Research Article

Cite This: ACS Appl. Mater. Interfaces 2018, 10, 20342−20355 www.acsami.org

Photothermal-Activatable Fe3O4 Superparticle Nanodrug Carriers

with PD-L1 Immune Checkpoint Blockade for Anti-metastatic Cancer
Immunotherapy
Rui Ge,† Cangwei Liu,‡ Xue Zhang,† Wenjing Wang,† Binxi Li,† Jie Liu,‡ Yi Liu,† Hongchen Sun,‡
Daqi Zhang,*,§ Yuchuan Hou,*,∥ Hao Zhang,*,† and Bai Yang†
†
State Key Laboratory of Supramolecular Structure and Materials, College of Chemistry, Jilin University, Changchun 130012, P. R.
China
‡
Department of Oral Pathology, School and Hospital of Stomatology, Jilin University, Changchun 130021, P. R. China
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

§
Department of Thyroid Surgery, China-Japan Union Hospital, Jilin University, Changchun 130033, P. R. China
∥
Department of Urinary Surgery, The First Hospital of Jilin University, Changchun 130021, P. R. China
*
S Supporting Information
Downloaded via SEOUL NATL UNIV on July 30, 2018 at 02:24:22 (UTC).

ABSTRACT: Checkpoint blockade immunotherapy has

shown great potential in clinical cancer therapy, but the
body’s systemic immune must be fully activated and generates
a positive tumor-specific immune cell response. In this work,
we demonstrate the design of the immune-adjuvant nanodrug
carriers on the basis of poly(ethylene glycol)-block-poly(lactic-
co-glycolic acid) copolymer-encapsulated Fe3O4 superparticles
(SPs), in which imiquimod (R837), a kind of Toll-like
receptor 7 agonist, is loaded. The nanodrug carriers are
defined as Fe3O4-R837 SPs. The multitasking Fe3O4-R837 SPs
can destroy the 4T1 breast tumor by photothermal therapy
(PTT) under near-infrared laser irradiation to generate the
tumor-associated antigens because of the high efficiency of tumor magnetic attraction ability and photothermal effect. The PTT
also triggers the release of R837 as the adjuvant to trigger a strong antitumor immune response. By further combining with the
checkpoint blockade adjusted by programmed death ligand 1 (PD-L1) antibody, the Fe3O4-R837 SP-involved PTT cannot only
eliminate the primary tumors but also prevent tumor metastasis to lungs/liver. Meanwhile, this synergistic therapy also shows
abscopal effects by completely inhibiting the growth of untreated distant tumors through effectively triggering the tumors
infiltrated by CD45+ leukocytes. Such findings suggest that Fe3O4-R837 SP-involved PTT can significantly potentiate the
systemic therapeutic efficiency of PD-L1 checkpoint blockade therapy by activating both innate and adaptive immune systems in
the body.
KEYWORDS: Fe3O4 superparticles, cancer immunotherapy, photothermal-activatable, checkpoint blockade therapy, anti-metastasis

■ INTRODUCTION
Cancer has become one of the major threats to human life for
centuries.1 Because of its high metastasis and recurrence, the
conventional treatments, such as surgery, chemotherapy and
radiotherapy, are difficult to cure thoroughly.2,3 Accordingly,
massive efforts are being devoted to the development of new and
effective cancer treatments with low side effects, high
specificities, and anti-metastasis.4 Recently, along with the deep
understanding of cancer and their relationship with the immune
system, cancer immunotherapy by stimulating and training the
body’s immune system to actively attack tumor cells for the
purpose of controlling metastatic tumor’s growth has attracted
great attention and become one of the most promising
treatments for cancer.5−8 Several kinds of cancer immunothera-
pies, such as chimeric antigen receptor/T-cell receptor-
engineered T-cell therapy,9−11 checkpoint-blockade ther-
apy,12−15 cytokine therapy,16,17 dendritic cell (DC)-based
immunotherapy,18,19 cancer vaccines and so forth,20−23 have
been established and achieved exciting results in clinical trials.
Despite cancer immunotherapy is far from being satisfactory, the
continuous improvements of this strategy will finally build an
alternative for cancer treatment.24,25
Among the aforementioned cancer immunotherapies, check-
point-blockade therapy, which utilizes a series of antibodies that
can target to the overexpressed T-cell suppressor checkpoint
signaling pathways, is a promising strategy to solve the adaptive
immune evasion in the tumor and has been approved by the U.S.
Food and Drug Administration (FDA) for cancer treatment.26,27
Received: April 11, 2018
Accepted: May 31, 2018
Published: June 7, 2018

