Hollow structured Y2O3:Yb/Er-CuxS nanospheres with controllable size for simultaneous chemo/photothermal therapy and bioimaging

Article abstract of DOI:10.1021/cm503647k

To integrate photothermal therapy (PTT) with chemotherapy for improved antitumor efficiency, we designed a novel multifunctional composite by attaching Cu_xS nanoparticles onto the surface of Y₂O₃:Yb/Er hollow spheres through a combined coprecipitation and subsequent hydrothermal route. By altering the initial pH values for the synthesis of precursors, the size and structure properties of the final composites can controllably be tuned. The conjugated folic acid (FA) makes the composite recognize the targeted cancer cells and the attached Cu_xS nanoparticles endow the composite with photothermal function. It is found that the release of doxorubicin (DOX) from the functional carrier could be triggered by both pH value and near-infrared (NIR) radiation. In particular, both PTT and chemotherapy can be simultaneously driven by 980 nm laser irradiation. The synergistic therapeutic effect based on PTT and chemotherapy can lead to low in vitro viability of 12.9% and highly strong inhibition of animal H22 tumor in vivo, which is superior to any individual therapy. Moreover, the composite exhibits the clear in vivo red up-conversion luminescence (UCL). This multifunctional nanocarrier can be applicable as bioimaging agent and effective antitumor agent for the synergistic interaction between PTT and the enhanced chemotherapy.

Full text of DOI:10.1021/cm503647k

Article
pubs.acs.org/cm
Hollow Structured Y2O3:Yb/Er−CuxS Nanospheres with Controllable
Size for Simultaneous Chemo/Photothermal Therapy and
Bioimaging
Ruichan Lv,† Piaoping Yang,*,† Fei He,† Shili Gai,† Guixin Yang,† and Jun Lin*,‡
†Key Laboratory of Superlight Materials and Surface Technology, Ministry of Education, College of Material Sciences and Chemical
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,
Harbin 130021, P. R. China
S
* Supporting Information
ABSTRACT: To integrate photothermal therapy (PTT) with
chemotherapy for improved antitumor efficiency, we designed
a novel multifunctional composite by attaching CuxS nano-
particles onto the surface of Y2O3:Yb/Er hollow spheres
through a combined coprecipitation and subsequent hydro-
thermal route. By altering the initial pH values for the
synthesis of precursors, the size and structure properties of the
final composites can controllably be tuned. The conjugated
folic acid (FA) makes the composite recognize the targeted
cancer cells and the attached CuxS nanoparticles endow the
composite with photothermal function. It is found that the
release of doxorubicin (DOX) from the functional carrier
could be triggered by both pH value and near-infrared (NIR)
radiation. In particular, both PTT and chemotherapy can be simultaneously driven by 980 nm laser irradiation. The synergistic
therapeutic effect based on PTT and chemotherapy can lead to low in vitro viability of 12.9% and highly strong inhibition of
animal H22 tumor in vivo, which is superior to any individual therapy. Moreover, the composite exhibits the clear in vivo red up-
conversion luminescence (UCL). This multifunctional nanocarrier can be applicable as bioimaging agent and effective antitumor
agent for the synergistic interaction between PTT and the enhanced chemotherapy.
to fabricate hollow structures which mainly contains two
routes: hard templates and soft templates. Hard-template
method using solid template, such as polystyrene (PS) spheres,
carbon spheres, SiO2 can be used to synthesize lots of hollow
materials with beautiful morphology and controllable size, but
the synthesis method is partly disadvantaged because of the low
yield and complicated process.23−25 While for the soft-template
process, different surfactants have to be used to control the
synthetic process and the obtained materials are unsatisfactory
with uncontrollable size.26−28
Specially, hollow structures can be achieved via the sacrificed-
template process with proper precursors, among which
Kirkendall effect plays an important and effective rule.29−31
The key to a successful method for fabricating hollow structure
utilizing Kirkendall effect is to find a proper diffusion couple
and it is still a challenge to scientists in the inorganic material
field. Thus, for the biological application, it is highly desirable
to obtain controllable and highly dispersed hollow spheres with
INTRODUCTION
■
In drug delivery systems (DDSs) and disease diagnosis fields,
lanthanide ion-doped particles have been widely used as drug
carriers, bioimaging, and biolabel because of their 4f electronic
shells, physical and chemistry stability, and relatively low
toxicity.1−5 Especially, up-conversion (UC) emissions within
near-infrared (NIR) irradiations located within the optical
transmission window of biological specimens have deeper light
penetration, decreased damage to normal cells, enhanced image
contrast, and less autofluorescence.6−17 Thus, NIR irradiated
UC emissions may have a wide application in the biological
field.18,19 Meanwhile, materials with hollow structures have
attracted special attention due to their attractive typical features
including large surface area, low density, tunable pore size, and
high loading capacity based on the large cavity.20−22 Compared
with the solid materials, the large interior space and the hollow
structure induced large surface area can store/adsorb more drug
molecules, and the mesoporous shells can provide accessible
channels for drug molecules diffusion and mass transfer. In
addition, they can control the permeability of the shells for
matter exchange between the inner cavity and the outer
environment. Template-directed synthesis is the main method
Received: October 3, 2014
Revised: December 7, 2014
Published: December 9, 2014
© 2014 American Chemical Society
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Scheme 1. Schematic Illustration for the Formation of Y2O3:Yb/Er−CuxS Hollow Spheres and Bioapplication
simple approach. Especially, hollow spheres of the dimension
under 100 nm and the average pore diameters larger than 5 nm
are essential for future enhanced intracellular uptake, intra-
nuclear drug loading, and prolonged blood circulation.32,33
Thus, it is a booming requirement to develop new available
strategy to produce hollow spheres with controllable-scale
quantities for the future clinic application.
Then, folic acid (FA) was conjugated onto the surface of
Y2O3:Yb/Er hollow spheres, which made the materials well
recognize the cancer cells in order to reach the targeted cells.
Especially, the CuxS nanoparticles as an acceptable PTT agent
were attached onto the surface of Y2O3:Yb/Er hollow
nanospheres through the electronic attraction. doxorubicin
(DOX) release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide (MTT) assay, NIR-light stimulated PTT effect
to tumor cells in vitro and in vivo were well-detected.
Especially, the surface area, the UCL properties, drug loading
amount, release rate, and cellular up-taken process were
dependent on the sizes of the hollow spheres, which could
supply a wide choice to disparate drug delivery.
