PartPartSystCharact-2017-Li-CoreShellBi2Se3mSiO2PEGasaMultifunctionalDrugDeliveryNanoplatformfor-merged.pdf

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1700337 (1 of 12) © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Core–Shell Bi2Se3@mSiO2-PEG as a Multifunctional
Drug-Delivery Nanoplatform for Synergistic Thermo-
Chemotherapy with Infrared Thermal Imaging
of Cancer Cells
Zhuo Li, Zhenglin Li, Lei Sun, Baosheng Du, Yuanlin Wang, Gongyuan Zhao,
Dengfeng Yu, Sisi Yang, Ye Sun,* and Miao Yu*
Z. Li, Z. L. Li, L. Sun, Y. Wang, G. Zhao, S. Yang, Prof. M. Yu
State Key Laboratory of Urban Water Resource and Environment
School of Chemical Engineering and Technology
Harbin Institute of Technology
Harbin 150000, P. R. China
E-mail: miaoyu_che@hit.edu.cn
B. Du, D. Yu, Prof. Y. Sun
Condensed Matter Science and Technology Institute
Harbin Institute of Technology
Harbin 150000, P. R. China
E-mail: sunye@hit.edu.cn
DOI: 10.1002/ppsc.201700337
bioavailability after high-dose systemic
administration, traditional chemotherapy
can inevitably lead to unsatisfied outcomes
with serious side effects.[3,4] Recently,
numerous efforts have been devoted to
the development of various nanocarriers,
such as liposomes,[5,6] dendrimers,[7] and
silica nanostructures,[8,9] for targeted drug
delivery into tumors. Employing “smart”
nanoparticles as drug delivery systems in
response to stimuli, e.g., the acidic pH,
temperature or the light stimulation, has
become a promising way to improve the
efficacy of chemotherapy, thanking the
on-demand drug release with spatial and
real-time control.[10] Although significantly
improved biodistribution and bioavail-
ability have been realized, the developed
carriers are to a large extent confined by
low drug loading and/or the fact that car-
riers themselves are not therapeutically
active which may cause undesired side
effects.[11]
In recent years, combining chemo-
therapy with other therapies, especially
photothermal therapy,[12,13] has become
a thriving direction to remedy the inef-
ficiency of single therapy. Photothermal therapy (PTT), which
converts near-infrared (NIR) optical energy into thermal energy
aiming at ablation of tumor cells,[14] is an emerging photo-
therapy for cancer treatments with many superiorities such as
simplicity, noninvasiveness, remote control, and rapid thera-
peutic effect with low side effects.[15–21] Unfortunately, suffering
from the limited light penetrability as well as the inevitable light
scattering in biological tissues, single PTT normally cannot
eliminate tumors completely,[22] thus easily leading to tumor
recurrence. Due to the fact that hyperthermia can increase cel-
lular metabolism and membrane permeability for enhanced
drug uptake, the combination of chemotherapy and PTT (i.e.,
thermo-chemotherapy) has been demonstrated to be effective
in optimizing the efficacy of cancer treatments.[23]
In addi-
tion, the photothermal effect can be also employed to enable
NIR-responsive on-demand release or improve drug delivery
into tumors, leading to a synergistically enhanced therapeutic
Thermo-chemotherapy combining photothermal therapy (PTT) with chemo-
therapy has become a potent approach for antitumor treatment. In this study, a
multifunctional drug-delivery nanoplatform based on polyethylene glycol (PEG)-
modified mesoporous silica-coated bismuth selenide nanoparticles (referred
to as Bi2Se3@mSiO2-PEG NPs) is developed for synergistic PTT and chemo-
therapy with infrared thermal (IRT) imaging of cancer cells. The product shows
no/low cytotoxicity, strong near-infrared (NIR) optical absorption, high photo-
thermal conversion capacity, and stability. Utilizing the prominent photothermal
effect, high-contrast IRT imaging and efficient photothermal killing effect on
cancer cells are achieved upon NIR laser irradiation. Moreover, the successful
mesoporous silica coating of the Bi2Se3@mSiO2-PEG NPs cannot only largely
improve the stability but also endow the NPs high drug loading capacity. As a
proof-of-concept model, doxorubicin (DOX) is successfully loaded into the NPs
with rather high loading capacity (≈50.0%) via the nanoprecipitation method. It
is found that the DOX-loaded NPs exhibit a bimodal on-demand pH- and NIR-
responsive drug release property, and can realize effective intracellular drug
delivery for chemotherapy. The synergistic thermo-chemotherapy results in a
significantly higher antitumor efficacy than either PTT or chemotherapy alone.
The work reveals the great potential of such core–shell NPs as a multifunc-
tional drug-delivery nanosystem for thermo-chemotherapy.
Theranostics
1. Introduction
Chemotherapy has been clinically accepted as one of the most
commonly used and effective methods for antitumor treat-
ments.[1,2] However, due to the lack of targeting and the poor
Part. Part. Syst. Charact. 2018, 35, 1700337

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efficacy compared with monotherapy.[24] Furthermore, thermo-
chemotherapy can significantly decrease systemic toxicity of
chemotherapeutic agents to normal tissues and potentially
avoid under- or overdosing.[25] Up to now, various nanomate-
rials have been developed for thermo-chemotherapy, such as
Au nanocage,[26] CuS hollow structure,[27] MoS2 nanosheets,[28]
Prussian blue (PB),[29] and so on. Unfortunately, certain con-
cerns of the existing chemo-photothermal agents have been
aware,[30] including (1) the deficient photothermal capability
induced by poor NIR absorbance, low photothermal conversion
efficiency and low photothermal stability;[31] (2) the limited drug
loading which causes the indispensable demand for large doses
hence increased potential toxicity;[32] (3) the complex and high-
cost synthesis as well as difficult functional modifications;[33,34]
(4) the questionable safety and biocompatibility.[35]
Very recently, bismuth-containing nanoagents,[30,36–42] espe-
cially bismuth chalcogenides (e.g., Bi2S3, Bi2Se3)[30,36,37] have
stimulated considerable interest as promising non-noble
metal-based nanoagents for biomedical applications, arising
from their remarkably high X-ray attenuation coefficient, cost
effectiveness, well-known biological tolerance, and long-cir-
culating half-lives.[37,39] In particular, Bi2Se3, one of the most
typical topological insulators with a relatively large bulk gap
of ≈0.3 eV, is superior for biological applications due to the
straightforward synthetic route without hydrophilic modifica-
tion process, the extra important biological roles and function
of Se in reducing cancer incidence or mortality, together with
the well-demonstrated biocompatibility, metabolizability, and
very low toxicity of Bi2Se3 in vivo.[43,44] In the recent work of
our group, we have fabricated and investigated two distinct
Bi2Se3-based nanocomposites in vitro/vivo, including poly
dopamine/human serum albumin coated Bi2Se3 nanoplates[30]
and highly porous Bi2Se3 spherical sponge,[36] demonstrating
that the nanostructured Bi2Se3 can act as excellent PTT agents
integrating the high-performance photothermal properties
with X-ray computed tomography, photoacoustic, and infrared
thermal (IRT) imaging. In particular, as new drug delivery plat-
forms, these agents showed high efficacy of tumor ablation
by the synergistic thermo-chemotherapy. Although the Bi2Se3
nanoplate has been reported as a powerful photothermal agent,
two inherent drawbacks have been revealed, i.e., (1) the Bi2Se3
agent is unstable and can be easily oxidized and degraded to
be less or even totally incapable on photothermal conversion
without proper coating; (2) the loading capability of the Bi2Se3
nanoplate is limited. Obviously, improving the stability mean-
while empowering a high loading capability by a single facile
step will be highly preferred for practical applications.
Herein, we report the synthesis and biomedical applica-
tion of a multifunctional drug-delivery nanoplatform for syn-
ergistic PTT and chemotherapy with IRT imaging based on
mesoporous silica-coated Bi2Se3 nanoparticles (Bi2Se3@ mSiO2
NPs). The nanocomposites adopt a typical core–shell structure
with the Bi2Se3 nanoplates as the core and mesoporous silica
as the shell, and then modified with polyethylene glycol (PEG)
(Scheme 1). Originated from the core, the resultant Bi2Se3@
mSiO2-PEG NPs show strong NIR absorption, high photo-
thermal conversion capacity, and stability. Upon NIR laser irra-
diation, the Bi2Se3@mSiO2-PEG NPs can provide high-contrast
IRT imaging and efficient photothermal killing effect on cancer
cells, as confirmed by both the live-dead cell staining and CCK-8
assay. On the other hand, compared with the bare Bi2Se3 nan-
oplates, the successful surface coating of mSiO2 can not only
largely improve their storage stability but also endow the NPs
high drug loading capacity. By loading doxorubicin (DOX, a
clinical-used chemotherapeutic drug) into the Bi2Se3@mSiO2-
PEG NPs via the nanoprecipitation method, the high loading
capability of the cargo is demonstrated. The obtained Bi2Se3@
mSiO2-PEG/DOX NPs exhibit a bimodal on-demand pH- and
NIR-responsive drug release manner, and can realize efficient
intracellular drug delivery for chemotherapy. Most importantly,
Scheme 1. Illustration of the synthesis and multifunctions of the Bi2Se3@mSiO2-PEG NPs.

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a prominent synergistic therapeutic effect on cancer cells is
achieved through the combination of PTT and chemotherapy,
showing a significantly improved efficacy than either mono-
therapy alone. Therefore, this multifunctional and high-load
Bi2Se3@mSiO2-PEG NPs fabricated by the facile method could
act as a “smart” drug-delivery nanosystem for multiple thera-
peutic treatments and diagnose on anticancer treatments.
2. Results and Discussion
2.1. Synthesis and Characterization
The fabrication of the Bi2Se3@mSiO2-PEG NPs is illustrated
in Scheme 1. In brief, the Bi2Se3 nanoplates were synthesized
via a simple solvothermal reduction route, followed by surface
coating of mesoporous SiO2 and PEG modification. After the
removal of the cetyltrimethylammonium bromide (CTAB), DOX
was loaded into the mesopores of the NPs via the nanoprecipi-
tation method (refer the details in the Experimental Section).
The morphology of the as-prepared Bi2Se3 core was character-
ized by transmission electron microscopy (TEM). As shown in
Figure 1A,B, the core adopts a near circular morphology with an
average lateral diameter of ≈43.5 nm. Consistent with the pre-
vious results,[30] the distinct half transparency is characteristic
of the Bi2Se3 nanoplate due to the small thickness. The crys-
tallization nature of the nanoplates was examined by powder
X-ray diffraction (XRD) (Figure 1C), showing that all the peaks
in the XRD pattern can be assigned to the Bi2Se3 rhombohe-
dral phase (JCPDS Card No. 33-0214). In addition, except the
signal of C and O primarily from the sample substrate for
the measurement, the energy dispersive spectroscopy (EDS)
analysis (Figure 1D) confirms the presence of Bi and Se with
the Bi:Se ratio of ≈2:3, in absence of impurities.
After the mesoporous silica coating and PEG modification,
the resultant Bi2Se3@mSiO2-PEG NPs showed an obvious
core–shell structured morphology (Figure 2A,C). The average
size of the NPs was increased to ≈60.5 nm (Figure S1, Sup-
porting Information). Dynamic light scattering measurements
showed that the size of the Bi2Se3@mSiO2-PEG NPs was nearly
identical in various physiological environments, including
water, phosphate buffer saline (PBS) as well as serum, and the
size was barely changed after one-week storage (Figure S2, Sup-
porting Information), revealing its good biological application
prospect. The surface coating was further explored by using the
high-angle annular detector dark-field scanning transmission
electron microscopy. As shown in Figure 2D–H, the line-scan
element spectra of the Bi2Se3@mSiO2-PEG NP (indicated in
the inset of panel (D)) revealed the elemental distribution of Se,
Bi, Si, and O. The signal of both Se and Bi was significantly
reduced compared with that of Si and O at the position of
14 nm, further confirming the successful coating of silica shell.
The surface area and total pore volume of Bi2Se3@mSiO2-PEG
NPs via Brunauer–Emmett–Teller analysis of nitrogen adsorp-
tion–desorption isotherms (Figure S3, Supporting Information)
were ≈451.29 m g−1 and ≈1.114 cm3 g−1, respectively. Clearly,
the pore size distribution reveals dominant mesopores, ranging
of 2.0–6.0 nm, consistent with the literature.[43] Such highly
porous nanostructure of the Bi2Se3@mSiO2-PEG NPs may
have promising potentials for high loading of drugs and small
biomolecules to coordinate with chemotherapy and other addi-
tional functions.
2.2. Optical Absorption and Photothermal
Properties
Strong optical absorbance in the NIR range is
prerequisite for the photothermal agents. To
evaluate the potential of the Bi2Se3@mSiO2-
PEG NPs for PTT application, ultraviolet–vis-
ible–near−infrared (UV–vis–NIR) absorption
spectrum was measured (Figure 3A). It is
revealed that the NPs exhibited broad absorp-
tion from 400 to 900 nm covering the NIR
region (from 700 to 900 nm), showing great
promise for NIR laser-driven PTT. Moreover,
the absorption spectrum of the Bi2Se3@
mSiO2-PEG NPs was barely changed when
exposed to air at room temperature for one
week, suggesting the excellent stability and
antioxidative property of the NPs.
In sharp contrast, the bare Bi2Se3 nano-
plates without coating would be easily oxi-
dized under the same conditions, as evi-
denced by the dramatically decreased optical
absorption and color change of the NPs
dispersion from black to yellowish-brown
in Figure S2 in the Supporting Informa-
tion. Besides, the Bi2Se3@mSiO2-PEG NPs
Figure 1. A) Typical TEM image, B) size distribution histogram, C) XRD, and D) EDS analysis
of the as-synthesized Bi2Se3 nanoplates.