© 2018 American Chemical Society 20342 DOI: 10.1021/acsami.8b05876

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Figure 1. Schematic illustration of Fe3O4-R837 SP PTT with PD-L1 checkpoint blockade for cancer immunotherapy. With the help of tumor magnetic
attraction, Fe3O4-R837 SPs can destroy the tumors effectively under NIR irradiation. In the presence of Fe3O4-R837 SPs, the generated tumor-
associated antigens can induce strong antitumor immune responses. By further combining with PD-L1 checkpoint blockade, this method can eliminate
primary tumors, prevent the metastasis to lungs/liver, and further inhibit the distant tumor growth after PTT.
Adaptive immune evasion mechanism is a self-protecting
behavior of tumor cells to defend against the body’s immune
response.28 Programmed death 1 (PD-1) and its ligand,
programmed death ligand 1 (PD-L1), are two important
immune checkpoint molecules related to immune resistance.29
PD-1, expressed on T cells surface, interacts with PD-L1
expressed on tumor cells, terminating immune responses by
inhibiting cytotoxic/effector T-cell function and delivering anti-
apoptotic signals to tumors.30 The elimination of either one can
lead to cytotoxic/effector T cells normal operation without
inhibition.31 Hence, antibody-mediated PD-1/PD-L1 pathway
specific blockade can enhance the potent antitumor activity of
the body.32 However, the clinical trials show that if the body’s
immune system cannot be fully activated and produce large
numbers of immune cells, the durable responses generated by
checkpoint blockade in most kinds of tumor cells are still very
low.33−35 Therefore, combining checkpoint blockade therapy
with other types of treatments that can effectively activate the
systemic immune responses to enhance T-cell infiltration may
enhance the antitumor response rates and broaden the
application of immunotherapy in metastatic tumor.36,37
Photothermal therapy (PTT) is a new type of cancer
treatment strategy developed in recent years and has been
proved to kill tumor cells by thermal ablation and produce the
tumor-associated antigens from ablated tumor cell residues.38−40
The thermal ablation process can also activate the host immune
system and trigger acute inflammation in the tumor area, thereby
increasing the presentation of tumor-associated antigens to T
cells.41 A variety of inorganic nanoparticles (NPs), such as noble
metals,42−45 metal chalcogenides,46,47 metal oxides, and so on,48
have been used for the delivery of PTT, showing obvious tumor
ablation in primary animal experiments. However, the strategy
for the combination of the PTT of inorganic NPs with
checkpoint blockade therapy by constructing a novel drug
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delivery system has not been reported because of the difficulty in
choosing proper NPs. In this scenario, Fe3O4 NPs have been
approved by FDA for the clinical treatment with the function of
magnetic targeting and T2-weighted magnetic resonance imaging
(MRI).49 However, individual Fe3O4 NPs show poor photo-
thermal performance due to the low molar extinction coefficient,
making them useless in PTT. In our previous works, spherical
superparticles (SPs) of Fe3O4 NPs have been fabricated by oil-in-
water (O/W) microemulsion template method, which signifi-
cantly improves the photothermal performance and makes
Fe3O4 a good agent for PTT.50 Meanwhile, the SPs can also load
drugs to perform controlled drug release, thus acting as
multifunctional nanodrug carriers for tumor theranostics.
In this work, to utilize near-infrared (NIR)-triggered PTT to
induce effective cancer checkpoint blockade therapy, Fe3O4-
based multifunctional nanodrug carriers are fabricated from three
FDA-approved agents, namely, poly(ethylene glycol)-block-
poly(lactic-co-glycolic acid) copolymer (mPEG-PLGA), Fe3O4
NPs, and imiquimod (R837). The nanodrug carriers are defined
as Fe3O4-R837 SPs. Fe3O4 SPs act as the NIR photothermal,
magnetic targeting, and T2-weighted contrast agents, and mPEG-
PLGA is an amphiphilic copolymer for encapsulating the SPs.
The loaded immune adjuvant R837, a kind of Toll-like receptor 7
(TLR7) agonist, can promote dendritic cells (DCs), phagocytize
tumor-associated antigens, and become mature, thus enhancing
the activation and proliferation of antigen-specific lymphocytes
in draining lymph nodes (DLNs). The tumor retention rate of
the SPs with external magnetic field is as high as 14.8%ID/g and
under NIR irradiation, the Fe3O4-R837 SPs can effectively
eliminate the primary tumors and release R837. After PTT, with
the help of immune adjuvant R837, the released tumor-
associated antigens can stimulate strong systemic antitumor
immune response, which can be promoted by PD-L1 checkpoint
blockade therapy to prevent the lungs/liver metastasis in a 4T1
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Figure 2. Characterization of Fe3O4 and Fe3O4-R837 SPs. (a) Schematic illustration of the synthesis of Fe3O4-R837 SPs. TEM (b) and SEM (c) images
of Fe3O4-R837 SPs. Inset in (b): high-magnification TEM image of one SP. The scale bar represents 50 nm. (d) T2 relaxation rates and concentration-
dependent T2-weighted MRI at pH 7.4 under 1.5 T magnetic field (inset) for the aqueous solution of Fe3O4-R837 SPs. (e) Temperature variation of
PBS, 150 μg/mL Fe3O4 SPs, and Fe3O4-R837 SPs irradiated by a 1.0 W/cm2 808 nm laser. (f) UV−vis absorption spectra of Fe3O4 and Fe3O4-R837 SPs
at the concentration of 150 μg/mL. (g) Fourier-transform infrared (FTIR) spectra of Fe3O4 NPs, mPEG-PLGA, Fe3O4 SPs, R837, and Fe3O4-R837 SPs.

