figure S9A) to further prove the energy
transfer process. As shown, the lifetime of TYY at 476 nm is
0.30 ms, which is much shorter than that of pure YOF:Yb,Tm
The schematic diagram for the synthesis process of TYY
UCNPs is depicted in Figure 1a. The SEM image (Figure 1a)
and TEM images (Figure 1bꢀd) reveal that the final TYY
UCNPs are wellꢀdispersed with an average size of 70 nm. The
HRTEM image (Figure 1e) shows different crystal lattices
which indicates the product is composited of several phases.
The corresponding FFT image of the e1 region shows a typical
tetragonal crystal. The respective interplanar spacing of 0.35
nm, 0.19 nm, and 0.17 nm corresponds to the (101), (200), and
(211) planes of tetragonal TiO2 (JCPDS No. 21ꢀ1272). In the
FFT image of the e2 region, the typical parallel lattices with
the distances of 0.58 nm, 0.29 nm, and 0.15 nm are well conꢀ
sistent with the (111), (222), and (444) planes of cubic
Y2Ti2O7 (JCPDS No. 42ꢀ0413). In the e3 region, typical paralꢀ
lel lattices of 0.31 nm and 0.16 nm match well with the (101)
and (202) planes of tetragonal YOF (JCPDS No. 06ꢀ0347).
The results clearly reveal the TYY UCNPs are composited
with TiO2, Y2Ti2O7, and YOF phases. Meanwhile, the TEM
images with the corresponding selected area electron diffracꢀ
tion (SAED) images of different areas for TYY UCNPs are
given in Figure S5. The SAED lattice rings of single nanoparꢀ
ticle (Figure S5a, b) and many nanoparticles (Figure S5c, d)
are well consistent except for a slight difference of the densiꢀ
ties, indicating the particles are composited with the same
phases. The highꢀangle annular darkꢀfield scanning TEM
(HAADFꢀSTEM) image shows the coreꢀshell structure (Fig-
ure 1f). EDS analysis (Figure 1g) indicates there are Y, Yb,
Ti, F, C and O elements. The crossꢀsection compositional proꢀ
file lines (inset) indicate the product has coreꢀshell structure
with YOF outside and TiO2@Y2Ti2O7 inside. The elemental
mapping images (Figure 1h) further shows the F element disꢀ
tributes in the thick shell and Y, Yb, and Ti distribute in the
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(0.76 ms). Figure 2jꢀl gives the absorbance spectra of the soꢀ electron and hole pairs may decrease the catalytic ability,
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lutions of three samples with DPBF solution added with difꢀ
ferent irradiated times. It is apparent that the absorbance of
DPBF decreases with prolonged time due to the generation of
ROS. Especially, the TYY UCNPs have obviously superior
photodynamic property. In a sum, the energy transfer from the
YOF:Yb,Tm to Y2Ti2O7 in the blue region may produce the
photodynamic effect, while the decrease of energy in the
whole wavelength may generate the photothermal effect.
Thus, the asꢀsynthesized TYY UCNPs should have potential
application in the antiꢀcancer therapy field.
which is affected by the phase, structure, and size of the
Y2Ti2O7. (c) The photoꢀgenerated electrons and pairs will miꢀ
grate from the inner region to the surfaces of Y2Ti2O7, resultꢀ
ꢂ
1
ing in the formation of high ROS: OH', O2 , H2O2, and O2. It
should be noted that this efficient transfer process is benefited
from the close attachment of YOF and Y2Ti2O7 due to the
master design of TYY UCNPs. As shown in TEM images
(Figure 1d,f), the TYY UCNPs surfaces are not smooth but
have channels and pores which may be caused by the released
CO2 by the decomposition of the precursor. That means, the
water and oxygen could be exchanged between the nanopartiꢀ
cles and the surrounding environment. Additionally, as shown,
the thermal effect generated under NIR irradiation can be due
to two reasons. First, during the second energy transfer proꢀ
cess from the two different crystals of YOF to Y2Ti2O7, phoꢀ
tons in all the energy levels of Tm3+ ions will relax nonꢀ
radiatively and the decreased UCL in the whole region may
result in thermal effect. Second, the recombination of the holes
and electrons may also lead to thermal effect. Through designꢀ
ing this catalytic core and the luminescent shell, two adꢀ
vantages are obtained: the UCL material as the shell is benefiꢀ
cial to emit the visible light for tracking and diagnosis, while
the ROS and thermal effect generated from the catalytic subꢀ
stance inside could cross the channels to kill the tumor cells.