Most recently, photothermal therapy (PTT) co-operated
with chemotherapy have attracted considerable attention, which
can kill tumor cells effectively irradiated by 980 nm laser.34,35 If
the photothermal conversion can be realized by NIR laser
irradiation, it can be beneficial to tissues and organs because the
PTT process may discriminate the normal cells and the
targeted cancer cells. Advanced agents play an important role in
the efficient PTT. Up to now, there are several types of PTT
agents: the organic compounds, the carbon compound, noble
metal nanostructures, and semiconductors.36−40 The most
widely used type is the noble metal crystals including Au
nanostructures. These structures present intense absorption
with strong molar extinction coefficient of 1 × 108 to 1 × 1011
M−1 cm−1 and relatively high photothermal conversion
efficiency due to their tunable surface plasmon resonance
effects.41−44 The used carbon compounds include carbon
nanotubes, grapheme, and carbon dots.45,46 However, these two
kinds of agents may have side effects to the photoluminescence
because of strong absorption in the visible region, which could
decrease the UC conversion emissions by fluorescence
resonance energy transfer (FRET).47 Meanwhile, the organic
compounds have some disadvantages, such as low photo-
thermal conversion efficiency and serious photobleaching
effect.48 Thus, copper chalcogenides (CuxS) as a booming
PTT agent have been considered as a prospective candidate for
PTT. Meanwhile, CuxS has been demonstrated to have long-
time low toxicity and high stability and to be biodegradable,
which has almost no obvious side effect to the visible
emissions.49−51
EXPERIMENTAL SECTION
■
Reagents and Materials. All the chemical reagents used in this
experiment are of analytical grade without any further purification,
including urea, nitric acid (HNO3), hydrochloric acid (HCl), copper
chloride dehydrate (CuCl2), sodium citrate (Na3Cit), sodium sulfide
(Na2S), Y2O3, Yb2O3, and Er2O3 (99.99%) (from Sinopharm Chemical
Reagent Co., Ltd.); phosphate buffered saline (PBS), potassium
hydrogen phthalate (PHP), and glutaraldehyde (from Tianjin Kermel
Chemical Reagent Co., Ltd.); 3-aminopropyltrimethoxysilane (APTS),
FA, 1-(3-dimethy laminopropyl)-3-ethylcarbodiimide hydrochloride
(EDC), n-hydroxysuccinimide (NHS), doxorubicin (DOX), 3−4,5-
dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide (MTT), di-
methyl sulfoxide (DMSO), 4′,6-diamidino-2-phenylindole (DAPI),
3′,6′-Di(O-acetyl)-4′,5′-bis[N,N-bis(carboxymethyl) aminomethyl]
fluorescein (calcein AM), propidium iodide (PI), and trypan blue
(from Sigma-Aldrich). mPEG-NH2 (2k) was obtained from Beijing
Kaizheng Biotech Development Co. Ltd.
Synthesis of Y(OH)CO3:Yb/Er Precursor. In a typical process, 1
M Ln(NO3)3 (Ln = Y, Yb, and Er) solutions were prepared by
dissolving corresponding calculated Ln2O3 into HNO3 with gradual
heating. A total of 1 mL of 1 M Ln(NO3)3 (89%Y, 10%Yb, and 1%Er)
and 3 g of urea were dissolved in 50 mL of deionized water in a beaker.
After continuous stirring for 5 min, heating of the mixture was
continued at 90 °C for 3 h through a water bath. The resulting mixture
was centrifuged at 6000 rpm for 4 min, and this process was repeated
three times. To obtain the precursors with different sizes, we regulated
the pH values of Ln(NO3)3 solutions to 1, 1.5, and 2 by the diluted
HNO3.
Synthesis of Y(OH)CO3:Yb/Er@C and Y2O3:Yb/Er hollow
spheres. In a typical process for the coating of carbon layer, 1.6 g
of glucose was dissolved in a mixture of 18 mL of deionized water and
12 mL ethanol. Then the as-prepared Y(OH)CO3:Yb/Er was added
into the solution, the resulting suspension was transferred to a 50 mL
autoclave and heated at 190 °C for 4 h. After that, the sample was
In this contribution, Y(OH)CO3:Yb/Er spheres, synthesized
by a coprecipitation process, were used as the sacrificed
precursor to prepare Y2O3:Yb/Er hollow spheres. The sizes of
the precursors were precisely controlled by tuning the initial
pH values of the solution. Meanwhile, the formation of hollow
spheres was achieved because of the coating absorption effect of
glucose under the hydrothermal environment. After calcination,
Y2O3:Yb/Er hollow spheres with different sizes were obtained.
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washed with deionized water and ethanol three times. Finally, the
dried Y(OH)CO3:Yb/Er@C was calcined in air at 800 °C for 3 h, and
then Y2O3:Yb/Er hollow spheres were obtained.
Synthesis of Y2O3:Yb/Er−NH2. The as-prepared Y2O3:Yb/Er
hollow spheres were dispersed in 60 mL of deionized water. 0.15 mL
of APTS was added to the solution and heated to 45 °C for 8 h under
stirring, and then the sample was collected by centrifugation, washed
with ethanol ,and finally dried at 60 °C for subsequent use.
Hemolysis Assay. Red blood cells were acquired by removing the
serum from the EDTA.K2 stabilized human blood through washing
with 1% normal saline, and centrifugated several times until the
supernatant were transparent. Then, the blood cells were diluted to 1/
10 with PBS solution (pH = 7.4). Diluted cells suspension (0.3 mL)
was then mixed with (1) 1.2 mL of deionized water as a positive
control; (2) 1.2 mL of PBS as a negative control; and (3) 1.2 mL of
materials suspensions with varying concentration (15.625, 31.25, 62.5,
125, 250, 500, and 1000 μg mL−1). The ten samples were shaken and
kept stable for 2 h at room temperature. Finally, the mixtures were
centrifuged and the absorbance of the upper supernatants was
measured by UV−vis. The percentage of hemolysis was calculated
using the following equation
Synthesis of Y2O3:Yb/Er−NH2−FA. FA is conjugated onto the
surface of Y2O3:Yb/Er−NH2 because of the presence of cationic
polymer −NH2. First, 1 mg of FA, 2 mg of NHS, and 6 mg of EDC
were added into 20 mL of deionized water and stirred for 2 h in dark.
Subsequently, 20 mg of Y2O3:Yb/Er−NH2 hollow spheres were added
and allowed to react with stirring overnight in dark at room
temperature. The material was recovered by centrifugation and
washed twice with deionized water and ethanol to remove the free
FA. The as-prepared product was labeled as Y2O3:Yb/Er−NH2−FA.
Synthesis of Y2O3:Yb/Er−CuxS. CuxS nanoparticles were
synthesized according to the reported procedure52 (see Scheme 1).
1 mmol of CuCl2·2H2O was dissolved in 90 mL of deionized water,
and then 0.02 g of sodium sulfide was added with stirring. After stirring
for 5 min at room temperature, the brown solution was put into a
water bath kettle at 90 °C and maintained for 15 min. Finally, the
solution was transferred to ice-cold water and the green solution of
CuxS nanoparticles was prepared for future use. Then, the as-prepared
Y2O3:Yb/Er−NH2−FA was dissolved by deionized water and then the
solution of CuxS nanoparticles were added and stirred for 2 h. After
centrifugation, the Y2O3:Yb/Er−NH2−FA−CuxS particles were
prepared, and then dispersed in 20 mL deionized water for further
PEG modification. Ten mg of mPEG−NH2 was added to the solution
as final modification for better cell recognition and biocompatibility.