metastatic triple-negative breast cancer murine model. Besides,
the synergistic therapy has an abscopal effect to attack and inhibit
the nonirradiated distant tumors remaining in the mouse body.
In vitro and in vivo experiments indicate that immunogenic
Fe3O4-R837 SP PTT in combination with PD-L1 blockade
therapy has significant efficacy in PTT-activated cancer
immunotherapy (Figure 1).
prepared (Figure S1a).54 Then, O/W microemulsion is
generated by mixing mPEG-PLGA chloroform solution, Fe3O4
NPs, R837 in dimethyl sulfoxide (DMSO), and water under
vigorous stirring. The evaporation of chloroform generates
Fe3O4-R837 SPs. The Fe3O4 SPs are synthesized with the same
method but without R837. The obtained Fe3O4-R837 SPs are
well dispersed in water and appear as quasispheres with good

■
RESULTS AND DISCUSSION
Preparation and Characterization of Fe3O4-R837 SPs.
Fe3O4-R837 SPs are synthesized by assembling Fe3O4 NPs in O/
W microemulsion templates with the presence of mPEG-PLGA
amphiphilic block copolymer and loading with R837 (Figure
2a).51−53 According to the conventional thermal decomposition
method, 5.8 nm oleic acid (OA)-stabilized Fe3O4 NPs are first
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monodispersity under transmission electron microscopy (TEM)
(Figure 2b). The size distribution of Fe3O4-R837 SPs is
calculated from TEM observation, which shows the mean
diameter of 149.8 nm (Figure S1b). The average number of
Fe3O4 NPs in a superparticle is 1.27 × 104 (Calculation S1). The
scanning electron microscopy (SEM) observation also shows the
homogeneous size distribution of the SPs (Figure 2c). Fe3O4-
R837 SPs exhibit an average hydrodynamic diameter of 157.3 ±
3.0 nm, a ζ potential of −26.9 ± 2.8 mV, and a polydispersity
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Figure 3. In vitro transwell coculture system experiments. (a) Schematic illustration of the design of the transwell coculture system experiments. DCs are
placed in the lower compartment, and 4T1 cells are cultured in the upper compartment. (b−h) The percentage of mature DCs (CD11c+CD80+CD86+)
is analyzed by flow cytometry after different treatments. (i) Quantification of the level of DC maturation after different treatments in the transwell system
experiments. “−” and “+” in the figure means treatment without or with irradiation, respectively. Error bars are based on standard deviations of three
parallel samples.