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showed excellent dispersion stability without any macroscopic
aggregation, which was confirmed by the gradually increased
optical absorbance and the perfect linear dependence of the
808 nm absorbance on the NPs concentrations (Figure S3, Sup-
porting Information).
We then investigated the photothermal conversion capability
of the Bi2Se3@mSiO2-PEG NPs by monitoring the temperature
rise of the NPs dispersions at gradient concentrations (from
0–400 µg mL−1) upon the 808 nm laser irradiation (2.0 W cm−2)
via a thermocouple probe. The 808 nm laser is the most com-
monly used NIR-light source for PTT, mainly due to its large
absorption coefficient and deep tissue penetration.[45] As shown
in Figure 3B, significant irradiation time- and concentration-
dependent photothermal effect of the Bi2Se3@mSiO2-PEG
NPs was observed. When the NPs concentration was gradually
increased from 50 to 400 µg mL−1, the corresponding tempera-
ture elevation (ΔT) after 10 min irradiation was 12.2, 19.9, 29.3,
37.9, and 44.9 °C, respectively. In particular, at 400 µg mL−1,
the system temperature can increase to as high as ≈71.9 °C
after 10 min irradiation; and at 200 µg mL−1, even a short irra-
diation for less than 3 min can increase the temperature to the
critical temperature (≈43 °C), which is known for inducing the
apoptosis of cancer cells.[37] In marked contrast, for pure deion-
ized (DI) water, the temperature showed negligible increase
Figure 2. A,B) Typical TEM images and C,D) high-resolution TEM images of the Bi2Se3@mSiO2-PEG NPs. E,H) Element line-scan analysis of the line
indicated in the inset of panel (D), showing the distribution of Se, Bi, Si, and O.
Figure 3. A) UV–vis–NIR absorption spectra of the fresh Bi2Se3@mSiO2-PEG NPs dispersion and the dispersion exposed to air at room temperature
for one week. B) Temperature elevation of the Bi2Se3@mSiO2-PEG NPs dispersions at various concentrations upon irradiation for 10 min. C) Infrared
thermal images of the Bi2Se3@mSiO2-PEG NPs dispersions during irradiation. D) Temperature elevation of Bi2Se3@mSiO2-PEG (200 µg mL−1
) upon
five repeated cycles of NIR laser irradiation. E) UV–vis–NIR absorption spectra of the Bi2Se3@mSiO2-PEG (200 µg mL−1
) before and after the irradiation
cycles. F) Heating and cooling curves of the Bi2Se3@mSiO2-PEG NPs dispersed in water (200 µg mL−1
) and pure DI water. G) Plot of cooling time as
the function of negative natural logarithm of the temperature driving force, where the time constant of the heat transfer is measured as τS = 438.1 s.

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(≈3.2 °C) upon identical irradiation. In addition, based on the
strong photothermal effect of the Bi2Se3@mSiO2-PEG NPs,
high-contrast IRT imaging was also realized with the imaging
intensity dependent on the NPs concentration and irradiation
duration (Figure 3C), which is capable to provide real-time
monitoring on the PTT process.[30] Moreover, to assess their
photothermal conversion stability, repeated irradiation cycles
by switching on/off the laser were conducted on the Bi2Se3@
mSiO2-PEG NPs dispersion. For each cycle, the dispersion was
irradiated for 3 min and then cooled naturally for 3 min with
the laser off (Figure 3D,E). The results indicate that both the
temperature elevation ability and the absorption spectrum of
the Bi2Se3@mSiO2-PEG NPs were negligibly changed upon
multiple irradiation cycles, suggesting their excellent photo-
thermal stability.
Next, we measured the photothermal conversion efficiency
(η) of the Bi2Se3@mSiO2-PEG NPs (Figure 3F,G). The NPs
aqueous dispersion (200 µg mL−1) was irradiated by the 808
nm laser until the system reached the maximum temperature,
followed by natural cooling to room temperature. The system
temperature was recorded every 20 s. The NPs dispersion
attained a maximum temperature elevation ΔTNPs of ≈33.4 °C,
which was much higher than that of DI water (ΔTwater ≈4.2 °C).
According to the calculation method reported in the previous
literature,[46] the photothermal conversion efficiency (η) of
the Bi2Se3@mSiO2-PEG NPs was determined to be ≈30.5%.
Overall, the high photothermal conversion efficiency, high
photo
thermal stability together with the high performance in
IRT imaging makes the Bi2Se3@mSiO2-PEG NP a promising
PTT nanoagent.
2.3. Cytotoxicity of the Bi2Se3@mSiO2-PEG NPs
Prior to studying the PTT effect of the Bi2Se3@mSiO2-PEG
NPs on cancer cells, we first evaluated the cytotoxicity of the
NPs, as good biocompatibility of the nanoagent is paramount
for biomedical applications. Human umbilical vein endothelial
cells (HUVEC), 4T1 and Hela cells were employed the normal
and cancer cell model, respectively. HUVEC, 4T1 and Hela cells
were incubated with the Bi2Se3@mSiO2-PEG NPs dispersions
at various concentrations for 24 and 48 h, and the cell viability
was measured by the Cell Counting Kit-8 (CCK-8) assay. As
shown in Figure 4, the cell viability of all cells remained over
95% after treated by the NPs dispersions at all tested concen-
trations (10, 20, 50, 100, 200, 300, 400, 600, and 800 µg mL−1),
suggesting rather low cytotoxicity of the NPs.[47,48]
2.4. IRT Imaging and PTT Effect on Cancer Cells
To realize real-time monitoring of thermal dynamics during
PTT process, we investigated the IRT imaging performance
of the Bi2Se3@mSiO2-PEG NPs on 4T1 cells. As indicated in
Figure 5A–D, 4T1 cells in the wells no. 1, 2, 5, 6 were incubated
with Bi2Se3@mSiO2-PEG NPs, while the cells in wells no. 3
and 4 only contained pure Dulbecco’s modified Eagle’s medium
(DMEM) culture medium. Selective laser irradiation (marked
with the white circle) was applied on the wells no. 3, 4, 5, and
6. As anticipated, all the wells exhibited the homogeneous blue
color related to the initial room temperature before irradiation.
However, during the irradiation, intensive IRT imaging con-
trast increased with extended irradiation was observed only in
the wells no. 5 and 6 in presence of the Bi2Se3@mSiO2-PEG
NPs, revealing that the combination of the NPs and irradiation
could lead to significant temperature elevation.
To quantitatively evaluate the PTT efficacy of the Bi2Se3@
mSiO2-PEG NPs, we then performed the CCK-8 assay to com-
paratively measure the cell viability of 4T1 cells after treated
with the NPs at different concentrations with/without laser
irradiation. As shown in Figure 5E, the cell viability of 4T1 cells
dramatically decreased after treated with the NPs plus laser irra-
diation, and such photothermal killing effect followed a promi-
nent irradiation time- and concentration-dependent manner.
For instance, upon 5 min irradiation, the cell viability was
32.7% for 50 µg mL−1 and 9.5% for 100 µg mL−1. At 50 µg mL−1,
the cell viability upon 0, 5, and 10 min was 98.6%, 32.7%, and
18.9%, respectively. In sharp contrast, the cell viability of 4T1
cells was barely affected by either the NPs or irradiation treat-
ment alone.
To further intuitively demonstrate the photothermal killing
effect on cancer cells, 4T1 cells were first incubated with the
Bi2Se3@mSiO2-PEG NPs suspensions (200 µg mL−1, 1.0 mL
per well) and then exposed to the 808 nm laser (2.0 W cm−2)
for 0, 3, and 10 min. Fluorescence staining by calcein acetoxy-
methyl ester (Calcein AM) and propidium iodide (PI) was per-
formed to visualize the live cells (vivid green) and dead cells
(red). As shown in Figure 6, 4T1 cells after treated with either
Figure 4. Cell viability of HUVEC, 4T1, and Hela cells after incubation with the Bi2Se3@mSiO2-PEG NPs dispersions at various concentrations for A)
24 and B) 48 h.