For better conjunction, 3 mg of EDC and 1 mg of NHS were added
into the above mixture with continuous stirring at room temperature.
The final product was labeled as Y2O3:Yb/Er−CuxS up-conversion
nanocrystals (UCNCs).
DOX Loading and Release Test. Thirty mg of Y2O3:Yb/Er−
CuxS hollow nanospheres was added into 5 mL of PBS (pH 7.4) and
dispersed by ultrasonication. After that, 2.5 mg of DOX was added into
the solution with slow stirring at room temperature for 24 h. The as-
prepared mixture was centrifugally separated at 6000 rpm for 4 min,
and then the supernatant solution was kept for ultraviolet visible
(UV−vis) analysis. The as-obtained sample was then washed with PBS
solution and separated by centrifugation. This process was repeated
three times. Subsequently, 10 mL of fresh PBS was replenished in the
centrifugal tube and set in water bath kettle at 37 °C with magnetic
stirring for 30 min, and then the supernatant solution was kept for
further analysis. The process was repeated and changed release time as
30 min, 1, 2, 4, 8, 12, 24, and 36 h, respectively. PBS (pH = 5.5) was
prepared by PBS (pH 7.4) with citric acid added, and PHP (pH 4) was
prepared directly by pH modifier. The loading amount and
concentration of DOX in the solution were determined at the
wavelength of 480 nm by UV−vis measurement.
hemolysis% = (Asample − Acontrol(−))/(Acontrol(+) − Acontrol(−)
)
where A is the absorbance of UV−vis spectrum.
Cellular Uptake. Cellular uptake by HeLa cancer cells was
examined using a confocal laser scanning microscope (CLSM). The
HeLa cells were seeded in a 6-well culture plate (a clean coverslip put
in each well) and grown overnight as a monolayer. Then, they were
incubated with as-prepared Y2O3:Yb/Er−CuxS+DOX at 37 °C for 30
min and 3 h, respectively. After that, the cells were rinsed with PBS
three times, fixed with 2.5% glutaraldehyde (1 mL well−1) at 37 °C for
10 min, and then rinsed with PBS three times again. To perform
nucleus labeling, we stained the nuclei with DAPI solution (20 μg
mL−1 in PBS, 1 mL well−1) for 10 min. The cells were then rinsed with
PBS three times. The coverslips were placed on a glass microscope
slide, and the samples were visualized using CLSM (Leica TCS SP8).
UC Luminescence Microscopy (UCLM) Observation. UC
luminescence imaging of HeLa cells (5 × 104 well−1) were seeded
in 6-well culture plates, and then incubated overnight as a monolayer.
After that, these cells were incubated for different time (0.5 h, 1 and 3
h) at 37 °C with Y2O3:Yb/Er−CuxS, respectively. The cells were
washed with PBS solution 3 times, fixed with 1 mL of 2.5%
glutaraldehyde in each well for 10 min at 37 °C. After that, the as-
prepared cells were washed with PBS three times in order to remove
attached nanoparticles. Meanwhile, we have detected the cell uptaken
process of Y2O3:Yb/Er−CuxS+DOX.
In Vitro Cytotoxicity of Y2O3:Yb/Er−CuxS+DOX. The cytotox-
icity of Y2O3:Yb/Er−CuxS+DOX were detected similar to the viability
MTT assay by incubation of HeLa cancer cells instead of L929
fibroblast cells. Meanwhile, the HeLa cells viabilities in vitro incubated
for 24 h with free DOX, Y2O3:Yb/Er−CuxS, Y2O3:Yb/Er−CuxS+DOX
with and without NIR irradiation (pump power of 0.6 W cm−2) with
different concentrations. Calcine AM and PI dyed HeLa cells were
detected for the cells incubated with culture only under NIR
irradiation and with Y2O3:Yb/Er−CuxS+DOX under NIR irradiation
(pump power of 0.6 W cm−2). HeLa cells (5 × 104 well−1) were
seeded in 6-well culture plates for two wells, and then incubated
overnight as a monolayer. After that, 1 mL culture was added to the
first well and 1 mL Y2O3:Yb/Er−CuxS+DOX (1 mg/mL) was added
to the second well, respectively. After irradiation for 5 min, the two
wells were both dyed with calcine AM and PI visualized using CLSM.
In Vivo Toxicity of Y2O3:Yb/Er−CuxS+DOX. First, the tumors
were established by subcutaneous injection of H22 cells (murine
hepatocarcinoma cell lines) in the left axilla of each female mouse
(20−25 g). After grown for 1 week, the tumors sizes reached the size
about 6−8 mm. The tumor-bearing mice were randomized into five
groups (n = 5, each group) and were treated by intratumoral injection
with Y2O3:Yb/Er−CuxS with NIR irradiation, pure DOX, Y2O3:Yb/
Er−CuxS+DOX, and Y2O3:Yb/Er−CuxS+DOX with NIR irradiation.
The first group were made for blank control without any injected. The
injected material is 100 μL (1 mg/mL) every 2 days, and the pure
DOX were consistent with the materials. For the NIR irradiation, the
tumor site was irradiated with 980 nm laser for 10 min. The tumor
sizes and body weights were monitored every 2 days after treatment.
Histology Examination. Histology analysis was performed at the
14th day after treatment. The typical heart, liver, spleen, lung, and
kidney tissues of the mice in the control group and treatment group
In Vitro Viability of Y2O3:Yb/Er−CuxS. Five mg mL−1 MTT
solution was prepared by PBS. Two hundred microliters of material
per well plated L929 fibroblast cells with the number of 6000−7000
were put in a 96-well plate. For blank control, there were 8 wells left
with culture only, and then incubated 24 h in order to make cells
attach to wells with 5% CO2 at 37 °C. After that, they were diluted
into respective concentration of 15.625, 31.25, 62.5, 125, 250, and 500
μg mL−1, and then the solutions were added to the wells and incubated
for another 24 h. After that, 20 μL of the as-prepared MTT solution
was added to each well. The plate was subsequently incubated for
another 4 h at 37 °C. Meanwhile, viable cells make MTT reduce into
formazan which can be dissolved by DMSO. Finally, 150 μL of DMSO
was added to each well and placed on a shaking table for 5 min of 150
rpm in order to blend the formazan and solvent completely. The
absorbance was recorded using a microplate reader regulating to 490
nm. Optical density which received no drug was regarded as 100%
growth.
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Figure 1. SEM and TEM images with different magnification of Y(OH)CO3:Yb/Er@C with particle diameter of (A1−A4) 90 nm, (B1−B4) 150
nm, and (C1−C4) 200 nm.
were isolated. After that, the organs were dehydrated using buffered
formalin, ethanol with different concentrations, and xylene. They were
then embedded in liquid paraffin. The sliced organs and tumor tissues
(3−5 mm) were stained with hematoxylin and eosin (H&E) and
examined by a microscope.
(LD) module (K98D08M-30W, China) as the irradiation source and
detected by R955 (HAMAMATSU) from 400−800 nm. DOX
concentration was detected by UV-1601 UV−vis spectrophotometer.