index (PDI) of 0.05 ± 0.016, as measured by dynamic light
scattering (DLS) (Figure S1c). The structural stability of Fe3O4-
R837 SPs is favorable in a physiological environment, as proved
by stable size and PDI when dispersed in phosphate-buffered
saline (PBS, pH 7.4) for up to 7 days (Figure S1d). According to
the previous reports,55 such SPs with negative surface charge,
high stability, and hydrophilic “stealth” polymer surface possess
prolonged blood circulation and efficiently accumulate into
tumor tissues by enhanced permeability and retention effect,
potentially applicable for the living body.
Recent studies have shown that the assembly of inorganic NPs
into SPs can significantly enhance MRI and the photothermal
performance.50 The self-assembly of NPs into Fe3O4-R837 SPs
does not change the superparamagnetic behavior of Fe3O4 NPs
in comparison with the magnetic curve of Fe3O4 NPs (Figure
S1e). The r2 of Fe3O4-R837 SPs is 178.1 mM−1 s−1 (Figure 2d),
which is higher than that of commercial MRI T2-weighted
contrast agents (Feridex, 93 mM−1 s−1; Resovist, 143 mM−1
s−1).56 The high r2 allows Fe3O4-R837 SPs to act as an effective
T2-weighted contrast, with fewer doses getting darker contrast
(Figure 2d inset). Compared with the final temperature of PBS
solution (32.9 °C), the temperature of SP solution increases to
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55.4 °C under the irradiation of 1.0 W/cm2 808 nm laser (Figure
2e). The photothermal conversion efficiency of Fe3O4-R837 SPs
is 68.2% (Figure S2 and Calculation S2). Both the UV−vis
absorption and Fourier-transform infrared (FTIR) spectra of
Fe3O4-R837 SPs show the characteristic absorption peak of
R837, indicating the effective encapsulation of R837 in the SPs
(Figure 2f,g). In water, the R837 concentration and the
absorbance in 2.5−10 μg/mL maintain a linear relation (Figure
S3a). The regression equation is y = 0.0698x − 0.03415 (R2 =
0.999) (Figure S3b). Thus, the R837 loading is calculated by
UV−vis absorption spectrum, and the relationship between
encapsulation/loading efficiency and the dosage of R837
solution in the reaction system are shown in Figure S3c,d.
Considering the encapsulation efficiency and safe dose in vivo
(0.4 mg/kg), the optimal feeding concentration of R837 is 200
μg/mL.57 In our system, R837 is loaded in mPEG-PLGA. Upon
NIR laser irradiation, the system temperature increases due to
the photothermal effect of Fe3O4-R837 SPs. According to the
previous literature,58 the temperature increment promotes the
degradation of PLGA-based NPs. So, NIR irradiation leads to
rapid R837 release from Fe3O4-R837 SPs. Figure S4a shows the
release of R837 with and without irradiation. Without irradiation,
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Figure 4. In vivo tumor magnetic attraction experiments. (a) Schematic illustration of the design of in vivo tumor magnetic attraction. (b) A photograph
showing how the magnet is applied during magnetic attraction. (c) Blood circulation kinetics of Fe3O4-R837 SPs without magnetic attraction. (d) Blood
circulation kinetics of Fe3O4-R837 SPs with magnetic attraction. (e) Biodistribution of Fe3O4-R837 SPs in 4T1-bearing mice at 24 h p.i. administration
with or without magnetic attraction. Error bars are based on standard deviations of three parallel samples. (f) T2-weighted MRI images of mice for
control, Fe3O4-R837 SPs and Fe3O4-R837 SPs with magnetic attraction. The red dotted lined circle points to tumors.