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Figure 3a,b present the UVꢀVis diffuse reflection absorpꢀ
tion spectra and Taucꢀplots (for indirect band gap transition)
calculated from the absorption spectra for band gap energy
calculations of different samples: pure TiO2 and the three
samples, which are well consistent with the UCL result. Fig-
ure 3a shows the absorbance of the KubelkaꢀMunk function
(F(R)) as a function of wavelength (λ). There is an obvious
difference between pure TiO2 and the samples synthesized
with different TiO2/Ln3+ molar ratio. Furthermore, in Figure
3b for the Taucꢀplots (for indirect band gap transition) calcuꢀ
lated from the absorption spectra for band gap energy calculaꢀ
tions, there is a relationship between the absorption coefficient
and the band gap energy: α = A × (hν ꢀ Eg)n/(hν), where α is
absorption coefficient which is proportional to F(R), A is a
constant, ν is the frequency, n = 2 for the indirect transitions,
and Eg means the band gap energy which could be changed
through different coꢀdoped ions. Thus, the Eg can be calculated
using intercept at (F(R) × hν)1/2 = 0 with the x axis of hν.13c
Table S1 summarizes the calculated band gap energy of Eg
using the SlopeꢀIntercept form of linear equation and the corꢀ
responding absorbance edge λ. Compared with pure TiO2, the
TYY UCNPs has an obvious red shift of the absorption edge
(from 387.5 nm to 484.1 nm). Interestingly, compared with the
other three curves, the curve of TYY UCNPs has two different
slopes which are well composited to the wavelength of 484.1
nm. This result may be due to the composition of TYY
UCNPs: two different photocatalysts of TiO2 and Y2Ti2O7. It
indicates the coꢀdopant of Y decreases the conducting band of
the TiO2 to Y2Ti2O7 and narrows the band gap, which is favorꢀ
able to the cross of the phosphor and the photoꢀcatalyst.
Cell Viability, Drug Release and MTT Cytotoxicity As-
say. Standard MTT assay was studied on L929 cells to detect
the shortꢀterm viability. Figure 4a demonstrates the cell viaꢀ
bility with particle concentrations from 15.63 to 500 ꢁg mL–1
incubated for 24 h. The biocompatibility of the sample in all
dosages is 101.9ꢀ118.0%. Even at the highest concentration of
500 ꢁg mL–1, the cell viability keeps as high as 106.5%, sugꢀ
gesting the good biocompatibility in vitro. Meanwhile, the
hemolysis assay on red blood cells was employed to guarantee
the potential intravenous administration in vivo. Figure 4b
shows the hemolytic result detected by UVꢀvis absorbance,
and the photograph of the red solution in the tube (inset) is
taken to show the presence of hemoglobin. There is no visualꢀ
ly red colour in the solution with added UCNPs, indicating the
negligible hemolysis in the controlled tubes. The highest heꢀ
molytic efficiency is 0.12% with particle concentration from
15.63 to 500 ꢁg mL–1, indicating almost no hemolysis occurs.
In conclusion, the MTT assay and the blood compatibility of
TYY UCNPs are excellent which is almost nontoxic to live
cells.