Inductively coupled plasma (ICP) was carried out on a Thermo
Electron X Series II ICP apparatus.
Biodistribution and Circulation of Y2O3:Yb/Er−CuxS in Mice.
Balb/c mice (20−25 g) were injected intravenously with 100 μL of
Y2O3:Yb/Er−CuxS UCNCs (20 mg/mL). The mice (n = 3) were
euthanized at different time points (10 min, 1 h, 4 h, 12 h, 1 day, 3
days, 7 days, and 14 days). The blood, tumor, and major organs (heart,
liver, spleen, lung, and kidney) were collected and dissolved with 5 mL
of HNO3 and HCl (v/v = 1:3), and then heated at 70 °C for 5 min to
obtain clear solutions. After that, the solutions were centrifuged and
the supernatants were kept for further ICP-OES analysis. For the
blood circulation, more dense time points (10 and 30 min, 1, 2, and 4
h, etc.) were acquired.
Characterization. Powder X-ray diffraction (XRD) measurements
were performed on a Rigaku D/max TTR-III diffractometer at a
scanning rate of 15° per min in the 2θ range from 20 to 80°, with
graphite monochromatized Cu Kα radiation (λ = 0.15405 nm). Images
were obtained digitally on scanning electron microscope (SEM, JSM-
6480A), transmission electron microscopy (TEM, FEI Tecnai G2 S-
Twin) and high-resolution transmission electron microscopy
(HRTEM). Fourier Transform Infrared Spectroscopy (FT-IR) spectra
were measured on a PerkinElmer 580B IR spectrophotometer using
the KBr pellet technique. N2 adsorption/desorption isotherms were
obtained on a Micromeritics ASAP Tristar II 3020 apparatus. Pore size
distribution was calculated by the Barrete-Jonere-Halenda (BJH)
method. UC emission spectra were acquired using 980 nm laser diode
RESULTS AND DISCUSSION
■
Phase, Structure, Morphology and UC Luminescence.
Figure S1 in the Supporting Information shows the XRD
patterns of the Y(OH)CO3:Yb/Er precursor, Y(OH)CO3:Yb/
Er@C, Y2O3:Yb/Er hollow spheres, and the final Y2O3:Yb/Er−
CuxS hollow spheres. There are no obvious diffraction peaks in
the precursor and Y(OH)CO3:Yb/Er@C, which indicate both
samples are amorphous. After calcination, the diffraction peaks
of the product are in good agreement with the cubic-phase of
Y2O3 (JCPDS No. 43−1036). The sharp and strong peaks
indicate the high crystallinity. The cell lattice parameter is
calculated to a = 10.585 Å, matching well with cell volume of
cubic-phased Y2O3 (a = 10.604 Å). The slight contraction of
the unit cell is due to the smaller ionic radius of doped Yb3+ and
Er3+ ions substituting for larger Y3+ ions. When CuxS
nanoparticles were attached onto the surface of Y2O3:Yb/Er
hollow spheres, the XRD pattern of as-obtained Y2O3:Yb/Er−
CuxS has almost not changed peaks because of the small
amount of CuxS nanoparticles and high crystallinity of
Y2O3:Yb/Er.
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Figure 2. SEM images of Y(OH)CO3:Yb/Er@C nanospheres prepared with reaction time of (A) 1, (B) 2, and (C) 4 h; (D) corresponding TEM
images; and (E) illustrated formation scheme of Y(OH)CO3:Yb/Er@C spheres prepared with different reaction time.
Figure S2A−C in the Supporting Information displays the
TEM images of the Y(OH)CO3:Yb/Er precursor spheres with
different diameters. They were obtained from the coprecipita-
tion of urea and Ln(NO3)3 with different initial pH values of 1,
1.5, and 2, respectively. The typical TEM images present all the
three samples consist of high-dispersed nano/micro-spheres.
From Figure S2D in the Supporting Information, we can see
that the size distributions are narrow with the controlled
average sizes of 60, 120, and 180 nm, respectively. In fact,
varied particle sizes can be obtained by simply regulating the
initial pH values of the lanthanide nitrate solutions. It is
significant to control the sizes of the precursor because the sizes
play an important role in the final morphology and sizes, which
could generate different luminescent and biological properties
for further choice.
When the Y(OH)CO3:Yb/Er precursors reacted with
glucose under hydrothermal environment, hollow Y(OH)-
CO3:Yb/Er@C nano/micro-spheres with different sizes were
obtained. Figure 1A−C present SEM and TEM images with
different magnification of the Y(OH)CO3:Yb/Er@C hollow
spheres. It is clear that the product contains light cores and dark
shells, indicating the formation of hollow spheres. Typical SEM
and TEM images with low magnification show the three hollow
samples are uniform with mean size of 90, 150, and 200 nm,
corresponding to the sizes of 60, 120, and 180 nm of the
precursors. The increased sizes of about 20−30 nm are
consistent with the thickness of the dark shells. The results
reveal that the shell are not simply composited with C layer,
because the hollow spheres would be much larger if the effect of
glucose is only physical superposition. The sphere size increases
because the precursors adhered to the glucose shell have strong
attraction to the outer glucose. The reaction changes the
structure of the spheres but keeps the shape with gradually
increased sizes. After that, the solid spheres change into hollow
spheres.
To investigate the formation mechanism of the as-prepared
hollow spheres, we carried out step-dependent experiments.
SEM images (Figure 2A−C) show the products are all uniform
with gradually enhanced sizes with increased reaction time.
Figure 2D shows the TEM images with high magnification of
Y(OH)CO3:Yb/Er@C with reaction time of 1, 2, 3, and 4 h.
When the reaction time is 1 h, there is little difference between
the obtained product and the solid precursor. We believe that
the C shell may turn up on the surface of the solid precursor.
Core/shell yolk-like spheres are generated with the time
increased to 2 h, and the cores are shrunken gradually with
prolonged reaction time. When the reaction is increased to 4 h,
the whole hollow spheres with only dark shell form (without
survived cores). Figure S3 in the Supporting Information gives
the XRD patterns of the samples with reaction time of 1, 2, and
4 h, respectively. It can be seen that the wide diffraction peaks
of different samples move with enhanced reaction time. The
formation process is schematically illustrated in Figure 2E,
which is ascribed to the Kirkendall effect. There are two
diffusion groups: OH− and CO32− ions of the precursors in the
core and CH2−OH group of the glucose in the shell. The two
groups form the shell because of the coating attraction, and the
hollow spheres are produced because of the different diffusion
rate of the two groups.
After further calcination, Y2O3:Yb/Er hollow spheres with
different sizes were prepared. It is known that the metallic oxide
Y2O3:Yb/Er solution and the metallic sulfide (CuxS) are both
negative-charged.53 To combine the two kinds of particles
together, several preliminary modifications have to be made.