the released R837 in water keeps a low concentration. Whereas, a
high release rate is achieved and maintained under 1 W/cm2 808
nm laser. The released percentage is nearly 34% in 28 min,
implying that R837 can be constantly released from Fe3O4-R837
SPs (Figure S4b). With higher laser power density, Fe3O4-R837
SPs release more R837 (Figure S4c).
PTT-Induced Apoptosis and/or Necrosis in Vitro. 4T1
metastatic triple-negative breast cancer cells are used to test the
photothermal therapy of Fe3O4-R837 SPs. Figure S5a shows that
Fe3O4 SPs/Fe3O4-R837 SPs are rapidly engulfed by 4T1 cells,
with most uptake occurring within 4 h and then remaining stable
within 24 h. High cellular uptake and low efflux ensure the high
cellular accumulation of SPs (Figure S5b). In the control group,
the structure of 4T1 cells is intact and smooth. With the
increment of incubation time, even though the cells maintain
structural integrity, more and more SPs are adhered and
internalized by the cells (Figure S5c). The high cellular
accumulation of SPs is beneficial to induce cells apoptosis/
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necrosis more efficiently when the cells are irradiated under the
808 nm laser.
The 24 h cytotoxicity of Fe3O4 SPs and Fe3O4-R837 SPs is
investigated by standard methyl thiazolyltetrozolium (MTT)
assay. Even when incubated with 400 μg/mL Fe3O4 SPs, the 4T1
cells’ viability is still maintained at a high level (>80%), (Figure
S6a). Although R837 can induce apoptosis in cancer cells, the cell
viability is still higher (>70%). Meanwhile, the cytotoxicity of
Fe3O4-R837 SPs incubated with 293 cells (human embryo
kidney cells) shows that the SPs also have low cytotoxicity in
normal cells. The Fe3O4-R837 SPs can effectively induce the
photothermal ablation of 4T1 cells in vitro (Figure S6b). 4T1
cells are cultured with Fe3O4-R837 SPs and then irradiated with
an 808 nm laser at different power densities for different times.
The cell viability is evaluated by MTT assay. The cell viability
decreases with the increment of laser power and irradiation time.
When the laser power density reaches 2 W/cm2 and the
irradiation time is 20 min, less than 5% of the 4T1 cells survive.
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Figure 5. In vivo Fe3O4-R837 SP PTT triggers DC maturation. (a) Schematic illustration showing the design of experiments to examine the immune
responses induced by Fe3O4-R837 SP PTT. (b) IR thermal images of 4T1 tumor-bearing mice performed by an IR camera. The tumors are irradiated by
a 0.33 W/cm2 808 nm laser for 5 min. “−” and “+” in the figure mean treatment without or with irradiation, respectively. (c) Quantification of the level of
DC maturation induced by control, Fe3O4 SPs, and Fe3O4-R837 SP group with or without irradiation on 4T1-bearing mice. (d−i) The percentage of DC
maturation after different treatments. After staining with CD11c, CD80, and CD86, cells in the tumor-draining lymph nodes are assessed by flow
cytometry. Error bars are based on standard deviations of three parallel samples.