Figure 3c schematically shows the generation of ROS and
the thermal effect caused by energy transfer process of TYY
UCNPs. Firstly, blue emissions could be generated due to the
twoꢀ/threeꢀphoton UCL process under NIR irradiation. The
absorption of pump photons from the NIR irradiation only
Cellular cytotoxicity and dead cells marked by trypan blue
were utilized to evaluate the potential application of antiꢀ
cancer therapy. The cytotoxicity of HeLa cells incubated with
TYY UCNPs, with pure DOX, with TYY UCNPs under NIR
irradiation, and irradiated with NIR only is given in Figure 4c.
When the culture was exposed to NIR irradiation, the pump
power was adjusted to 0.72 W cm–2 and kept for 5 min. As
shown in Figure 4c, when only TYY UCNPs is added, more
than 96.0% of HeLa cells are viable under a varying concenꢀ
tration range from 15.63 to 500 ꢁg mL–1. The results indicate
that TYY UCNPs have no cytotoxicity to cancer cells. When
the cells are incubated with culture only under the NIR irradiaꢀ
tion, the cell viability is lower (73.0%ꢀ83.5%) than that withꢀ
out NIR irradiation, which may be due to the slight absorbance
of culture to NIR laser. When incubated with the anticancer
drug of DOX, the cells viability is 34.5%ꢀ53.4% with DOX
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populates photons from the ground state F7/2 to F5/2 level of
Yb3+. Then, through successive energy transfer process, the
excited photons transfer from Yb3+ ions to Tm3+ ions and popꢀ
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ulate the G4, D2, and H4 levels of Tm3+ ions. After all these
process, the excited Tm3+ ions will fall to lower energy levels:
1D2 → F4 (449 nm), G4 → H6 (476 nm), G4 → F4 (646
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nm), and H4 → H6 (803 nm). Secondly, ROS are generated
from the Y2Ti2O7 photocatalyst, which is excited by the blue
emissions generated from Yb3+/Tm3+. During the following
process, there are three steps: (a) as shown in the absorbance
spectra in Figure 3a, the Y2Ti2O7 photocatalyst is sensitive to
the blue light. Then, Y2Ti2O7 will produce holes and electrons
in the valence band (VB) and conduction band (CB), respecꢀ
tively. (b) The excited holes and electrons separate and miꢀ
grate to the surface of Y2Ti2O7. Note that the recombination of
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concentration of 3.13ꢀ100 ꢁg mL–1. The calculated IC50 value
be found. With respect to potential clinical application, the
of pure DOX to HeLa cells is 4.47 ꢁg mL–1. In the last group,
the viability of the HeLa cells incubated with UCNPs with
concentrations from 15.63 to 500 ꢁg mL–1 under NIR irradiaꢀ
tion is 28.1%ꢀ38.4%, which is lower than that of the cells inꢀ
cubated with pure DOX. The calculated IC50 value of UCNPs
with NIR irradiation is 5.06 ꢁg mL–1, which is almost as low
as the value of pure DOX.
detection of the bioꢀdistribution and blood circulation of the
particles with different intravenous injection times is necesꢀ
sary. As shown in Figure 5f, in the early stages (10 min, 1 h, 4
h, and 12 h after the injection), the nanoparticles accumulate in
the livers, spleens, lungs, and targeted tumors. In the whole
detection time, Y concentrations still keep low in kidney and
heart. After three days of postꢀinjection, the Y concentrations
reduce obviously in all the organs. In the 7th and 14th day after
injection, the nanoparticles in the liver, spleen, and lung alꢀ
most disappear. The results reveal that the injected TYY
UCNPs can be excreted from the mice with prolonged times.
Figure 5g shows the pharmacokinetics of blood circulation
curve. The fitted curve presents a twoꢀcompartment model
with the first and second phase blood circulation halfꢀlives at
0.018± 0.013 day (about 0.432 h) and 0.437 ± 0.069 day
(about 10.5 h), respectively.