First, the amino group (−NH2) of APTS, which is positive-
charged, was grafted onto the surface of Y2O3:Yb/Er by
electrostatic adsorption. Meanwhile, folic acid was conjugated
on Y2O3:Yb/Er−NH2 through coupling agent of EDC and
NHS and a portion of amino groups. Most of the −NH2 groups
were used to adsorb negatively charged CuxS nanoparticls by
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Figure 3. (A−C) TEM images with different magnification, (D) HRTEM image of Y2O3:Yb/Er−CuxS, (d1−d3) the corresponding FFT images of
the three rectangle zones in panel D, and (E) the elemental mapping of Y2O3:Yb/Er−CuxS nanospheres. The particle size of Y2O3:Yb/Er−CuxS
hollow spheres is 90 nm.
electrostatic interaction, forming the Y2O3:Yb/Er−CuxS
composite. Figure S4 in the Supporting Information indicates
that Y2O3:Yb/Er−CuxS hollow spheres keep the similar size to
the corresponding Y(OH)CO3:Yb/Er@C, which is 90, 150,
and 200 nm, respectively. Notably, we can see the dispersibility
and homogeneity of the products are improved due to the
burning of the carbon layer during the annealing process.
TEM images with different magnifications of Y2O3:Yb/Er−
CuxS hollow spheres with an average size of 90 nm are
presented in Figure 3A−C. We can see the samples are
composed of hollow spheres with attached hexagonal nano-
particles. HRTEM image with three rectangle zones (d1, d2,
and d3) and the corresponding FFT images are shown in
Figure 3D and Figure S5 in the Supporting Information.
Among all of the lattice distances, 0.64 and 0.43 nm (Figure
3d1) are two typical distances of (111) and (022) planes of the
monoclinic Cu2S (JCPDS No. 33−0490). The interplanar
spacing of 0.43 and 0.31 nm (Figure 3d2) corresponds to (211)
and (222) planes of cubic Y2O3 (JCPDS No. 43−1036). In
Figure 3d3, the lattice distance of 0.82 nm is consistent with
(002) plane of hexagonal-phased CuS (JCPDS No. 06−0464).
These results indicate that the CuxS nanocrystals have been
successfully attached onto the surface of Y2O3:Yb/Er hollow
spheres. In the STEM element mapping of Y2O3:Yb/Er−CuxS
(Figure 2E), Y, Yb, O, and Cu element are detected and well-
distributed in the hollow spheres, further confirming the
formation of the composite of Y2O3:Yb/Er and CuxS.
FT-IR spectra of the Y(OH)CO3:Yb/Er precursor, Y(OH)-
CO3:Yb/Er@C, Y2O3:Yb/Er treated with APTS, and the final
Y2O3:Yb/Er−CuxS hollow nanospheres are presented in Figure
S6 in the Supporting Information. The two similar bands at
3402 and 1082 cm−1 in the spectra of the precursor and
Y(OH)CO3:Yb/Er@C are assigned to the −OH stretching and
bending vibrations of water and hydroxyl group.54 For
Y(OH)CO3:Yb/Er, the characteristic absorption bands of O−
2−
C−O (1525 and 1402 cm−1) and CO3 (843, 753, and 688
cm−1) indicate the existence of the Y(OH)CO3:Yb/Er
precursor.54 After the hydrothermal reaction with glucose,
besides the peaks of the precursor, three new peaks around
2980, 1650, and 1125 cm−1 are ascribed to respective −C−H,
−CHO, and −C−O,55 which should be induced by the
introduction of glucose. After calcination, the bands of −C−H,
−CHO, and −C−O disappear and new transmittance bands
occur. That is Y−O (565 cm−1), −CO (953, 1530 cm−1),
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Figure 4. (A) L929 fibroblast cell viability incubated with Y2O3:Yb/Er−CuxS with different concentrations for 24 h, (B) the hemolytic percentage of
Y2O3:Yb/Er−CuxS to human red blood cells, (C) DOX release efficiency of Y2O3:Yb/Er−CuxS+DOX at different pH values with and without NIR
irradiation, (D) different sizes, (E) the corresponding luminescence intensity of Y2O3:Yb/Er−CuxS as a function of the increase time (pH 4), (F) the
integrated intensity of the UC emission and the red/green ratio as a function of the release time. The particle sizes of all the Y2O3:Yb/Er−CuxS
samples are 90 nm except for panel D.
−CH2 (2881 cm−1), and −NH2 (1398 cm−1), indicating the
successful modification of −NH2 group.56 After being
conjugated with FA, CuxS, and PEG, the peak at 1695 cm−1
is attributed to the −CO amide stretching of the α-carboxyl
group from the FA molecules, and new peak at 3463 cm−1 is
assigned to −OH group because of the modification of PEG.
N2 adsorption/desorption isotherms of Y2O3:Yb/Er−CuxS
hollow spheres with different diameters are given inFigure S7A
in the Supporting Information. The three samples exhibit
typical IV-type isotherms with H1 hysteresis loops, which are
characteristic of typical mesoporous materials. The BET surface
areas of the three samples are calculated to be 36.0, 28.0, and
24.4 m2 g−1, respectively. As shown in the pore size distribution
(Figure S7B in the Supporting Information), the mesopore size
range is from 2 to 50 nm. The big pores are due to the particle
stacking, and the small pores (4.52, 4.14, and 2.26 nm) from
the BET results should be the actual pore distribution on the
particles.
Figure S7C in the Supporting Information shows UC
emission spectra of Y2O3:Yb/Er−CuxS hollow spheres with
different particle sizes. The three samples have same UC
conversion emission regions in 520−560 nm and 650−700 nm,
2
4
4
4
which correspond to H11/2/4S3/2 → I15/2 and F9/2 → I15/2
transitions of Er3+ ions.57−61 Meanwhile, the emission intensity
of the samples increases with the enhanced particle size, which
should be due to the decreased surface defect. The inset of
Figure S7B in the Supporting Information shows the UC
emission photograph of Y2O3:Yb/Er−CuxS with the particle
size of 90 nm, in which obvious red light can be observed.
Cell Viability, Drug Release, and MTT Cytotoxicity
Assay. For potential biological application, it is essential to
evaluate the biocompatibility of Y2O3:Yb/Er−CuxS UCNCs.
Standard MTT cell assay was carried on L929 cell lines to
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Figure 5. (A) Confocal laser scanning microscopy (CLSM) images of HeLa cancer cells incubated with Y2O3:Yb/Er−CuxS+DOX for 0.5, 3, and 6 h
at 37 °C; (B) CLSM images of HeLa cancer cells incubated with Y2O3:Yb/Er−CuxS+DOX hollow spheres with particle size of 90, 150, and 200 nm
for 1 h. Scale bars for all images are 50 μm.
detect the short-term viability. Figure 4A demonstrates the cell
viability with varying concentrations of the particles from
15.625 to 500 μg mL−1 incubated for 24 h. It is obvious that the
biocompatibility of the as-prepared sample in all dosages is up
to 101.6−127.0%, indicating the good biocompatibility in vitro.
To guarantee the potential intravenous administration in vivo,
it is essential to detect the biocompatibility with red blood cells.