Then, 4T1 cells are stained with fluorescein diacetate (FD) and
propidium iodide (PI), as the living cells can be stained into
green and apoptotic cells into red.50 Figure S6c shows the
confocal fluorescence and bright microscope images of 4T1 cells
incubated with Fe3O4-R837 SPs after irradiation for 0, 5, 10, and
20 min. The decrease in green and increase in red along with the
irradiation time indicate the effective photothermal therapy of
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SPs in vitro. To investigate the contribution of Fe3O4-R837 SPs
during the apoptosis/necrosis process, the apoptosis process on
PBS, R837, Fe3O4 SPs, and Fe3O4-R837 SP group is monitored
by flow cytometric analysis. As shown in Figure S7a−h, the cells
in upper left, upper right, bottom left, and bottom right areas
represent necrosis, late apoptosis, viability, and early apoptosis,
respectively. In comparison with the control group, R837 cannot
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cause cell necrosis distinctly but can lead to partial cell apoptosis
(Figure S7b,f). It is consistent with the results of the MTT assay
that the cell viability of Fe3O4-R837 SPs is a little lower than that
of Fe3O4 SPs. Figure S7c,d,g,h shows that both Fe3O4 SPs and
Fe3O4-R837 SPs have low toxicity and can induce the early
apoptosis of 4T1 cells effectively after PTT (1 W/cm2, 10 min).
The apoptosis ratios are as high as 68.7 and 71.6%, respectively.
Fe3O4-R837 SP-Induced DC Maturation in Vitro. As one
of the most crucial antigen presenting cells (APCs), DCs provide
a pivotal functional link between innate and adaptive immune
responses.18 The immature DCs can engulf and process antigens
in the tissue and peripheral blood, then undergo maturation and
migrate to lymphoid nodes where they present the antigens to
̈ T cells. The mature DCs can upregulate the costimulatory
naive
molecules such as CD80 and CD86 on the APC surface to
̈ CD8+ cytotoxic T cells and naive
activate both naive ̈ CD4+ helper
18
T cells. Hence, the immunological effects of Fe3O4-R837 SPs
toward bone-marrow-derived DCs by analyzing the upregulation
of CD80 and CD86 are first investigated. A transwell system is
introduced to investigate the effect at the in vitro level. The upper
compartment contains 4T1 cells after different treatments,
whereas the lower compartment is placed with bone-marrow-
derived DCs from Balb/c mice (Figure 3a). It is found that in the
absence of R837, compared with the 4T1 cells cocultured with
Fe3O4 SPs without irradiation (Figure 3e), the released tumor-
associated antigens after PTT can notably enhance the DC
maturation from 26.3 to 39.2% (Figure 3g) and with the help of
R837, Fe3O4-R837 SPs with irradiation (89.6%, Figure 3h)
further promote in vitro DC maturation even higher than free
R837 at the same dose (75.7%, Figure 3d). Note that although
free R837 works well for DC maturation in vitro, the low
solubility in water greatly limits the applications for a living body.
Therefore, it must be loaded into amphiphilic drug carriers like
Fe3O4 SPs. Meanwhile, the DC-secreted immune related
cytokines, such as tumor necrosis factor α (TNF-α) and
interleukin 6 (IL-6), which are also indicators of DC maturation,
are measured by enzyme-linked immunosorbent assay (ELISA)
kits.18 The secretion levels of TNF-α and IL-6 from DCs are
distinctly enhanced after Fe3O4-R837 SP PTT and higher than
that in the treatment with free R837 (Figure S8). Both the flow
cytometry and ELISA data prove that the tumor-associated
antigens released by tumor cells after PTT, especially with the
help of R837-loaded SPs adjuvant, can induce DC maturation
effectively.
In Vivo Magnetic Attraction-Enhanced Photothermal
Tumor Ablation for Immune System Activation. The above
results indicate that Fe3O4-R837 SPs are an effective immune
stimulating reagent in vitro. Furthermore, the PTT of Fe3O4-
R837 SPs is considered to induce enhanced tumor-specific
immune responses in vivo. Before this, because of the
superparamagnetism of Fe3O4-R837 SPs, we test if adding an
external magnetic field can effectively enhance tumor accumu-
lation of SPs, thereby increasing the treatment efficiency of PTT.
The blood circulation kinetics and biodistribution studies are
performed on orthotopic 4T1 tumor-bearing Balb/c mice. When
the 4T1 tumors grown on Balb/c mice reach ∼200 mm3 on day
10, Fe3O4-R837 SPs are intravenously (iv) injected into the mice.
The mice are divided into two groups. The tumors of one group