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the NIR laser or the Bi2Se3@mSiO2-PEG NPs alone showed
vivid green without red fluorescence signal, indicating the
unaffected cell viability upon these treatments. Remarkably,
combining the Bi2Se3@mSiO2-PEG NPs and laser irradiation
caused remarkable cell death. Under 3 min irradiation, 4T1
cells in the irradiation spot (marked by the white circle) were
accurately killed. When the irradiation time was extended to
10 min, the cells of the whole well were completely extermi-
nated due to heat transfer. All these results clearly indicate the
potent PTT efficacy of the Bi2Se3@mSiO2-PEG NPs on ablation
of cancer cells.
2.5. Drug Loading and Release
The surface coating of mesoporous silica not only increased
the stability of the Bi2Se3 core but also endowed the Bi2Se3@
mSiO2-PEG NPs great potential as a drug delivery carrier due to
the well-addressed loading capacity of mSiO2. As a conceptual
validation experiment, we chose DOX, a clinical-used chemo-
therapeutic drug, as a model drug to be loaded into the NPs.
The Bi2Se3@mSiO2-PEG NPs dispersion was simply mixed
with DOX solution overnight, followed by repeated washing to
remove the unbound DOX. Similar to the previously reported
cases,[29,30,36,37] DOX can be transformed from hydrophilia to
hydrophobicity upon loading and attached by the strong inter-
action between the NH2 group of DOX and with the cargo.
After DOX loading, the color of the resultant Bi2Se3@mSiO2-
PEG/DOX NPs dispersion was changed from pure black to
purple-black. The loading of DOX was confirmed by the Fou-
rier transform-infrared (FT-IR) spectra. As shown in Figure 7A,
compared with the Bi2Se3@mSiO2-PEG NPs, the spectrum
of the DOX-loaded NPs showed characteristic DOX vibration
peaks at ≈1731 cm−1 attributed to carbonyl, ≈1573 cm−1 attri
buted to the CC stretching vibration in aromatic ring, and
≈1108 cm−1 attributed to the CO stretching vibration.[49,50]
In addition, the successful DOX loading into the NPs was fur-
ther supported by the photoluminescence spectra (Figure 7B)
The characteristic emission of DOX was observed from the
Bi2Se3@mSiO2-PEG/DOX NPs, while the Bi2Se3@mSiO2-
PEG showed no fluorescence. We measured the loading con-
tent of DOX in the Bi2Se3@mSiO2-PEG/DOX NPs using the
corresponding fluorescence standard calibration curve, and the
DOX loading capacity of the NPs was calculated to be as high
as ≈50.0%. The variation of loading capacity with the increased
feeding DOX during the synthesis was examined. As shown in
Figure S4 in the Supporting Information, the loading capacity
reached nearly maximum at an applied DOX:Bi2Se3@mSiO2-
PEG weight ratio of 1.25, and was barely increased with a larger
amount of feeding DOX. In favor of low-cost and environment-
friendly synthesis, we chose the ratio of 1.25 in the following
experiments. These results indicate that the Bi2Se3@mSiO2-
PEG NPs have great potential as a drug delivery nanoagent.
The drug release from the Bi2Se3@mSiO2-PEG/DOX was
studied in PBS solutions at different pH, including pH = 7.4, 5.2,
and 4.8, to simulate the normal physiological environment (neu-
tral) and the tumor microenvironment (acidic), respectively. As
shown in Figure 7C,D, the DOX release was strongly depended
on the pH value and the releasing time. The drug release at
Figure 5. A) Digital photo and B–D) infrared thermal images of a 96-well
cell-culture plate containing 4T1 cells and the Bi2Se3@mSiO2-PEG (wells
no. 1, 2, 5, and 6) or 4T1 cells only (wells no. 3 and 4) upon irradiation for
0, 5, and 10 min, respectively, where the irradiated region is marked by the
circle. E) Cell viability of 4T1 cells after treated by the Bi2Se3@mSiO2-PEG
NPs dispersions at various concentrations for different duration.
Figure 6. Photothermal destruction of 4T1 cells treated with A) laser
only, B) the Bi2Se3@mSiO2-PEG NPs only, and C,D) the combination of
the Bi2Se3@mSiO2-PEG NPs and NIR laser irradiation for 3 and 10 min,
respectively. Scale bar is 1000 µm.

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pH = 4.8 and 5.2 were significantly higher than that at pH = 7.4.
While barely increased after 4 h at pH = 7.4, the released DOX
was increased gradually with time during the whole studied
duration at pH = 5.2 and 4.8. At 7 h, the total DOX release was
of ≈58.0% and ≈65.6% at pH = 5.2 and pH = 4.8, respectively.
As a sharp contrast, at pH = 7.4, the release was kept as low
as ≈15.4%. Such a pH-sensitive release manner was mainly due
to the amino protonation and the increased solubility of DOX
molecule at low pH.[51] Importantly, eruptible DOX release was
observed over multiple NIR irradiation cycles (5 min irradia-
tion for each cycle), indicating that the drug release could also
be sensitively triggered by the irradiation. For instance, at pH =
4.8 and 1 h, a release of ≈32.0% was detected upon irradiation,
which is 128% as that without irradiation. In fact, the local tem-
perature rise caused by the NPs could promote thermal vibra-
tion and weaken the interaction between the NPs and DOX,
thus ultimately accelerating DOX release.[52] This bimodal pH-
and NIR-responsive drug release property may be beneficial and
favorable for on-demand drug delivery at the tumor site, as the
acidic tumor microenvironment and the external stimulus of
laser irradiation can trigger sufficient DOX for accurate chemo-
therapy without unwanted risk to normal cells.
2.6. Chemotherapy Effect on Cancer Cells
To intuitively show the intracellular drug delivery, 4T1 cells
were incubated with the Bi2Se3@mSiO2-PEG/DOX dispersions
for different duration (1, 3, and 5 h), and then stained with
4′,6-diamidino-2-phenylindole (DAPI), followed by observa-
tion under an inverted fluorescence microscope. As shown in
Figure 8, unlike the control group, remarkable red DOX fluores-
cence signals in 4T1 cells after treated with the Bi2Se3@mSiO2-
PEG/DOX were clearly observed around the cell nucleus (blue),
showing the effective intracellular delivery of DOX, and also
implying the efficient cellular uptake of the NPs. In addition,
such cellular drug delivery became more pronounced when the
incubation time was extended from 1 to 5 h, suggesting a time-
dependent delivery manner. The uptake efficacy of the Bi2Se3@
mSiO2-PEG/DOX by 4T1 cells was examined (Figure S5, Sup-
porting Information), with free DOX as the control group. It
is found that the uptake was increased with incubation time
for both the NP and free DOX. The uptake efficiency of the
NP versus free DOX was also increased gradually, i.e., 33.3%,
50.5%, and 52.2% for incubation time of 1, 3, and 5 h. There-
into, the small increase from 3 to 5 h suggests that an uptake
efficiency of 52.2% may have been the maximum. These results
further confirm not only the efficient cellular internalization
of the Bi2Se3@mSiO2-PEG/DOX but also the time-dependent
manner of the uptake. These results indicate that the Bi2Se3@
mSiO2-PEG NPs can be used as an effective drug-delivery cargo
for effective intracellular drug delivery.
We then investigated the chemotherapy effect of the Bi2Se3@
mSiO2-PEG/DOX NPs. The 4T1 cells were incubated with the
Bi2Se3@mSiO2-PEG/DOX dispersions for 24 h at gradient
concentrations, then stained with DAPI and observed using
Figure 7. A) FT-IR spectra of the Bi2Se3@mSiO2-PEG, Bi2Se3@mSiO2-PEG/DOX, and free DOX. (Inset: the digital photos of the sample before/after
DOX loading.) B) Fluorescence emission spectra of DOX, the Bi2Se3@mSiO2-PEG, and Bi2Se3@mSiO2-PEG/DOX NPs suspensions with excitation at
480 nm. C) DOX released from the Bi2Se3@mSiO2-PEG/DOX at various pH and selected time points. D) DOX released from the Bi2Se3@mSiO2-PEG/
DOX triggered by NIR irradiation at selected pH and selected time points.

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an inverted fluorescence microscopy. As shown in Figure 9,
4T1 cells incubated with the Bi2Se3@mSiO2-PEG/DOX NPs
showed significant karyopyknosis with distinct abnormal
nucleus fragments (as pointed by the yellow arrows), which is
characteristics of cell apoptosis. And such phenomenon was
more pronounced at higher doses. In comparison, for the cells
after treated by the Bi2Se3@mSiO2-PEG NPs without DOX
loading, no apparent change was observed on nuclei mor-
phology. These results indicate that the DOX delivered from
the Bi2Se3@mSiO2-PEG/DOX NPs can effectively induce apop-
tosis of cancer cells.
The CCK-8 assay was further used to quantitatively assess
the chemotherapy efficacy. 4T1 cells were incubated with the
Bi2Se3@mSiO2-PEG/DOX and Bi2Se3@mSiO2-PEG NPs dis-
persions at gradient concentrations for 24 h. It was found that
the Bi2Se3@mSiO2-PEG NPs hardly affected the cell viability,
while the DOX-loaded NPs caused a remarkable killing effect
on 4T1 cells (Figure 10A). In particular, more than ≈86%
of 4T1 cells were killed after incubation with the Bi2Se3@
mSiO2-PEG/DOX NPs at a concentration of 100 µg mL−1 for
24 h, suggesting the powerful chemotherapeutic effect on
cancer cells.
2.7. Thermo-Chemotherapy
Finally, we studied the synergistic therapeutic efficacy of the
Bi2Se3@mSiO2-PEG/DOX NPs by combining chemotherapy
and PTT (thermo-chemotherapy) on cancer cells. 4T1 cells were
incubated with the Bi2Se3@mSiO2-PEG, Bi2Se3@mSiO2-PEG/
DOX, and free DOX (the concentration was normalized to be
the equivalent DOX content in the Bi2Se3@mSiO2-PEG/DOX)
for 12 h, and then washed with PBS, followed by laser irradia-
tion for 5 min. After further 24 h incubation, the CCK-8 assay
was employed to evaluate the therapeutic efficacy. As shown
in Figure 10B, the group treated by the thermo-chemotherapy
(Bi2Se3@mSiO2-PEG/DOX NPs plus laser irradiation) showed
a much higher killing efficacy on cancer cells than either
chemo
therapy (free DOX) or PTT (Bi2Se3@mSiO2-PEG NPs)
PTT (Bi2Se3@ mSiO2-PEG NPs) alone at all the tested concen-
trations. For example, at the same concentration of 50 µg mL−1,
the killing efficacy of the thermo-chemotherapy was as high
as ≈92%, significantly higher than that of the chemotherapy
(≈69%) or PTT (≈75%). In fact, the largely improved thera-
peutic effect by thermo-chemotherapy can be attributed to the
increased cellular uptake and drug release caused by the local
hyperthermia.[53]
To explore the cell death mechanism upon thermo-chemo-
therapy treatment, flow cytometric measurement has been con-
ducted by an Annexin-V-FITC/PI method on 4T1 cells treated
by the Bi2Se3@mSiO2-PEG NPs with/without laser (NPs group
and NPs+Laser group), DOX with laser (DOX+Laser group)
and Bi2Se3@mSiO2-PEG/DOX with/without laser (NPs/DOX
group, and NPs/DOX+Laser group) (Figure 11). The quanti-
ties of living cells, early-apoptosis cells, and late-apoptosis/
necrosis cells can be directly determined by the percentage
of AnnexinV−/PI−, Annexin V+/PI−, Annexin V−/PI+, and
AnnexinV+/PI+, respectively. Consistent with the results by
CCK-8 essay, the inhibition efficacy of the group of NPs/
DOX+Laser is most pronounced among all treatments. There-
fore, our results support that the Bi2Se3@mSiO2-PEG/DOX
NPs can perform as a promising thermo-chemotherapy agent
for anticancer treatment.
Figure 8. Fluorescence images of 4T1 cells treated with A) the Bi2Se3@
mSiO2-PEG and B–D) the Bi2Se3@mSiO2-PEG/DOX NPs for various
durations, where the red fluorescence signal is attributed to DOX, and
cell nuclei are in blue after DAPI staining. Scale bar is 100 µm.
Figure 9. Fluorescence images of 4T1 cells incubated with A) the
Bi2Se3@mSiO2-PEG (50 µg mL−1) and B–D) the Bi2Se3@mSiO2-PEG/
DOX NPs at a concentration of 0, 20, and 50 µg mL−1 for 24 h, respec-
tively. Scale bar is 100 µm. Abnormal nuclear morphology is indicated
by the yellow arrows.