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The viability of HeLa cells with different treatments marked
with trypan blue was also detected. The dead cells can be dyed
with blue colour. As shown in Figure 4d, the cells almost
survive completely when only incubated with UCNPs without
NIR irradiation, while most of the cells are dead when incuꢀ
bated with UCNPs under NIR irradiation, which is well coinꢀ
cident with above cytotoxicity MTT assay. The confocal laser
scanning microscopy (CLSM) images of HeLa cells incubated
with added UCNPs with and without NIR irradiation dyed by
AM and PI (Figure S10) further confirm the PDT/PTT effect
of UCNPs triggered by NIR light. The results indicate that the
asꢀsynthesized TYY UCNPs should be an efficient anticancer
candidate triggered by a single NIR laser.
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CT imaging has been proved an effective diagnostic techꢀ
nique owing to the high resolution and deep tissue penetration.
Because rare earth ions doped products can be used as contrast
agents for CT imaging, we measured the imaging properties of
TYY UCNPs in vitro and in vivo, which are presented in Fig-
ure 6. It is obvious that the signal and CT value increases with
the increase of UCNPs concentration (Figure 6a, b), and there
is a wellꢀfitted linear relationship between them with the slope
of 10.66 ± 0.61. In vivo CT imaging was further measured by
intratumoral injection of tumorꢀbearing Balb/c mice (Figure
6cꢀf). As shown, the CT value after injection is 959.5 HU
(Hounsfield units), which is markedly higher than that (39.8
HU) of the control (without injection). The in vitro and in vivo
results show the asꢀsynthesized TYY UCNPs are effective
contrast agent for CT imaging.
The cell upꢀtaken property was also studied. HeLa cells
were incubated with TYY UCNPs for 1 h, then dyed by DAPI
and detected by CLSM. As discussed above, the emission
wavelength of TYY UCNPs is 476 nm of blue region and 803
nm of NIR region. We utilized 488 nm as the excitation waveꢀ
length, and red emission can be interestingly detected. Thus,
there are blue emissions emitted by DAPI in the nuclei and red
emissions emitted by the UCNPs. Figure 5a reveals the TYY
UCNPs are evident in the intracellular region, and no superꢀ
fluous luminescent signals outside is found. The result indiꢀ
cates the asꢀsynthesized UCNPs are internalized into the cells
rather than merely stained in the membrane surface. Furtherꢀ
more, Figure S11 indicates that stronger red fluorescence
emission of UCNPs is observed in the cytoplasm with the enꢀ
hanced incubation time, suggesting that more naoparticles are
localized in the cells. Particularly, it is apparent that the UC
luminescence microscopy (UCLM) images (Figure 5b) of
HeLa cells incubated with TYY UCNPs presents strong blue
emissions, and there are stronger emissions and more internalꢀ
ized nanoparticles inside of HeLa cells with prolonged times
from 0.5 h to 3 h (Figure S12). The results further confirm
that TYY UCNPs can be effectively taken up by tumor cells
and emit blue light under 980 nm irradiation.
The good biocompatibility and synergistic antiꢀcancer effect
in vitro stimulate us to study the in vivo antiꢀtumor effect. As a
proof of principle experiments, the H22 tumorꢀbearing mice
were treated with different conditions: without treatment as the
control group, with TYY UCNPs intravenously injected only,
with NIR irradiation on the tumor site, with pure DOX, and
with TYY UCNPs under NIR irradiation. Here, the NIR irraꢀ
diation is carried for 10 min with the pump power of 0.72 W
cm–2. The mice were under treatment when the tumor size
grew to 4ꢀ8 mm in size (one week). After different treatments
of the five groups for two weeks, the result of each group is
given in Figure 7a and b. It is obvious that the tumor size is
very large (about 27 mm) without any treatment. When
UCNPs are injected only, the tumor size keeps similar to the
control group (24 mm). The tumor is inhibited a little with
NIR irradiation with the tumor size of 19 nm, which may be
due to the photothermal effect absorbed by the tumor cells.