Figure 4B presents the hemolytic results, and the red solutions
in the tubes (inset of Figure 4B) are attributed to the presence
of released hemoglobin. No visual red color occurred, which
indicates there is negligible hemolysis in the controlled tubes
with different solution added. The highest hemolytic efficiency
with different particle concentration from 15.62 to 1000 μg
mL−1 is 0.08%, indicating that the as-prepared Y2O3:Yb/Er−
CuxS is almost not hemolytic. In conclusion, the blood
compatibility of the as-synthesized material is excellent, and
Y2O3:Yb/Er−CuxS is almost nontoxic to live cells.
DOX was chosen as a typical anticancer drug to evaluate the
loading and release behaviors of the hollow nanospheres. After
being continuously stirred for 24 h, DOX has been almost
completely absorbed onto the surface of Y2O3:Yb/Er−CuxS
(90 nm) with a loading efficiency of 78.3% (1.96 mg). We have
also detected the loading efficiency and amount of the other
two samples with mean size of 150 and 200 nm, which is 1.89
mg and 1.74 mg, respectively. The slightly enhanced loading
amount should be due to the decreased BET surface area.
Usually, the pH value of normal cells in the human body is
nearly neutral, while the pH value of cancer cells is decreased
which is attributed to excess metabolic product by the fast
proliferation of cancer cells. Here, pH values of 4, 5.5, and 7.4
are selected to investigate the release efficiency of the DOX
loaded carriers. As shown in Figure 4C, only 12.2% of DOX is
released in the first 1 h, and 24.9% of DOX has been released
after 36 h when the pH value is 7.4. In contrast, for the release
process with the pH value of 4, the carrier shows a much faster
and higher drug-release rate and efficiency. During the
beginning 1 h, 45.9% of DOX is released, and 70.7% of DOX
has been released after 36 h. While the release efficiency is
42.4% after 36 h for pH value of 5.5. Because the enhanced
positive charge of the material under the low pH values
weakens the electrostatic interaction with positively charged
DOX, a much faster release rate is achieved in acidic
surrounding (pH 4). The result indicates the release efficiency
of Y2O3:Yb/Er−CuxS is pH-dependent. In particular, to
confirm whether the photothermal effect derived from attached
CuS nanoparticles can be used to promote DOX release from
the carrier, Y2O3:Yb/Er−CuxS+DOX hollow spheres in PBS
solutions at pH value of 4, 5.5, and 7.4 were irradiated by 980
nm NIR laser at density power of 0.6 W/cm2. It is found that
much faster DOX release has been achieved upon the NIR
irradiation under all pH conditions, indicating that the release
of DOX can be triggered by an external NIR laser due to the
thermal effect. Figure 4D shows the release efficiency of
Y2O3:Yb/Er−CuxS+DOX hollow spheres with different sizes.
The release efficiency after 36 h (pH 4) of the samples with size
of 90, 150, and 200 nm is 70.0, 72.6, and 79.9%, respectively.
This reveals the release efficiency is also size-dependent, which
provides a wide choice to deal with the different anticancer
therapy. We can see that all the release profiles have a similar
tendency: an initial fast release and a slower rest release. The
initially rapid release of anticancer drug is essential to the
treatment because it can inhibit the growth of cancer cells. After
that, the slow release of the rest of the drug molecules can be
continued for killing the survived cells from the initial stage.
The UC emission intensities of the composite as a function
of the release time are shown in Figure 4E. As known, DOX
can absorb green visible light at the wavelength of 400−550 nm
(Figure S8 in the Supporting Information), the UC emission
intensities of drug carriers in the green emission region will be
largely inhibited due to fluorescence resonance energy transfer
(FRET) effect. With the increased release efficiency, the FRET
effect is decreased because of the reduced amount of DOX.
Meanwhile, it is known that the FRET effect is significantly
affected by the distance. When the distance between DOX (the
acceptor) and drug carrier (the donor) is longer than 10 nm,
the FRET effect will disappear.62,63 Figure 4F depicts the
integrated emission intensities and green/red ratios versus the
release time, and we can see that the intensities almost have a
linear relationship within the initial 12 h. This further indicates
that red emission may not be enhanced greatly with increased
release efficiency compared with green regions. This property
can also be utilized to track the position of the carrier when
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used for targeted lesion therapy. Additionally, in Figure 4F, the
red/green ratio decreases from 15.8 to 2.9 with prolonged
release time due to the FRET effect. In conclusion, due to the
high drug loading amount, pH, size, and especially thermal-
dependent release, Y2O3:Yb/Er−CuxS hollow nanospheres with
UC emission can be used as potential candidates for targeted
therapy and imaging simultaneously.
Figure 5A shows the CLSM images of Hela cancer cells
incubated with Y2O3:Yb/Er−CuxS+DOX (90 nm) for 0.5, 3,
and 6 h in order to verify the cell uptake process. The blue
fluorescence is from the DAPI used to mark the nuclei, and the
red emission is from the loaded DOX, which is used to track
the carrier. The merge of the two channels is also given
correspondingly. In the first 0.5 h, there is little red emission,
which reveals only a few of Y2O3:Yb/Er−CuxS+DOX particles
has been taken up by HeLa cells. Stronger red fluorescence
emission of DOX is observed in both the cytoplasm and the cell
nucleus with the enhanced incubation time, suggesting that
more particles are localized in the cells. The results further
reveal that Y2O3:Yb/Er−CuxS+DOX carrier can be effectively
taken up by tumor cells.
It is significant to control the size of the precursor as the size
plays an important role in the final size of the product obtained
through the sacrifice route. Y2O3:Yb/Er−CuxS+DOX hollow
spheres with average size of 90, 150, and 200 nm should
present different cellular untaken properties. In Figure 5B, it is
obvious that the CLSM images of HeLa cells incubated with
Y2O3:Yb/Er−CuxS+DOX with 90 nm size show stronger red
emissions than those of samples with larger particle sizes. The
number of particles internalized by cells is different for each size
and exhibits an interesting size-dependent phenomenon.64 The
increased size of the material slows the cellular uptaken process
and thus weakens the red emission. The smaller-sized hollow
spheres of 90 nm are more easily internalized by cells compared
with lager spheres of 150 and 200 nm.
microscope equipped with a 980 nm NIR laser. It is found that
the UC luminescence of Y2O3:Yb/Er−CuxS hollow nano-
spheres increase with the enhanced time. Meanwhile, the
luminescence is evident in the intracellular region, whereas no
signal outside of the cells is found, which further proves that the
product has been internalized into the cells rather than merely
stained the membrane surface. The results also indicate that the
Y2O3:Yb/Er−CuxS hollow nanospheres are promising candi-
dates for high-contrast in vitro bioimaging with negligible
background. Furthermore, UC luminescence of DOX loaded
carrier is needed virtually for antitumor application. We have
taken the inverted florescence microscope images of HeLa cells
incubated with Y2O3:Yb/Er−CuxS+DOX for 1 h (Figure 6B).
It is obvious the red luminescence inside of the cells kept as or
even higher than that of Y2O3:Yb/Er−CuxS, revealing that the
product can act as proper candidate of drug carrier and
antitumor bioimaging probe.