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3. Conclusion
In conclusion, we have successfully synthesized the mul-
tifunctional Bi2Se3@mSiO2-PEG core–shell NPs. The NPs
show an excellent effect for synergistic PTT and chemotherapy
with high-contrast IRT imaging on cancer cells. Moreover,
the product exhibits low/no cytotoxicity, high stability, high
photo
thermal conversion efficiency (≈30.5%), and photo-
thermal stability, high IRT imaging contrast, as well as a rather
high loading capacity (≈50.0%). By loading DOX into the NPs,
a bimodal on-demand pH- and NIR-responsive drug release
and effective intracellular drug delivery for chemotherapy have
Figure 10. A) Cell viability after incubation with the Bi2Se3@mSiO2-PEG and Bi2Se3@mSiO2-PEG/DOX NPs at various concentrations for 24 h.
B) The synergistic therapeutic efficacy the Bi2Se3@mSiO2-PEG/DOX NPs upon external stimulus of NIR irradiation (P values are calculated by Tukey’s
post-test, **p < 0.01 or *p < 0.05).
Figure 11. Flow cytometric profiles of 4T1 cells treated by the Bi2Se3@mSiO2-PEG NPs with/without laser, DOX with laser and Bi2Se3@mSiO2-PEG/
DOX with/without laser at a NPs concentration of 10 µg mL−1.

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been achieved. Most importantly, it has been demonstrated
that the thermo-chemotherapy effect on the ablation of cancer
cells is much more efficient than either PTT or chemotherapy
alone. Therefore, such multifunctional Bi2Se3@mSiO2-PEG
NPs can act as a powerful drug-delivery PPT agent for high-
performance thermo-chemotherapy treatments and real-time
diagnose.
4. Experimental Section
Materials: The chemicals used in this study were polyvinylpyrrolidone
(PVP, Mw ≈ 55 000, Sigma-Aldrich), bismuth nitrate pentahydrate
(Bi(NO3)3·5H2O, ≥99.99+%, Aladdin), ethyl acetate (≥99.5%, Sinopharm
Group Chemical Reagent Co., Ltd.), sodium selenite (Na2SeO3, ≥97.0%,
Shenyang Huadong reagent), hydroxylamine (NH2OH, 50 wt% in
H2O, Sigma-Aldrich), sodium hydroxide (NaOH, ≥97.0%), acetone
(≥99.9%, Aladdin), ethylene glycol (EG > 99%, Aladdin), doxorubicin
hydrochloride (98%, Aladdin), dimethyl sulfoxide (>99%, Aladdin),
tetraethoxysilane (TEOS, >99%, Aladdin), CTAB (≥99.0%, Sinopharm
Group Chemical Reagent Co., Ltd.), ammonium nitrate (NH4NO3, AR,
Tianjin Fengchuan Chemical Reagent Technologies Co., Ltd), mPEG-
Silane (95%, Shanghai ToYong Biological Co., Ltd.), Cell Counting Kit-8
(CCK-8, Dojindo Laboratories), PI (Dojindo Laboratories), Calcein AM
(>90.0%, Dojindo Laboratories), and DAPI (Dojindo Laboratories).
Unless otherwise stated, all the chemicals and reagents were analytical
grade and used as received. DI water with a resistivity of ≈18.2 MΩ cm
was obtained from Milli-Q water purification system.
Characterization: TEM (Tecnai G20, FEI Co., USA) and EDS were used
to characterize the morphology and analyze the elements of the sample.
Powder XRD analysis was carried out on Bruker Advanced D8 Discover
with Cu Kα radiation. UV–vis–NIR absorption and FT-IR spectra were
measured by using Evolution 300 UV–vis–NIR spectrophotometer
(Thermo Scientific, USA) and Nicolet 6700 FT-IR spectrometer
(Thermo Scientific, USA), respectively. Drug release was determined
using fluorescence spectrophotometer (Cary Eclipse, Varian, USA).
The NIR irradiation was performed with a continuous-wave diode laser
with a center wavelength of 808 ± 10 nm and an output power of 2 W
(Beijing Kaipulin Optoelectronic Technology Co., China). The solution
temperature was measured via a thermocouple microprobe (STPC-510P,
Xiamen Baidewo Technology Co., China).
Synthesis of the Bi2Se3 Nanoplates: Briefly, PVP (0.75 g) was dissolved
in EG (120.0 mL), then Na2SeO3 (180 mg) and Bi(NO3)3·5H2O
(337.5 mg) were added upon magnetic stirring at room temperature.
The mixture was then heated to 160 °C in a nitrogen atmosphere.
Subsequently, the reaction was triggered by rapid injection of 1.8 mL of
hydroxylamine solution. After 10 min, the solution was cooled to room
temperature. The final products were precipitated by centrifuging and
washing with a mixture of acetone and DI water, followed by drying in an
oven at 50 °C in vacuum for 12 h.
Synthesis of the Bi2Se3@ mSiO2-PEG Nanoparticles: The synthesized
Bi2Se3 nanoplates (16 mg) and CTAB (0.06 g) were dispersed in DI
water (10.0 mL) in a three-necked flask via ultrasonication for 5 min,
followed by the addition of DI water (30.0 mL) and sodium hydroxide
(1.0 mol L−1
, 300 µL). After heated to 70 °C, the mixture solution
was dropwise added with TEOS (1.0 mL) and ethyl acetate (3 mL)
and reacted upon stirring for 24 h. For the PEG coating, the product
was further reacted with mPEG-Silane (16 mg) for 2.5 h, and then
centrifuged (13 000 rpm, 10 min) and washed with ethanol for three
times. To remove the CTAB and achieve the mesopores, the above
synthetic product was dispersed in ethanol (40.0 mL) and mixed with
ammonium nitrate (100 mg), and then maintained at 50 °C for 2 h.
The final Bi2Se3@ mSiO2-PEG NPs were purified by centrifugation and
washing with DI water, and finally stored at 4 °C for further use.
Photothermal Experiments: The photothermal conversion capacity
of the Bi2Se3@mSiO2-PEG NPs was measured by monitoring the
temperature of the NPs aqueous dispersions (1.0 mL) at various
concentrations (0, 50, 100, 200, 300, 400 µg mL−1) upon irradiation by
the NIR laser (808 nm, 2.0 W cm−2) for 10 min. The system temperature
was measured every 1 s by a thermocouple microprobe submerged in the
solution. For comparison, DI water (1.0 mL) under the same radiation
was used as a control. In order to calculate the photothermal conversion
efficiency (η), the Bi2Se3@mSiO2-PEG NPs suspension (200 µg mL−1,
1.0 mL) was irradiated by the 808 nm laser (2.0 W cm−2) until the system
temperature reached equilibrium. Then, the laser was turned off, and the
system was allowed to cool to the ambient temperature. The solution
temperature was measured every 20 s. To study the photothermal
conversion stability, the Bi2Se3@mSiO2-PEG NPs dispersion
(200 µg mL−1, 1.0 mL) was repeatedly irradiated by the 808 laser for five
cycles (3 min irradiation and 3 min cooling for each cycle) Then, the
Bi2Se3@mSiO2-PEG NPs dispersion after the repeated irradiation cycles
was collected for UV–vis–NIR absorption measurement.
Cellular Uptake: To study the cellular uptake, 4T1 cells (5 × 105 cells
per well) were incubated with the Bi2Se3@mSiO2-PEG/DOX dispersions
(100 µg mL−1) on a 12-well cell-culture plate for 1, 3, and 5 h, respectively.
Subsequently, the cells were washed three times with PBS, then fixed
with paraformaldehyde (4%) and stained with DAPI (2.0 µmol L−1). The
cells were then visualized using the inverted fluorescence microscope
(IX71, Olympus, Japan). To study the cellular uptake efficacy, 4T1 cells
(5 × 105
cells per well) were incubated with the Bi2Se3@mSiO2-PEG/
DOX dispersions (100 µg mL−1
) with free DOX as the control group.
The concentration was normalized to be the equivalent DOX content in
the Bi2Se3@mSiO2-PEG/DOXon a 12-well cell-culture plate for 1, 3, and
5 h, respectively. The culture medium was collected to test the uptake
efficacy. After centrifugation, the collected supernatant was analyzed by
the fluorescence spectrum measurement (excitation: 480 nm; emission:
590 nm) to determine the released DOX.
Cytotoxicity: To study the cytotoxicity, human umbilical vein
endothelial cells (HUVEC, a normal cell line) and murine breast cancer
4T1 cells (1 × 104
cells per well) were first seeded on 96-well cell-culture
plates overnight, and then incubated with the Bi2Se3@mSiO2-PEG NPs
dispersions at selected concentrations (0, 10, 20, 50, 100, 200, 300,
and 400 µg mL−1
) for 24 and 48 h, respectively. The cell viability was
then measured using the CCK-8 assay according to the manufacturer
suggested protocol.
Photothermal Effect on Cancer Cells: To evaluate the photothermal
killing effect of the Bi2Se3@mSiO2-PEG NPs on cancer cells, 4T1
cells (5 × 105
cells per well) were first seeded on a 12-well plate
for 48 h, and then incubated with the Bi2Se3@mSiO2-PEG NPs
dispersion (200 µg mL−1
, 2.0 mL per well) for 12 h. The cells were
then exposed to the NIR irradiation (808 nm, 2.0 W cm−2
) for 0, 3,
and 10 min, respectively. After the irradiation, the cells were washed
three times with PBS, incubated with fresh culture medium at 37 °C
for 30 min, and then stained with calcein AM (2.0 µmol L−1) and
PI (3.0 µmol L−1) to observe the live and dead cells. CCK-8 assay
was also used to study the photothermal efficacy of the Bi2Se3@
mSiO2-PEG NPs on cancer cells. Briefly, 4T1 cells seeded on 96-well
plates (1 × 104 cells per well) were incubated with the Bi2Se3@
mSiO2-PEG NPs dispersions at different concentrations (0, 5, 100,
200 µg mL−1) for 12 h, followed by irradiation with the 808 nm laser
(2.0 W cm−2) for 0, 5, and 10 min, respectively. The cell viability was
finally measured by the CCK-8 assay.
DOX Loading and Release Experiments: In a typical experiment, the
Bi2Se3@mSiO2-PEG NPs (8.0 mg) dispersed in PBS (35 mL) was fully
mixed with DOX-HCl solution (2.0 mg mL−1, 5.0 mL), followed by the
addition of NaOH solution (1 mol L−1, 75 µL) to neutralize the HCl
molecule. After stirred in the dark room for 24 h, the obtained Bi2Se3@
mSiO2-PEG/DOX NPs were centrifuged (13 000 rpm, 10 min) at
4 °C and washed with DI water. For the DOX release experiment, the
Bi2Se3@mSiO2-PEG/DOX (500 µg) was dispersed in PBS (2.0 mL)
with different pH (7.4 and 4.8) and then stirred in the dark room at
37 °C. The solution was centrifuged at 4 °C (13 000 rpm, 5 min) at
predetermined time intervals and the supernatant was replaced with
2.0 mL fresh PBS with the same pH. For the NIR-triggered DOX