The pure DOX was also injected intravenously with 100 ꢁL (1
mg/mL) every two days, and the tumor size is 15 mm. It is
noted that the mouse of this group has the thinner body which
may be caused by the side fact of the anticancer drug. For the
last group injected with UCNPs under NIR irradiation, the
tumor size (9 mm) is obviously smaller than that of the other
four groups. Figure 7c shows the body weights of all of the
five groups increase with prolonged time, and the result also
indicates the weights are almost caused by the increased tumor
volumes (Figure 7b). Notably, the body weight of the best
treated group increases slightly, which indicates the tumor can
be efficiently inhibited due to the simultaneous PDT and PTT
As discussed above, the photothermal effect is generated
due to the energy transfer process between YOF:Yb,Tm and
Y2Ti2O7 and the recombination of holeꢀelectron pairs in
Y2Ti2O7. The in vivo infrared thermal images of tumorꢀbearing
mice after injection of saline and TYY UCNPs versus irradiaꢀ
tion time with the pump power of 0.72 W cm–2 are displayed
in Figure 5c, and the temperature curves as a function of irraꢀ
diation time is depicted in Figure 5d. It is obvious that the
second group with UCNPs injected has the obviously inꢀ
creased temperature (up to 49.7 °C) than that (42.1 °C) injectꢀ
ed with saline, which proves the photothermal effect caused by
the TYY UCNPs. The controllable temperature is beneficial to
therapy which can supply various choices and has a synergisꢀ
tic therapy with PDT. Figure 5e presents the digital photoꢀ
graph of a mouse subcutaneously injected with TYY UCNPs
under 980 nm NIR irradiation, and apparent purple light can
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effect. Figure 7d presents the tumor histologic section acꢀ
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quired from different groups. There are obvious degenerative
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marked karyolysis in control group, and the conditions beꢀ
come better from the left to the right. For the best inhibition
group, due to the markedly increased apoptotic and necrotic
tumor cells, the cells in this group seems to be almost regular
with normal cells.
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In order to evaluate the nanotoxicity of the asꢀsynthesized
material, the H&E stained images of heart, liver, spleen, lung,
and kidney from different groups are given in Figure 8. In the
control group, necrosis is found in the histological samples,
and there are symptoms of inflammation and glomerulus atroꢀ
phy. Compared with the control group, the organs with best
treatment show the following result: no pulmonary fibrosis is
detected in all the lung samples. Hepatocytes in the liver samꢀ
ples are found normal, and the glomerulus structure in the
kidney section is observed clearly. The results clearly demonꢀ
strate the potential clinical applicability of the asꢀsynthesized
TYY UCNPs as antiꢀtumor system.
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CONCLUSIONS
In summary, uniform coreꢀshell structured TiO2@Y2Ti2O7
@YOF with UCL and photothermal/ photodynamic properties
under NIR irradiation were synthesized by a facile coꢀ
precipitation method. Different composites were obtained by
simply adjusting the TiO2/Ln3+ ratio. The reactive oxygen
species (ROS) and thermal effect could be generated under
NIR irradiation due to the energy transfer from YOF:Yb,Tm to
the photocatalyst of Y2Ti2O7. The results reveal that the asꢀ
synthesized TYY UCNPs have good biocompatibility. The
composite exhibit obvious cytotoxicity to HeLa cancer cells,
which were evidenced by MTT assay and CLSM images of
HeLa cells dyed by trypan blue and AM/PI. In particular, the
product also shows clear photoꢀthermal imaging, UCL imagꢀ
ing and CT imaging in vivo. The final animal experiment and
the histologic section assay also prove the antiꢀcancer therapy
efficiency of the asꢀsynthesized product.
ASSOCIATED CONTENT
Figure S1-S12 and Table S1 can be seen from the supporting
information. This material is available free of charge via the Inꢀ
ternet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
yangpiaoping@hrbeu.edu.cn (P. Y.)
jlin@ciac.ac.cn (J. L.)
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
Financial supports from the National Natural Science Foundaꢀ
tion of China (NSFC 21271053, 21401032, 51472058,
51332008) are greatly acknowledged.