The absorbance properties of CuxS aqueous solution with
concentration of 1000, 500, 250, 125, 62.5, and 31.25 μg mL−1
were examined by UV−vis spectroscopy, as shown in Figure S9
in the Supporting Information. There is a strong absorption in
the NIR region even under the lowest concentration. The
strong absorption in the NIR region of CuxS nanoparticles may
play a potential important role in photothermal cancer therapy
co-operated with Y2O3:Yb/Er UC phosphor under 980 nm
excitation at the plasmon band. The Y2O3:Yb/Er−CuxS
composite was synthesized based on this consideration. The
photograph of HeLa cells in the 96-well plate incubated with
Y2O3:Yb/Er−CuxS+DOX was taken and shown in Figure 7A,
and in vitro and in vivo NIR measurement are given in Figure
S10 in the Supporting Information. After irradiated by 980 nm
laser (0.6 W cm−2) for 5 min, the infrared thermal image
(Figure 7B and Figure S11 in the Supporting Information)
indicates the temperature of the cell supernatant increases to
42.3 °C. It is known that the tumor cells can be effectively killed
kept at temperature of 40−60 °C for a few minutes.65
Here, calcein AM (which could mark living cells with green
color) and PI (which could mark died cells with red color) were
used to dye the HeLa cells under different conditions detected
by CLSM to prove the killing effect of the irradiation. Figure
7C shows CLSM image of HeLa cancer cells in culture
irradiated with NIR laser, we can see that almost all of cells are
green, implying that the pure culture has no absorbance of NIR
irradiation. However, when HeLa cancer cells were incubated
with Y2O3:Yb/Er−CuxS+DOX and irradiated by NIR laser for
5 min (0.6 W cm−2), there are few green ones, indicating that
the tumor cells can be effectively killed by the chemo- and
photothermal synergistic effect (Figure 7D). Figure 7E shows
in vitro HeLa cells viabilities incubated for 24 h with free DOX,
Y2O3:Yb/Er−CuxS, Y2O3:Yb/Er−CuxS+DOX with different
concentrations with and without NIR irradiation. For HeLa
cells incubated with pure Y2O3:Yb/Er−CuxS, the viability of the
cells is 94.3−109.7%, indicating that the pure composite is
nontoxic. When there is NIR irradiation, the viability is reduced
to 42.5−72.9%, which depends on the particle concentration of
Y2O3:Yb/Er−CuxS. When the concentration is high as 500 μg
mL−1, the viability is 42.5%, while the viability is 72.9% for the
low concentration of 31.25 μg mL−1. This reveals that pure
NIR irradiation may have no damage to the pure cells and the
thermal effect is generated from Y2O3:Yb/Er−CuxS under NIR
laser. For HeLa cells incubated with free DOX and Y2O3:Yb/
Er−CuxS+DOX, there is obvious decreased viability due to the
chemotherapy of the antitumor DOX. Interestingly, a large
Figure 6A gives the respective inverted UC florescence
microscope images of HeLa cells incubated with Y2O3:Yb/Er−
CuxS for 0.5, 1, and 3 h at 37 °C, measured by a confocal
Figure 6. (A) Inverted fluorescence microscope images of HeLa cells
incubated with Y2O3:Yb/Er−CuxS for 0.5, 1, and 3 h; (B) inverted
fluorescence microscope images of HeLa cells incubated with
Y2O3:Yb/Er−CuxS+DOX for 1 h. Scale bars for all images are 50 μm.
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Figure 7. (A) Digital photograph of a 96-well HeLa cell-culture plate incubated with Y2O3:Yb/Er−CuxS without NIR irradiation. (B) Infrared
thermal photograph of 96-well HeLa cell-culture plate incubated with Y2O3:Yb/Er−CuxS after NIR irradiation for 5 min. (C) CLSM image of HeLa
cancer cells dyed with Calcein AM and PI without NIR irradiation. (D) CLSM image of HeLa cells incubated with Y2O3:Yb/Er−CuxS dyed with
Calcein AM and PI under NIR irradiation for 5 min (0.6 W/cm2). (E) In vitro HeLa cell viability of HeLa cells incubated for 24 h with pure
Y2O3:Yb/Er−CuxS, Y2O3:Yb/Er−CuxS+DOX, and pure DOX at varied concentration with and without NIR-laser irradiation. (F) In vivo infrared
thermal images of a tumor-bearing mouse after injection of Y2O3:Yb/Er−CuxS as a function of the irradiation time under 980 nm irradiation (0.6 W/
cm2). The UCL images of a tumor-bearing mouse after injection of Y2O3:Yb/Er−CuxS under 980 nm irradiation with pump power of 0.6 W/cm2:
(G) bright field, and (H) merged image. The particle sizes of all the used Y2O3:Yb/Er−CuxS samples are 90 nm.
difference of the viability between them is found, and the cells
viability of Y2O3:Yb/Er−CuxS+DOX (43.1%) is much higher
than pure DOX (29.4%) when the concentration is low (31.25
μg mL−1), which may be related with the different cell uptake
mechanism. When the concentration is low, the smaller DOX
molecules enter the cells by diffusion process with a rapid rate.
However, Y2O3:Yb/Er−CuxS+DOX enters the cells with a
much lower rate by endocytosis. When the concentration is
high, the two groups present unconspicuous difference, in both
of which the viabilities are low. It should be noted that the
viabilities with all different concentrations decrease to 12.9−
15.1% when irradiated by NIR laser for 5 min, indicating both
chemotherapy and PTT take effect in the incubation process.
After confirming the synergistic effect in vitro, we further
investigated the NIR light induced PTT effects and chemo-
effect in vivo. Before in vivo experiment, we have done
intravenous injection of Y2O3:Yb/Er−CuxS and subcutaneous
injection of Y2O3:Yb/Er−CuxS+DOX into five mice, respec-
tively. There is no abnormal reaction of mice with intravenous
injection, which further proves the good in vivo compatibility of
Y2O3:Yb/Er−CuxS.66−68 Under 980 nm laser irradiation with
the pump power of 0.6 W cm−2, the infrared thermal images of
a tumor-bearing mouse after injection of Y2O3:Yb/Er−CuxS
reveals that the composite shows an obvious thermal effect with
the increase of the irradiation time (Figure 7F). Furthermore,
for the mice with subcutaneous injection, the obvious red
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Figure 8. (A) Representative photographs of mice and liver tumor after various treatments: with nothing, Y2O3:Yb/Er−CuxS with NIR-laser
irradiation, pure DOX, Y2O3:Yb/Er−CuxS+DOX without and with NIR-laser irradiation, and photographs of tumor tissue were obtained after 14
days. (B) Body weight and (C) tumor size of H22 tumor in different groups after treatment.
Figure 9. H&E stained images of heart, liver, spleen, lung, and kidney collected from different treated groups.
emission can be detected in dark under 980 nm irradiation
(Figure 7G, H), suggesting the effective tissue penetration of
NIR laser and good UC luminescence of the as-synthesized
material.