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release, the Bi2Se3@mSiO2-PEG/DOX dispersion was irradiated by the
808 nm laser for 5 min at selected time points. After centrifugation,
the collected supernatant was analyzed by the fluorescence spectrum
measurement (excitation: 480 nm; emission: 590 nm) to determine the
released DOX.
Chemotherapy on Cancer Cells: To assess the chemotherapy effect
of the Bi2Se3@mSiO2-PEG NPs on cancer cells, 4T1 cells seeded on a
96-well cell-culture plate (1 × 104 cells per well) were treated with the
Bi2Se3@ mSiO2-PEG/DOX NPs at gradient concentrations (0, 10, 20,
50, 100 µg mL−1) for 24 h. Cell viability was measured using the CCK-8
assay. The data represented the average of triplicate measurements.
Meanwhile, 4T1 cells treated with the Bi2Se3@mSiO2-PEG NPs (without
DOX loading) were used as the control. For the nuclear morphology
study, 4T1 cells (1 × 105 cells per well) were first seeded on the 12-well
cell-culture plate, and then incubated with the Bi2Se3@mSiO2-PEG
(50 µg mL−1) and Bi2Se3@mSiO2-PEG/DOX NPs (0, 20, and 50 µg mL−1)
for 24 h at 37 °C. The cells were fixed with 4% paraformaldehyde for
20 min, and stained with DAPI (2 µmol L−1) for 15 min. Afterward, the
cells were washed with PBS (pH 7.4) and examined using an inverted
fluorescence microscope.
Thermo-Chemotherapy on Cancer Cells: In order to evaluate the
synergistic PTT and chemotherapy effect of the Bi2Se3@mSiO2-PEG/
DOX NPs on cancer cells, 4T1 cells seeded on a 96-well plate at
a density of 1 × 104
cells per well were incubated with free DOX, the
Bi2Se3@mSiO2-PEG NPs dispersions, the Bi2Se3@mSiO2-PEG/DOX
NPs dispersions, respectively, at 37 °C for 12 h. Thereafter, the cells were
washed with PBS and incubated with fresh DMEM containing 10% fetal
bovine serum, followed by irradiation with the 808 nm laser (2 W cm−2
)
for 5 min. After laser irradiation, the cells were further incubated at
37 °C for 24 h. Finally, the cell viability was determined using the CCK-8
assay. Furthermore, cells incubated with the Bi2Se3@mSiO2-PEG NPs
with/without laser, DOX with laser and Bi2Se3@mSiO2-PEG/DOX with/
without laser were collected, washed three times with PBS, dyed with
Annexin-V-FITC/PI kit, and then examined by flow cytometry to explore
the cell death mode.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
Z.L., Z.L.L., and L.S. contributed equally to this work. This work was
financially supported by the National Natural Science Foundation of
China (Grant Nos. 21473045 and 51401066), the Fundamental Research
Funds from the Central University (PIRS OF HIT A201503), and the
State Key Laboratory of Urban Water Resource and Environment (Grant
No. 2018DX04).
Conflict of Interest
The authors declare no conflict of interest.
Keywords
bismuth selenide, chemotherapy, drug delivery, mesoporous silica,
photothermal therapy
Received: September 12, 2017
Revised: October 5, 2017
Published online: November 27, 2017
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ISSN 1998-0124 CN 11-5974/O4
2019, 12(8): 1770–1780 https://doi.org/10.1007/s12274-019-2341-8
Research
Article
Immune-adjuvant loaded Bi2Se3 nanocage for photothermal-improved
PD-L1 checkpoint blockade immune-tumor metastasis therapy
Yilin Song, Yidan Wang, Siyu Wang, Yu Cheng, Qianglan Lu, Lifang Yang, Fengping Tan, and Nan Li ()
Tianjin Key Laboratory of Drug Delivery & High-Efficiency, School of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072,
China
© Tsinghua University Press and Springer-Verlag GmbH Germany, part of Springer Nature 2019
Received: 31 October 2018 / Revised: 9 February 2019 / Accepted: 15 February 2019
ABSTRACT
Checkpoint blockade based immune therapy has shown to be effective but benefit only the minority of patients whose tumors have been
pre-infiltrated by T cells. To overcome this obstacles, a PEG-modified Bi2Se3 nanocage (NC) loaded with imiquimod (R848), which could
efficiently destroy the tumors thus producing enough tumor-associated antigens (TAA) and with the existence of R848, a toll-like-
receptor-7 agonist, could generate strong anti-cancer immune responses is reported in this study. Moreover, immunogenic Bi2Se3
NC-PEG/R848 mediated photothermal therapy (PTT) sensitizes tumors to checkpoint inhibition mediated by a PD-L1 antibody, not only
ablating cancer cells upon NIR laser but also causing strong anti-cancer immunity to suppress distant tumor growth post PTT. Both in
vitro and in vivo experiments demonstrate that the Bi2Se3 NC-PEG/R848 could effectively activate a PTT-induced immune response as
well as silence immune resistance based on PD-L1 checkpoint blockade to ablate the primary tumor and further inhibit the tumor
metastasis. Bi2Se3 NC reported here exhibits high photothermal conversion efficiency and stability, as well as competent drug loading
capacity with large hollow structures and high surface area. Our study not only provides a facial way to synthesize Bi2Se3 NC, but also
offers an alternative strategy for tumor metastasis.
KEYWORDS
Bi2Se3 nanocage, R848, checkpoint blockade, photothermal-immune therapy, anti-tumor metastasis
1 Introduction
On account of the highly complex biological procedure in cancer
formation, tumor metastasis is yet hard to be cured and causes the
majority of cancer deaths. Considering the treatment strategies,
conventional radio- and chemo-therapy are both suffered by limited
efficiency and serious side-effect [1–4]. Thus, it is significant to
uncover efficient ways to eliminate and inhibit tumor metastasis.
Due to the stimulation of specific immune-therapy and long-term
immune memory capacities [5–7], cancer immune-therapy have
drawn great attentions in recent years, particularly for checkpoint
blockade therapy and adoptive T cell transfer. Cancer immune-therapy
could eliminate the primary tumor, moreover, offer great chances to
tumor metastases [8]. Immune checkpoint blockade therapy is a
hopeful strategy against tumor metastatic through tumor specific T
cells activation, especially tumor-infiltrating cytotoxic T lymphocytes
(CTLs) [9–11]. Nevertheless, the activation of anti-cancer immune
response and evasion has limited the efficiency of immune
checkpoint blockade therapy. Cancer cells could grow against
immune response of the host based on a self-protecting mechanism,
immune evasion [12]. In the associated processes, programmed death 1
(PD-1) and corresponding receptor (PD-L1) are important immune
checkpoint molecules [13, 14]. PD-1 is a type of immunosuppression
molecule expressed on the surface of many types of cells, including
T cells. After specifically bound with PD-L1, a trans-membrane
protein over-expressed in many malignant tumors [15], which may
transmit suppressed signal thus suppressing cytokines excretion as
well as causing T cells apoptosis [16–18]. Nevertheless, the efficiency
of anti-cancer immune-therapy is yet limited by ineffective immune
activation, in spite of efficient decreased immune evasion by PD-L1
checkpoint blockade.
Recently, photothermal-immune therapy has gain lots of attentions
due to promising use in PTT-induced anti-cancer immune response
activation [19–21]. Through producing hyperthemia, PTT may ablate
the cancer cells as well as release tumor-associated antigens (TAA),
which will be presented to CTLs and thus activate anti-cancer immuno-
therapy. Still, immune evasion has negative influence on PTT in
situ of tumor sites [22, 23]. As a consequence, it is promising to
combine PTT and PD-L1 checkpoint blockade immune therapy
to increase the efficacy of tumor metastases therapy [21, 23, 24].
Besides, compared with CTLA4, PD-L1 can be expressed on many
cell type, including T cells, epithelial cells, endothelial cells, and
tumor cells after exposure to the cytokine IFN-γ, produced by
activated T cells [25]. This has led to the notion that rather than
functioning early in T cell activation, the PD-1/PD-L1 pathway acts
to protect cells from T cell attack. Thus, the existence of a T cell
infiltrate and select biomarkers, such as expression of PD-L1, which
indicate a “hot” tumor microenvironment, does correlate with clinical
benefit for patients treated with anti-PD-1 or anti-PD-L1 [13].
Herein, we first introduce a hollow photothermal agent, bismuth
selenide nanocage (Bi2Se3 NC) based nanomedicine for anti-tumor
photothermal-immune therapy, which integrates enhanced immune
response activation and PD-L1/PD-1 suppression. As a representative
topological insulators, Bi2Se3 possess distinguished thermo-electric
and photo-electric performance, thus is applying for photothermal and
CT contrast agent in vivo [26]. Among reported Bi2Se3 nanoparticles,
Address correspondence to linan19850115@163.com

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Bi2Se3 NC with stronger NIR region optical absorption has not been
studied yet. Such high tissue absorption coefficient enables Bi2Se3
NC with higher in vivo PT conversion efficiency. Meanwhile, compared
with other reported Bi2Se3 nanoparticles, Bi2Se3 NC is a desired
drug carrier with larger free volume. Due to these interesting
characters, in this study, we synthesized PEG-modified Bi2Se3 NC
with good bioactivity and biocompatibility to load Resiquimod (R848),
a toll-like receptor 7 and 8 (TLR7/TLR8) agonist to enhance immune
responses activation [27, 28]. Under NIR laser irradiation, Bi2Se3
NC-PEG/R848 induced PTT could ablate primary tumors then
expose TAA, which would display vaccine-like properties with the
help of nanoparticles loading R848 immune adjuvant. Subsequently,
professional antigen presenting cells, such as dendritic cells (DCs)
would present these exposed TAA to activate CTLs. In addition,
anti-PD-L1 checkpoint blockade could enhance and protect the
activity of CTLs. At last, CTLs could move to distant cancer cells
without NIR laser irradiation and regulate cell immune response to kill
metastasizing cancer cells particularly. Moreover, NC-PEG/R848-based
PTT in combination with anti-PD-L1 therapy is able to protect
treated mice from tumor re-challenging 40 days after primary
tumors ablation, verifying a strong immune-memory effect to save
mice from tumor relapse. Thus, we hypothesize that our Bi2Se3
NC-PEG/R848 nanoparticle could synergistically inhibit the primary
and distant tumor growth through PTT improved PD-L1 immune-
therapy.
Scheme1 Illustration of (a) synthesis procedure of Bi2Se3 nanocage (NC)-PEG/R848,
(b) Bi2Se3 NC-PEG/R848 induced combined photothermal- and immune-therapy.
2 Experimental
2.1 Materials
Oleylamine (OM, 70%, Sigma-Aldrich), oleic acid (OA, 90%, Sigma-
Aldrich), 1-octadecene (ODE, > 90%, Sigma-Aldrich), decanoic
acid (> 90%, Sigma-Aldrich), 1-dodecanethiol (1-DDT, ≥ 98%, Sigma-
Aldrich), Mn(CH3COO)2·4H2O (99%, Alfa Aesar), Se (> 99.5%,
Alfa Aesar), bismuth neodecanoate (98%, Sigma-Aldrich), N-(3-
dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride crystalline
(EDC) and N-hydroxysuccinimide (NHS) (Sigma-Aldrich),
poly(maleic anhydride-alt-1-octadecene) (C18PMH) (Sigma-Aldrich)
and mPEG-NH2 (MW = 5K) (Biomatrik Co., Ltd). All chemicals
were used as received.
2.2 Synthesis of Bi2Se3-PEG/R848 nanoparticles
We first synthesized the MnSe nanocube template based on a reported
way [29]. Briefly, we first added manganese acetate tetrahydrate
(22.5 mg) into a mixture of 1-octadecene (ODE, 3.2 mL), oleylamine
(OM, 8.75 mL) and OA (1.07 mL) at room temperature. After that,
we heated the mixture to 120 °C and maintained this temperature for
60 min, thus producing transparent mixture (solution 1). Afterwards,
selenium (40 mg) was dispersed in a solution of OM (3 mL) and
1-dodecanethiol (1-DDT, 0.1 mL), then injected swiftly this mixture
into solution 1 at 220 °C. Further kept at 220 °C for 120 min before
cooling down to 50 °C.
After MnSe nanocube was cooled down to 50 °C, ODE (2.5 mL)
containing bismuth neodecanoate (63 μL) was then injected into
the mixture under constant flow of nitrogen. Following, we heated
the mixture to 180 °C gradually and then kept for 30 min under
vigorous stirring (1,000 rpm). After cooling down to room temperature,
Bi2Se3 NC was gained by centrifugation (8,000 rpm, 10 min), and
then washed twice with ethanol and chloroform [30].
OM ligand coated Bi2Se3 NC was then decorated with C18PMH-
PEG via an amphiphilic polymer modifying strategy [31]. We
dispersed the Bi2Se3 NC (20 mg) in chloroform (2 mL) contained
C18PMH-PEG (50 mg) and constantly stirred for 18 h. Next, the
chloroform was evaporated and then added water (10 mL) into this
solution under ultrasonication (15 min). Bi2Se3 NC-PEG was thus
obtained through centrifugation (12,000 rmp, 10 min).
2.3 R848 loading and releasing
As for R848 loading, 30 μL R848 solutions in DMSO (3 mg/mL)
was added to 1 mL Bi2Se3 NC-PEG (4 mg/mL). The unloaded R848
was filtered and washed after 24 h constant stirring. For R848
release study, we packed Bi2Se3 NC-PEG/R848 (1 mL) and PBS (19 mL)
into a dialysis bag (MWCO: 13 kDa). Then we took out the
incubation medium (2 mL) at diverse time points and refreshed
with new ones.To study R848 release property induced by NIR laser,
we adopted an 808 nm laser to irradiate the Bi2Se3 NC-PEG/R848
solution at diverse time point.
As for Cy5.5 labeled Bi2Se3 NC-PEG/R848, 30 μL R848 solutions
in DMSO (3 mg/mL) and 10 μL Cy5.5 solution in DMSO (3 mg/mL)
was added to 1 mL Bi2Se3 NC-PEG (4 mg/mL). The unloaded R848
and Cy5.5 were filtered and washed after 24 h constant stirring.
2.4 Photothermal performance
We irradiated H2O (0.5 mL) and various concentrations of Bi2Se3
NC/R848 (0.5 mL) with NIR laser (808 nm, 0.8 W/cm2
, 5 min). We
monitored the solution temperature via a digital thermometer at
certain time points. The real time thermal images for PBS (0.5 mL)
and Bi2Se3 NC-PEG/R848 ([NC-PEG] = 80 μg/mL, [Cy5.5] = 6 μg/mL,
[R848] = 0.8 μg/mL, 0.5 mL) was taken via an infrared thermal
camera as well. In order to calculate the PT conversion efficiency (η),
we monitored the temperature changes of Bi2Se3 NC-PEG/R848
solution (60 μg/mL, 0.5 mL) upon NIR laser irradiation (808 nm,
0.8 W/cm2
) at designed time points. We then calculated the η by the
following the formula: max surr s
A808
( )
100%
(1 10 )
hS T T Q
η
I -
- -
= ´
-
, hS was
obtained from Fig. 3(g) [30].
2.5 Cellular experiments
Murine breast cancer 4T1 cells were cultured under recommended
conditions with 1% penicillin/streptomycin and 10% fetal bovine
serum (FBS) at 37 °C in a 5% CO2-containing condition. As for
cytotoxicity experiments in vitro, 4T1 cells were seeded into 96-well
plates at a density of 5 × 104
cells/well until adherent. After incubating
with diverse concentrations of Bi2Se3 NC-PEG/R84, they were further
kept in dark at 37 °C for another 24 h. As for PT treatment group, we
irradiated the cells with NIR laser (808 nm, 0.8 W/cm2
, 3 min) after
6 h incubated. Then we demonstrated the cell viability via the MTT
assay.