(9) (a) Ostrowski, A. D.; Chan, E. M.; Gargas, D. J.;
Katz, E. M.; Han, G.; Schuck, P. J.; Milliron, D. J.; Cohen,
B. E. ACS Nano 2012, 6, 2686. (b) Zeng, S.; Yi, Z.; Lu, W.;
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Scheme 1. Schematic diagram of the antiꢀcancer therapy mechanism for TYY UCNPs under a single
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NIR irradiation .
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Figure 1. (a) Schematic diagram of the synthetic process, (b) SEM image, (c, d) TEM images, (e)
HRTEM image, (e1ꢀe3) the corresponding FFT images, (f) HAADFꢀSTEM image, (g) EDS with crossꢀ
section compositional line profiles inset, and (h) elemental mapping images of TYY UCNPs.
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Figure 2. (aꢀc) SEM images of TiO2@YOF:Yb,Tm, TiO2@Y2Ti2O7@YOF:Yb,Tm and
Y2Ti2O7@YOFꢀYOF:Yb,Tm. Insets are the corresponding structure sketches. (dꢀf) The UCL emission
spectra of the samples under 980 nm irradiation for 0.5 min and 3 min. (gꢀi) The UC emission stability
of Tm3+ at 476 nm under 980 nm excitation within 10 min with each reꢀirradiation for 5 min. (jꢀl) The
absorbance spectra of TiO2@YOF:Yb,Tm, TiO2@Y2Ti2O7@YOF:Yb,Tm and Y2Ti2O7@YOFꢀ
YOF:Yb,Tm with DPBF solution added under 980 nm irradiation.
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Figure 3. (a) UVꢀvis diffuse reflection absorption spectra and (b) Taucꢀplots (for indirect band gap tranꢀ
sition) calculated from the absorption spectra for band gap energy calculations of pure TiO2 and the
three samples synthesized with different TiO2/Ln3+ molar ratio. (c) Schematic diagram of the energy
transfer process of TYY UCNPs between the Yb/Tm ions and the Y2Ti2O7 photoꢀcatalyst.
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Figure 4. (a) L929 fibroblast cell viability incubated with TYY UCNPs with different concentrations
for 24 h. (b) The hemolysis property of TYY UCNPs to human red blood cells. (c) In vitro viability of
HeLa cells after various treatments: incubated with TYY UCPCs with different concentration for 24 h,
with NIR irradiation only, incubated with pure DOX, and incubated with UCNPs with different concenꢀ
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Figure 5. (a) CLSM images and (b) UCLM images of HeLa cells incubated with TYY UCNPs for 1 h.
(c) In vivo infrared thermal images of tumorꢀbearing mice after injection of saline, and TYY UCNPs
versus irradiation time under 980 nm NIR irradiation with the pump power of 0.72 W cm–2. (d) Temperꢀ
ature profiles of the mice after intravenous injection with saline and UCNPs as a function of irradiation
time. (e) UCL photograph of a mouse after injection of TYY UCNPs under 980 nm irradiation with the
pump power of 0.72 W cm–2. (f) The biodistribution and (g) blood circulation of TYY UCNPs in H22
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Figure 6. (a) In vitro CT images of TYY UCNPs with different concentrations. (b) CT value of aqueous
solution of TYY UCNPs as a function of the particle concentration. CT imaging of tumorꢀbearing
Balb/c mouse (c, d) before intratumor injection and (e, f) after injection.
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Figure 7. (a) Representative photographs of tumorꢀbearing mice after various treatments: without any
treatment as the control group, injected with TYY UCNPs only, with NIR irradiation only, injected with
pure DOX, and injected with UCNPs under NIR irradiation, all of the materials were injected intraveꢀ
nously; digital photographs of tumor tissues after different treatments for 14 days. (b) The normalized
tumor size, and (c) body weight of H22 tumor in different groups after different treatments. (d) H&E
stained images of tumors from the different groups.
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Figure 8. H&E stained images of heart, liver, spleen, lung, and kidney from different groups at 14th day.
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