As a proof of principle experiments, the H22 tumor-bearing
mice were intratumorally injected Y2O3:Yb/Er−CuxS nano-
spheres. The mice were investigated when the tumor size grew
to 6−8 mm in size. It is found that the results are much
different after different treatment. In Figure 8A-C, the tumor
size is very large without any injection. When treated with
Y2O3:Yb/Er−CuxS under NIR irradiation or with pure DOX,
the tumor is inhibited to some extent. When Y2O3:Yb/Er−
CuxS+DOX is injected, the tumor is markedly inhibited
compared with pure DOX because of the targeted effect of
FA. It is obvious that the tumor can markedly be inhibited after
injection with Y2O3:Yb/Er−CuxS+DOX under NIR irradiation.
All the results prove the Y2O3:Yb/Er−CuxS hollow nano-
spheres are proper candidate as drug carrier for dual-modal
chemo- and PTT carriers. Histological analysis of the heart,
lung, kidney, liver, and spleen are given in Figure 9.
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Hepatocytes in the liver samples are found normal. No
pulmonary fibrosis is detected in all the lung samples. The
glomerulus structure in the kidney section is clearly observed.
Necrosis cannot be found in any of the histological samples
analyzed. The glomerulus structure in the kidney section is
clearly observed in the best treated group.
In addition, for the potential clinical application, the
biodistribution and blood circulation of the particles with
different intravenous injection times are detected. As shown in
Figure 10A, in the early stages (10 min and 1, 4, and 12 h after
area, the UC emission intensities, drug loading amount and
release efficiency, and cellular up-taken process are dependent
on the particle sizes of hollow spheres. The as-synthesized
composite has good biocompatibility and exhibits high ablation
efficiency to tumor cells because of the synergistic therapeutic
effect between chemotherapy and PTT, which arises from the
loaded DOX and attached CuxS nanoparticles. The UCL
property as an imaging nanoprobe endows the composite with
in vivo imaging ability. The results suggest this kind of
Y2O3:Yb/Er−CuxS hollow spheres can be used as potential
candidate to bioimaging, chemotherapy, and PTT carriers
simultaneously.
ASSOCIATED CONTENT
■
S
* Supporting Information
XRD patterns of the samples obtained in different steps; TEM
images of the precursor with different particle diameter; XRD
patterns of Y(OH)CO3:Yb/Er@C obtained for different
reaction time; TEM images of Y2O3:Yb/Er−CuxS with different
particle size; magnified TEM image of Y2O3:Yb/Er−CuxS; FT-
IR spectra of the samples obtained at different steps; Nitrogen
adsorption/desorption isotherms and UC emission spectra of
Y2O3:Yb/Er−CuxS with different particle size; Absorbance of
DOX solution; Absorbance of CuxS solution with different
particle concentration; the measurement of the NIR irradiation
to MTT assay of HeLa cells in vitro and the mouse in vivo;
Infrared thermal photographs of a 24-well HeLa cells-culture
plate after laser irradiation with pump power of 0.6 W/cm2 with
different irradiation time; TEM image and N2 adsorption/
desorption isotherm of solid Y2O3:Ln. DOX release profiles of
the solid and hollow Y2O3:Ln-DOX. The viability of HeLa cells
incubated with pure DOX, the solid and hollow Y2O3:Ln-DOX.
CLSM images of the HeLa cells dyed by trypan blue after
incubated with the solid and hollow Y2O3:Ln-DOX for 1 h. The
digital photographs of H22 tumor-bearing Balb/c mice
intravenously injected with the hollow and solid Y2O3:Ln-
DOX and the corresponding tumor sizes. The normalized
tumor sizes of the tumor-bearing mice after intravenously
injection of the solid and hollow Y2O3:Ln-DOX; Confocal laser
scanning microscopy (CLSM) images of HeLa cells incubated
with DOX-Y2O3:Yb/Er−NH2−FA and DOX-Y2O3:Yb/Er−
NH2 for different times of 1 and 3 h. All the scale bars are
50 μm. The ICP-OES result of the Y3+ concentration in HeLa
cells after incubated with the two samples. The cell viability of
HeLa cells incubated with the two samples. The digital
photographs of the H22 tumor-bearing Balb/c mice injected
intravenously with DOX-Y2O3:Yb/Er− NH2−FA and DOX-
Y2O3:Yb/Er−NH2 and the corresponding tumor sizes. This
material is available free of charge via the Internet at http://
pubs.acs.org.
Figure 10. (A) Biodistribution and (B) blood circulation of Y2O3:Yb/
Er−CuxS UCNCs in H22 tumor-bearing mice.
the injection), the nanoparticles accumulated in the livers,
spleens and targeted tumors. In the whole detection times, Y
concentrations keep low in lung, heart, and kidney. After 24 h
of postinjection, the Y concentrations reduce in liver, spleen,
and lung. In the seventh and fourteenth day after injection, the
nanoparticles in the liver, spleen, and lung are much lower than
the first 1 day. The results reveal that the injected UCNCs can
be excreted from the mice with prolonging times. Figure 10B
shows the pharmacokinetics of blood circulation curve. The
fitted curve presents a two-compartment model with the first
and second phase blood circulation half-lives at 0.032 0.007
day (0.77 0.17 h) and 0.958 0.212 day (23.0 5.1 h),
respectively. The longer circulation of UCNCs is also beneficial
to biomedical diagnosis and therapy, such as hepatic metastases
and angiography. These results clearly demonstrate the
potential clinical applicability of Y2O3:Yb/Er−CuxS as anti-
tumor carriers.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: yangpiaoping@hrbeu.edu.cn.
*E-mail: jlin@ciac.ac.cn.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
CONCLUSIONS
■
In summary, we successfully synthesized uniform Y2O3:Yb/Er−
CuxS hollow nanospheres with controllable size by sacrifice
route, followed by attaching CuxS nanoparticles. The surface
Notes
The authors declare no competing financial interest.
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(33) Zhang, F.; Shi, Y.; Sun, X.; Zhao, D.; Stucky, G. D. Chem. Mater.
2009, 21, 5237−5243.
ACKNOWLEDGMENTS
■
Financial supports from the National Natural Science
Foundation of China (NSFC 21271053, 21401032,
51472058, 51332008), Research Fund for the Doctoral
Program of Higher Education of China (20112304110021),
Natural Science Foundation of Heilongjiang Province
(B201403), and Harbin Sci.-Tech. Innovation Foundation
(2014RFQXJ019) are greatly acknowledged.
(34) Li, W.-P.; Liao, P.-Y.; Su, C.-H.; Yeh, C.-S. J. Am. Chem. Soc.
2014, 136, 10062−75.
(35) Tao, Y.; Ju, E.; Liu, Z.; Dong, K.; Ren, J.; Qu, X. Biomaterials
2014, 35, 6646−56.
(36) Huang, X. H.; El-Sayed, I. H.; Qian, W.; El-Sayed, M. A. J. Am.
Chem. Soc. 2006, 128, 2115−2120.
(37) Jain, P. K.; Huang, X.; El-Sayed, I. H.; El-Sayed, M. A. Acc.
Chem. Res. 2008, 41, 1578−1586.
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