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For Calcein AM/PI co-stained study, we seeded 4T1 cells with a
density of 5 × 105
cells per well in CLSM culture dishes and then
incubated them in PBS and different formulations. After 6 h, NC-
and NC-PEG/R848-treated culture dishes were irradiated with an
808 nm laser (0.8 W/cm2
, 3 min), with further incubation for 24 h,
respectively. After removed the culture medium, we added the AO
(10 ng/mL, 1 mL) and PI (10 ng/mL, 1 mL) respectively into culture
dishes then further incubated for 20 and 30 min. Finally, we washed
the dishes several times with PBS (pH 7.4) and then observed through
CLSM.
In order to learn the NC-PEG/R848 cellular uptake profile in vitro,
we seeded the cells into the CLSM dishes at a density of 1 × 105
cells
per well. Post 12 h incubation, we added free Cy5.5 and Cy5.5
labled-Bi2Se3 NC-PEG/R848. After another 4 h incubation, we
irradiated the cells under NIR laser (808 nm, 0.8 W/cm2
, 3 min),
followed by washing with PBS and fixing with 4% paraformaldehyde.
Next, we stained the nuclei with DAPI (10 μg/mL) and washed cells
with PBS to remove unloaded Cy5.5 for CLSM observation.
2.6 Animal models
Balb/c mice were purchased from Huafukang Biological Technology
Co., Ltd (Beijing, China). The animal study protocol was approved
by the Institutional Animal Care and Use Committee at Tianjin
University. To develop the 4T1 tumor model, 4T1 cancer cells (1 × 107
)
were subcutaneously injected on the oxter of each Balb/c mouse.
2.7 In vivo images
For in vivo FL images, we i.v. injected free Cy5.5 (100 μL) or Cy5.5
labeled NC-PEG (100 μL, equivalent 100 μM Cy5.5), respectively,
into 4T1 tumor-bearing Balb/c nude mice tail veins. The FL images
were obtained via a in vivo imaging system. Then, we sacrificed the
mice post imaged in vivo and collected major organs and tumors
for quantitative bio-distribution assessment and ex-vivo images.
As for in vivo CT imaging, 100 μL of NC-PEG/R848 (5 mg/mL)
was i.t. injected into 4T1 tumor bearing Balb/c nude mice. We then
collected CT imaging before and after injection. The images were
taken by micro CT scanner (Quantum FX, PerkinElmer, Hopkinton,
MA, USA). Image analysis software: Analyze 12.0 (AnalyzeDirect,
Overland Park, KS, USA) was utilized to analyze images. (Main
parameters: Scan Time: 4.5 min, field of view (FOV): 73 mm, Current:
180 μA, Voltage: 90 kV.)
To evaluate the quantitative bio-distribution of Bi2Se3 NC-PEG/R848,
major organs conclude heart, liver, spleen, lung, kidney and tumors
collected from Bi2Se3 NC-PEG/R848 treated mice were solubilized
for ICP-MS measurement to confirm Bi content after 4, 8, 12 and
24 h.
2.8 In vivo animal model
To develop the 4T1 tumor metastasis model, 4T1 cancer cells (1 × 107
)
were injected into both left and right flanks of mice, respectively.
After the volume of tumors came up to ~ 100 mm3
, NC-PEG or
NC-PEG/R848 was i.v. injected into mice at day 6 ([NC-PEG] =
80 μg/mL, [Cy5.5] = 6 μg/mL, [R848] = 0.8 μg/mL). And then we
exposed the mice to 808 nm laser irradiation (0.8 W/cm2
, 10 min) in
order to kill cancer cells at day 7. Then, we i.v. injected anti-PD-L1
antibody (BioXcell, product number: BE0101, clone number: 10F.9G2)
into mice from diverse treated groups at a dose of 750 μg/kg at day
8, 9, 10, 11. We measured the mice body weight and tumor size
every two days.
2.9 Ex vivo analysis
We the evaluated the infiltrated cytotoxic T lymphocytes (CTL)
both in distant and primary tumors at day 18 after diverse
treatments through flow cytometry post stained with anti-CD8-PE
and anti-CD3-APC (BD Biosciences). Cells were further stained
with anti-NKp46 to analyze NK cell via flow cytometry. Secondary
tumor cells were further stained with anti-CD3-FITC (eBioscience),
anti-CD4-PerCP (Biolegend), and anti-Foxp3-PE (eBioscience)
antibodies to analyze CD4+
helper T cells. Lymph nodes harvested
from mice post different treatment were further stained with
anti-CD44-PE (eBioscience), anti-CD62L-APC (eBioscience), anti-
CD8-PerCP-Cy5.5 (eBioscience) and anti-CD3-FITC (eBioscience)
to analyze memory T cells. Subsequently, we assessed the pro-
inflammatory cytokines in sera and DC medium supernatants, such
as TNF-α, IFN-γ, and IL-12p40 (eBiosciences) through utilizing
ELISA kits under standard protocols.
3 Results and discussion
3.1 Preparation and characterization
The Bi2Se3 NCs have been gained via a one-pot synthesis method
which is shown in Fig. 1(a). We first synthesized the MnSe template
via a hot injection way. The morphology of the obtained MnSe
template showed a cubic phase, which was demonstrated through
transmission electron microscopy (TEM, Fig. 1(b)), field emission
scanning electron microscopy (SEM, Fig. 1(c)) and the powder
XRD analysis (Fig. S1 in the Electronic Supplementary Material
(ESM)). All these results confirmed that the synthesized MnSe
monodisperse possessed a relatively high morphological purity
yield with the average particle size of around 36 nm. Then we
adopted a cation exchange method to produce Bi2Se3 nanocage
from pre-made MnSe template by injecting bismuth neodecanoate
into the MnSe reaction system at 180 °C. Due to the cation exchange,
MnSe@Bi2Se3 core-shell structure was produced to further reaction
through ions diffusion. The outward diffusion of the core Mn2+
was
much faster than the inward diffusion of Bi3+
, as a result, an inward
flux of vacancies accompanied the outward Mn2+
flux to balance the
diffusivity difference. The hollow Bi2Se3 structure (Bi2Se3 nanocage)
was thus formed through coalescence of the vacancies based on
the nanoscale Kirkendall effect. The obtained Bi2Se3 nanocage
(NC) displayed a well-maintained shape of MnSe template with the
average size and shell thickness around 37 and 6 nm, respectively.
As expected, TEM images (Fig. 1(d) and insert), SEM images
(Fig. 1(e)), elemental mapping by the high-angle annular dark-field
scanning TEM (HAADF-STEM) (Figs. 1(f)–1(i)) and XRD data
(Fig. 2(b)) further demonstrated the successful synthesis of Bi2Se3
NC. Besides, the specific surface area of Bi2Se3 NC was measured to
be 68.3 cm2
/g (Fig. 2(c)), which enabled the NC with efficient drug
loading capacity.
Considering the bulk Bi2Se3 NC dispersion might display slow
deposition and oxidization at room temperature over one week, we
modified the Bi2Se3 NC surface with a PEG grafted amphiphilic
polymer. The average hydrodynamic size of coated NC was increased
slightly with a relatively low PDI as assessed through dynamic
light scattering (Fig. 2(a)), verifying that the NC has been coated
with PEG successfully. In addition, compared with the bulk NC,
NC-PEG displayed good dispersion and stability in water, which
was demonstrated in diverse formulations photos on Day 1 and
Day 7 (Figs. S2 and S3 in the ESM). Compared with bulk NC, the
appearance of NC-PEG after 7 days (water solution, room tem-
perature) kept unchanged, showing the remarkable stability of the
modified nanoparticles, which was promising for in vivo biomedical
applications.
Moreover, NC-PEG owned strong NIR light absorbance, thereby
exhibiting a desirable PT performance upon 808 nm laser irradiation
with a relatively low concentration of NC-PEG (80 μg/mL) (Figs. 2(f)
and 2(i)). The photothermal conversion efficiency of NC-PEG
was calculated to be 36.8% (Fig. 2(g)), which was higher than the
normally adopted photothermal agent. The PT stability of NC-PEG

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Figure 1 (a) Illustration of the synthesis process of Bi2Se3 NC-PEG/R848. (b) TEM image and (c) SEM image of MnSe nanocubes. (d) TEM image and (e) SEM image of
Bi2Se3 NC ((d) insert: high magnification TEM of the Bi2Se3 NC). (f)–(i) Energy dispersive X-ray (EDX) elemental mapping analysis of Bi2Se3 NC-PEG/R848 and the
corresponding elemental mappings of Bi and Se.
Figure 2 Characterization of Bi2Se3 NC-PEG/R848. (a) Size distribution of Bi2Se3 NC, Bi2Se3 NC-PEG and Bi2Se3 NC-PEG/R848. (b) XRD patterns of Bi2Se3 NC. (c) N2
adsorption-desorption isotherms of Bi2Se3 NC (inset: pore size distribution). (d) UV–vis absorption spectra of Bi2Se3 NC, Bi2Se3 NC-PEG and Bi2Se3 NC-PEG/R848.
(e) Cumulative release of R848 from the Bi2Se3 NC-PEG/R848 with or without NIR laser irradiation (0.8 W/cm2
, 5 min). (f) Temperature curves of different Bi2Se3
NC-PEG/R848 concentrations over a period of 5 min exposed to 808 nm laser. (g) Photothermal effect Bi2Se3 NC-PEG/R848 with NIR laser irradiation (808 nm, 0.8 W/cm2
).
(inset: linear time data versus −ln(θ) obtained from the cooling period of (g)). (h) Temperature elevations of Bi2Se3 NC-PEG/R848 (80 mg/mL) cycles. (i) Thermographic
images of PBS and Bi2Se3 NC-PEG/R848 (80 mg/mL) at determined time points (0, 1, 3, and 5 min, respectively).

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was also assessed by four laser on/off cycles. As demonstrated in
Fig. 2(h), the similar increased temperature of diverse cycles verified
excellent PT stability. Results above all verified that the NC-PEG
possessed a remarkable PT conversion efficiency and PT stability.
3.2 R848 loading and in vitro NIR triggered R848 release
profile
Since R848 is an effective immunologic adjuvant, it is important to
delivery R848 into tumor sites instead of distributing in whole body.
The loading ability of R848 was measured through a UV–vis
spectrum, which showed a typical peak at 325 nm (Fig. 2(d)). In
order to assess the release behaviors of R848 from NC-PEG based
drug delivery systems, the NIR triggered R848 release was investigated
in vitro. As illustrated in Fig. 2(e), without a 808 nm laser, the
amount of R848 released from NC-PEG/R848 showed only 10.7%
and 13.1% at 8 and 24 h, respectively. On the contrary, when
exposed to 808 nm laser, the release amount reached to 32.1% at
the first 8 h, and further enhanced to 59.0% at 24 h, demonstrating
that NIR laser could finely controlled the release profile of R848
from NC-PEG/R848, which is promising for decreasing the
systemic toxicity.
3.3 In vitro cytotoxicity and photothermal effect
We then evaluated the cytotoxicity of diverse formulation to 4T1 cells
with various conditions through the MTT assay. As demonstrated
in Fig. 3(a), the cell viability of diverse formulations were over 80%
without NIR irradiation, even with the highest concentration (NC-
PEG: 80 μg/mL and R848: 0.8 μg/mL). However, all of the treatments
displayed concentration-dependent cancer cells ablating ability when
we exposed the cells to NIR irradiation(Fig. 3(b)).
In anti-tumor therapy, it is important for nanoparticles to be
uptake and internalized by cancer cells. Thus, in order to assess the
cellular uptake capacity of NC-PEG/R848, we labeled NC-PEG/R848
with an identical Cy5.5 labeled concentration ([NC-PEG] = 80 μg/mL,
[Cy5.5] = 6 μg/mL, [R848] = 0.8 μg/mL). 4T1 cells were then treated
with diverse conditions. As demonstrated in Fig. 3(c), post 4 h
incubation, most Cy5.5 labeled NC-PEG/R848 was distributed in the
cytoplasm. The fluorescence intensity of free Cy5.5 was obviously
weaker than the final formulation upon the same conditions,
demonstrating the remarkable cellular uptake and internalization
capacity of our nanoparticles. Moreover, we could see an enhanced
Cy5.5 fluorescence intensity inside of the cells under a short time
NIR irradiation (808 nm, 0.8 W/cm2
, 3 min), which might due to
the NIR irradiation controlled release profile.
Furthermore, we also adopted fluorescence co-staining of live/dead
cells to evaluate therapeutic efficacy of our nanoparticle in Fig. 3(d).
Compared with PBS (–NIR), NC-PEG/R848 (–NIR) or NC (+NIR)
treated group, the optimal treatment strategy NC-PEG/R848 (+NIR)
showed the strongest red color, demonstrating good anti-cancer
efficiency of our nanoparticles.
Figure 3 (a) Cell viability of 4T1 cells incubated with diverse concentrations of Bi2Se3 NC-PEG, free R848 and Bi2Se3 NC-PEG/R848 for dark toxicity. Data were
presented as means SD (n = 5). (b) Cell viability of 4T1 from various treatment groups after being incubated with diverse concentrations of Bi2Se3 NC-PEG, free R848
and Bi2Se3 NC-PEG/R848 with 808 nm laser irradiation. Data were presented as means SD (n = 5), *p < 0.05, **p < 0.01. (c) CLSM images of 4T1 cells after incubated
with free Cy5.5 or Bi2Se3 NC-PEG/R848 for 4 h. NIR means 808 nm (0.8 W/cm2
) laser irradiation for 2 min. (d) CLSM images of Calcein AM and PI co-staining 4T1
cells incubated with diverse formulations. Scale bar indicated 100 μm.

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3.4 In vitro and in vivo CT and FL
For evaluating the bio-distribution of our nanoparticle, we adopted
Cy5.5 labeled NC-PEG/R848 for FL imaging in vivo using a 4T1
tumor-bearing nude mice model. Post i.v. injected free Cy5.5 or
Cy5.5 labeled NC-PEG/R848, we recorded the fluorescence signals
at 0, 4, 8, 12, and 24 h time intervals, respectively (Figs. 4(a) and
4(b)). At the early period after injection, we observed widely
distributed fluorescent signals over the whole body. Continuous
accumulation of the Cy5.5 labeled NC-PEG/R848 in the tumor site
was observed and achieved the maximum at 8 h compared with any
other units of the body (Fig. 4(b), top panel). On the contrary,
under the same conditions, the fluorescent signals of free Cy5.5
treated group showed no obvious tumor contrast. We excised the
major organs and tumors 24 h post-injection to gain a clearer sight
of the bio-distribution. Obviously, most of the Cy5.5 labeled
NC-PEG/R848 was accumulated in the tumor site, the majority of
free Cy5.5 was distributed in the liver and kidney by contrast (Fig. 4(a),
bottom panel). The ex-vivo images of different organs further
verified higher tumor retention of Cy5.5 labeled NC-PEG/R848
compared with other major organs (Fig. 4(c)).
Encouraged by the high cancer cell uptake of the Cy5.5 labeled
NC-PEG/R848 as evaluated by FL images, we further studied the
CT images because of the large X-ray attenuation of Bi. We gained
phantom images of NC-PEG/R848 with diverse concentration in
vitro to assess the CT contrast capacity (Fig. 4(d)). We noticed that CT
images gradually became brighter with the enhanced concentration and
showed a linear increase between the concentration of nanoparticles
and the gained CT value (Fig. 4(e)). We then assessed the profile of
CT images in vivo using a 4T1 tumor-bearing nude mice model. We
i.v. injected the NC-PEG/R848 (5 mg/mL, 100 μL) into the tumor
site of the mice to obtain the images via a small animal X-ray CT
imaging system at diverse time points. A strong tumor contrast was seen
post-injection, compared with the images before injection (Fig. 4(f)
and Fig. S4 in the ESM), demonstrating the remarkable CT imaging
ability of NC-PEG/R848. All of these results verified that the NC-
PEG/R848 could serve as a promising multi-model contrast agents
for images in vivo, which might be applied to direct the laser
irradiation in PTT.
To further confirm tumor uptake of Bi2Se3 NC-PEG/R848, we
quantitatively measured the biodistribution of the nanoparticles in
the mice body. Bi levels in major organs and tumors were measured
Figure 4 Fluorescence images of Balb/c nude mice at diverse time points after administration of (a) free Cy5.5 and (b) Cy5.5 labeled Bi2Se3 NC-PEG/R848, the
bottom panel shows the ex vivo images examined at 8 h post-injection. (c) Average fluorescence signals of tumors at diverse time points after administration of free
Cy5.5 and Cy5.5 labeled Bi2Se3 NC-PEG/R848. Data were presented as mean ± SD (n = 5), **p < 0.01. (d) In vitro CT images and (e) corresponding CT intensity of the
Bi2Se3 NC-PEG/R848 with diverse concentrations. (f) In vivo 3D, 2D CT images of Bi2Se3 NC-PEG/R848 in the tumor before and after i.t. injection. (g) The
biodistribution of Bi2Se3 NC-PEG/R848 measured at 4, 8, 12 and 24 h post i.v. injection.

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through inductively coupled plasma mass spectrometry (ICP-MS).
As Fig. 4(g) demonstrated, high Bi content was detected in tumors
at all designed time points, also verifying that Bi2Se3 NC-PEG/R848
could be accumulated to, and resided in the tumors. Moreover,
post 8 h injection, the content of Bi in the tumors have reached
maximum, which was in accordance with FL images results,
demonstrating tumor-targeting ability of our nanoparticles.
3.5 Photothermal tumor ablation for immune system acti-
vation
Recent years, cancer immune-therapy has shown many exciting
clinical results in various cancer treatments [31, 32]. However, the
immune-therapy clinical responses are yet limited due to complex
tumor microenvironment and heterogeneity. Very recently, some
researchers have verified that PTT could stimulate the tumor-specific
immune responses through generating TAA from cancer cell
residues, which subsequently could be processed by APCs such
as DCs and then presented to T cells. Therefore, we experienced
PTT could triggered enhanced immunological responses based on
NC-PEG/R848. In our in vivo experiments, 100 μL of NC-PEG or
NC-PEG/R848 ([NC-PEG] = 80 μg/mL, [R848] = 0.8 μg/mL) was
respectively i.v. injected into the tail vein of the mice when 4T1
tumors grown on Balb/c mice reached around 100 mm3
. After 12 h,
we irradiated the tumor sites with 808 nm NIR laser at 0.8 W/cm2
for 10 min. Five days after PTT (day 12), mice were sacrificed to cut
off the draining lymph nodes, which were utilized to analyze DC
maturation level via flow cytometry analysis (Figs. 5(b) and 5(c)). It
was found that NC-PEG/R848 induced PTT showed a much higher
DC maturation level compared with single NC-PEG or NC-PEG/R848
treated group. In conclusion, after the tumor was damaged by PTT,
DCs could be recruited to the ablated tumor site as APCs to
activate immune responses. In the same time, TAA in tumor debris
after PTT could be converted to lymph nodes nearby and then
simulated DC maturation, particularly under the assistant of
adjuvant nanoparticles.
Cytokines secretion is important in the immune responses as
well. In a parallel experiment, various cytokines changes including
TNF-α, interferon γ (IFN-γ) and interleukin 12 (IL-12p40, sera
from mice of day 12) were studied by ELISA assay. Similarly,
although PTT with NC-PEG or NC-PEG/R848 injection alone was
able to increase pro-inflammatory cytokines secretion, their
secretions induced by NC-PEG/R848 induced PTT were obviously
higher, which was favorable for activating anti-tumor immune
response (Figs. 5(d)–5(f)). These results demonstrated that NC-PEG/
R848 induced PTT could stimulate the immunological system in vivo.
The in vivo adjuvant activities of such nanoparticles combined with
Figure 5 Bi2Se3 NC-PEG/R848-based in vivo PTT induces DC maturation and activates the pro-inflammatory cytokines expression. (a) Schematic illustration of our
experiment design to assess immune responses triggered by Bi2Se3 NC-PEG/R848-based PTT. (b) and (c) DC maturation induced by Bi2Se3 NC-PEG/R848-based PTT
on mice bearing 4T1 tumors. (d)–(f) Cytokine levels of IL-12p40 (d), IFN-γ (e), and TNF-α (f) in sera from mice isolated on day 12. Data were presented as mean ± SD
(n = 5), *p < 0.05, **p < 0.01, ***p < 0.001.

Nano Res. 2019, 12(8): 1770–1780
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PTT induced TAA released might act as safe “tumor vaccine”, which
was promisingly useful for tumor immune-therapy.
3.6 PTT plus PD-L1 checkpoint blockade to inhibit growth
of distant tumors
In this study we further combined NC-PEG/R848 mediate PTT
with PD-L1 checkpoint blockade, which could efficiently increase
the anti-cancer immune activity of CTLs through preventing their
depletion. The method reported here might offer an alternative
method to ablate primary tumors and further kill spreading metastatic
cancer cells. In this study, Balb/c mice were inoculated with 4T1 cells
on the left and right flanks, respectively. The left tumor was selected as
primary tumor to be treated with PTT, while right tumor was chosen
as distant tumors (1–2 cm away) without direct therapy. Mice were
divided into six groups: PBS (Group 1), NC-PEG/R848 (Group 2),
PBS + NIR (Group 3), NC-PEG/R848 + anti-PD-L1 (Group 4),
NC-PEG/R848 + NIR (Group 5) and NC-PEG + NIR (Group 6),
NC-PEG + NIR + anti-PD-L1 (Group 7), NC-PEG/R848 + NIR +
anti-PD-L1 (Group 8). After 12 h diverse therapeutic agents i.v.
injection, the left tumors of mice from Group 3, Group 5 and Group
6 were exposed to 808 nm laser irradiation (0.8 W/cm2
, 10 min). At
day 8, 9, 10 and 11, we i.v. injected anti-PD-L1 antibody into mice in
Group 4 and Group 6 at a dose of 750 μg/kg after laser irradiation
(Fig. 6(a)). We found that NC-PEG + NIR + anti-PD-L1 treatment
(Group 7) could inhibit the primary tumor growth more effectively than
NC-PEG/R848 + NIR (Group 5), whereas NC-PEG/R848 +
anti-PD-L1 administration group (Group 4) showed no remarkable
therapeutic efficiency at applied anti-PD-L1 dose (Figs. 6(b) and
6(c)), verifying PTT alone could improve PD-L1 immune therapy.
Moreover, NC-PEG/R848 + NIR + anti-PD-L1(Group 8) treated group
showed higher anti-tumor efficiency than Group 7, demonstrating
the R848 could contributed to improve the PD-L1 therapy combined
with PTT.
Additionally, the body weights exhibited no remarkable changes
of different groups (Fig. 6(k)). Moreover, 90 percent of mice in
Group 6 survived over 40 days after inoculation of tumors (Fig. 6(l)),
which was in marked contrast to other treatment groups. All results
above indicated that our NC-PEG/R848 induced PTT combined
anti-PD-L1 blockade could synergistically cause highly efficient
anti-tumor immune responses to both destroy tumors with direct
PTT therapy strategy as well as inhibit tumors growth without direct
laser irradiation.
3.7 The mechanism study
To study the mechanism of synergistic anti-tumor ability triggered
by NC-PEG/R848 mediate PTT plus anti-PD-L1 therapy, NK cells, the
subspecies of leukocytes in the distant tumors were studied (Fig. S5 in
the ESM). In comparison with the control group (7.02% ± 0.61%),
the percentage of NK cells increased to about 51% in NC-PEG/R848
based PTT treated group, which demonstrated that, in comparison
with anti-PD-L1 alone, the percentage of NK cells is more affected
by NC-PEG/R848 based PTT group. These results suggest that
PD-L1 checkpoint blockade plays an important role in promoting
the dramatically increased NK cell infiltration and accumulation in
the distant tumor sites.
Cytotoxic T lymphocytes (CTL) in tumors were also tested to
study the mechanism of PTT combined with anti-PD-L1 therapy.
Different from other therapy strategies, only PTT + anti-PD-L1
treatment induced robust CD8+
cytotoxic T lymphocytes (CTL)
infiltration (over 4 folds than others) in the primary tumor (Figs. 6(d)
and 6(e)). Further more we noticed that only PTT plus anti-PD-L1
treatment could inhibit the growth of non-irradiated distant tumors
as well, whose progressing was not influenced in any other groups
(Figs. 6(g) and 6(h)). In addition, compared to other treated group,
remarkable CTL infiltration increase was also shown in the distant
tumors post the combined therapy strategies (Figs. 6(f) and 6(i)).
The robust interferon gamma (IFN-γ) production in the serum
samples with PTT plus anti-PD-L1 treatment was measured at day
18 post tumor incubation, which demonstrated the highly efficient
cellular immune responses mediated by the combined therapy
strategy (Fig. 6(j)). However, regulatory T cells (Tregs) could impede
efficient anti-tumor immune responses. Thus, Tregs in secondary
tumors were also collected for further study post co-staining with
CD4 and Foxp3. It was found that the percentage of Tregs was
greatly reduced in secondary tumors post PD-L1 blockade therapy
(Fig. S6 in the ESM). Moreover, comparing groups 4 and 8 in Fig. S6
in the ESM, PTT combined with anti-PD-L1 could induce the lowest
Tregs percentages, which was mainly major responsible for cell
immunity in tumor immune-therapy.
3.8 Long-term immune-memory effects
Remembering pathogens for few decades is an essential character of
immune systems, which is important for disease prevention. Thus,
evaluating immune memory induced by NC-PEG/R848 mediate
PTT is of great importance. In this study, the 2nd
tumors were
inoculated 40 days post surgery or NC-PEG/R848 mediated PTT
removing the 1st
tumors. Then, mice were i.v. injected with
anti-PD-L1 at diverse days (750 μg/kg every time) for two turns of
treatment, the first turn was injected right behind the 1st
tumor was
removed (Day 1 and 5), and then the 2nd
turn was injected at Day
41, 44 and 47 (Fig. 7(a)). Effector memory T cells (TEM) locate in
non-lymphoid as well as lymphoid tissues, which could induce
immediate protection through generating cytokines such as IFN-γ
[33–36]. In this case, we analyzed the TEM cells proportion at Day
40 post the 1st
tumor removal under various treatments. We noticed
that TEM cells percentage in NC-PEG/R848 mediated PTT treated
group was much higher (Fig. 7(b)) than other treated groups.
Moreover, seven days post the 2nd
tumor incubated, we analyzed the
cytokines in sera under various treatments by ELISA. It is reported
that IFN-γ and TNF-α [37] are cellular immunity typical markers,
playing important roles in immune therapy against tumors. The
TNF-α and IFN-γ serum levels were obviously increased in
NC-PEG/R848 mediated PTT treated group, especially for those
under PTT combined the 2nd
turn of anti-PD-L1 treatment (post),
demonstrating the successful performance of anti-cancer immune
responses triggered by the re-challenging of tumor cells 40 days
after in this group (Figs. 7(c) and 7(d)).
4 Conclusions
In conclusion, we demonstrated that the multifunctional Bi2Se3
NC-PEG/R848 integrating PT agent and immune-adjuvant is able
to stimulate vaccine-like immune responses, which could be
combined with PD-L1 checkpoint blockade to achieve efficient
anti-tumor photothermal-immune therapy. The Bi2Se3 NC here with
hollow interiors is desirable for stronger NIR region optical absorption
and higher tissue penetration. Meanwhile, NC is also a desired drug
carrier with larger free volume. Our Bi2Se3 NC-PEG/R848 can be
utilized for NIR-induced PTT to damage cancer cells directly, as
well as trigger the DCs maturation to activate immune responses
thus secret cytokines. In combination with PD-L1 checkpoint
blockade strategy to inhibit tumor cells immune escape, such Bi2Se3
NC-PEG/R848 based PTT could ablate primary tumors directly
and suppress distant tumors via activating strong anti-cancer
immune responses. Furthermore, a strong immune-memory effect
is observed after NC-PEG/R848 based PTT in combination with
anti-PD-L1 therapy could efficiently protect mice from tumor
re-challenge.

Nano Res. 2019, 12(8): 1770–1780
1778
Figure 6 Anti-tumor metastasis effect of PTT with Bi2Se3 NC-PEG/R848 in combination with checkpoint blockade immune-therapy. (a) Schematic illustration of
experimental design to combine PTT with anti-PD-L1 therapy. (b) Thermo-graphic images of mice 12 h post i.v. injection of PBS and Bi2Se3 NC-PEG/R848 under a
808 nm laser (0.8 W/cm2
, 5 min). (c)–(e) The tumor growth curves (c), average tumor weights at day 18 (d), and percentages of CTL infiltration at day 18 (e), for
primary tumors (left) after various treatments. (f)–(h) The tumor growth curves (f), average tumor weights at day 18 (g), and percentages of CTL infiltration at day
18 (h), for non-irradiated tumors (right) after various treatments. Data were presented as mean ± SD (n = 5), *p < 0.05, **p < 0.01, ***p < 0.001. (i) The IFN-γ levels in
sera from mice detected at 18 days after various treatments. (j) Changes in body weight of mice during treatment. (k) Percent survival for different treatment groups
during 42 days. Data were presented as mean ± SD (n = 5), **p < 0.01, ***p < 0.001.

Nano Res. 2019, 12(8): 1770–1780
1779
Acknowledgements
This work was supported by the National Basic Research Project (973
Program) of China (No. 2014CB932200), the National Natural Science
Foundation of China (Nos. 81503016, 81771880, and 81401453), and
the Application Foundation and Cutting-edge Technologies Research
Project of Tianjin (Young Program) (No. 15JCQNJC13800).
Electronic Supplementary Material: Supplementary material (XRD
of MnSe nanocube, hydrodynamic diameter and polydispersity
index (PDI) of Bi2Se3 NC-PEG in PBS in 4 days, digital photos of
Bi2Se3 and Bi2Se3 NC-PEG at day 7, corresponding HU value of
Bi2Se3 NC-PEG/R848 in the tumor before injection and post i.t.
injection, the distant tumors were harvested for flow cytometry,
the percentages of NK cells and proportions of tumor-infiltrating
regulatory T cells) is available in the online version of this article at
https://doi.org/10.1007/s12274-019-2341-8.
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Figure 7 Long-term immune-memory effects. (a) Schematic illustration of NC-PEG/R848-mediated PTT in combination with anti-PD-L1 therapy to inhibit cancer
relapse. (b) Proportions of effector memory T cells (TEM) in the spleen analyzed by flow cytometry before re-challenging mice with 2nd
tumor at Day 40. (c) IFN-γ
level in sera of mice isolated 7 days post re-challenging with the 2nd
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Surgery+NC-PEG/R848+anti-PD-L1 (pre & post); Group 4: NC-PEG/R848+NIR; Group 5: NC-PEG/R848+NIR+anti-PD-L1 (post); Group 6: NC-PEG/R848+
NIR+anti-PD-L1 (pre & post)).

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