are attached to a magnet for 24 h (Figure 4a,b). Blood samples
are collected at different time intervals and decomposed in aqua
regia solution to determine the Fe3+ concentration by inductively
coupled plasma optical emission (ICP-OES). As shown in Figure
4c,d, although adding the magnetic field does not significantly
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extend the blood circulation half-life (from 1.94 ± 0.16 to 2.48 ±
0.23 h), the tumor retention rate of the magnetic attraction group
increases almost 68% (from 8.8 to 14.8 of the injected dose per
gram tissue (%ID/g)) at 24 h postinjection (p.i.) administration
(Figure 4e). Compared with control and Fe3O4-R837 SP group,
T2-weighted MRI reveals a darkening effect with the magnet
attached, also suggesting the effective accumulation of SPs in the
tumor. Such high tumor retention caused by magnetic attraction
can make a contribution to improve PTT efficiency.
On the basis of in vitro experiments, the 4T1 tumor cell
residues post NIR-induced PTT with Fe3O4-R837 SPs can
enhance the level of DC maturation, higher than individual
Fe3O4-R837 SPs, or cell residues ablated by Fe3O4 SPs without
R837. The results suggest that R837-loaded SPs serve as the
adjuvant to induce the immune response from the tumor-
associated antigen in the cell residues. In further in vivo
experiments (Figure 5a), when the 4T1 tumors grown on Balb/c
mice reach ∼200 mm3 on day 10, the mice are iv injected with
PBS, Fe3O4 SPs, or Fe3O4-R837 SPs and attached to a magnet.
After 24 h on day 11, the tumors are exposed to a 0.33 W/cm2
808 nm NIR laser for 30 min. The infrared (IR) thermal images
show that the temperature increment caused by Fe3O4-R837 SP
PTT is sufficient to ablate the cells and prevent their malignant
proliferation (Figure 5b). To investigate the levels of DC
maturation by flow analysis, the draining lymph nodes of mice are
removed 3 days after PTT. Compared with the PBS without
irradiation group, the percentage of matured DCs increases from
6.58 to 31.7% after PTT with Fe3O4-R837 SPs (Figure 5d,i). For
the group of Fe3O4 SPs with PTT or Fe3O4-R837 SPs without
PTT, the percentages of DC maturation only increase to 17.6
and 22.8%, respectively (Figure 5h,f). These mean that after
Fe3O4-R837 SP PTT treatment for tumors, more DCs are
recruited to the tumor site as APCs to engulf the tumor-
associated antigens released by apoptotic tumor cells, then
transported to the nearby lymph nodes and matured with the
help of released R837 (Figure 5c).
The secretion of cytokine is another marker of immune
activation and indicates acute inflammation, a crucial mechanism
to evoke antitumor immunity.21 Hence, the changes of various
cytokines, including IL-6 (a kind of marker in the activation of
humoral immunity), TNF-α (a kind of marker in the activation of
cellular immunity), and interferon γ (IFN-γ) (a kind of marker in
the activation of innate immunity) in sera after different
treatments are analyzed by ELISA (Figure S9).21 Similarly,
even though the individual Fe3O4-R837 SPs or PTT with Fe3O4
SPs can promote the secretion of cytokine, the increment of
Fe3O4-R837 SPs PTT group is the highest, which is most
favorable for inducing antitumor immune responses. Such results
suggest that with the help of R837-loaded SPs, the tumor-
associate antigens released from tumor ablation therapy can
induce the systemic innate and adaptive immune systems
effectively in vivo.
In Vivo Fe3O4-R837 SP PTT with PD-L1 Blockade To
Eradicate Primary Tumor and Prevent Lungs/Liver
Metastasis. The main reason of a high cancer fatality rate is
that it is prone to metastasis, which is difficult to treat with
conventional treatments once they have metastasized.2,3 Hence,
we investigate if PTT-activatable immunotherapy with Fe3O4-
R837 SPs can make a contribution to treat the metastatic tumor.
As approved by FDA for clinically used immunotherapy, PD-L1
plays a pivotal role for tumor cells to evade the host’s immune
system and can be blockaded by antibodies (anti-PD-L1).31
Therefore, in the animal experiments, PD-L1 blockade therapy is
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Figure 6. In vivo Fe3O4-R837 SP PTT with PD-L1 blockade eliminates primary tumors and prevents lungs/liver metastasis. (a) Schematic illustration
showing the design of experiments. (b−e, j−m) Photographs showing the tumor nodules in the lungs/livers. (f−i, n−q) Representative lungs/liver
sections stained with H&E. The scale bar is 200 μm.
introduced to enhance the antitumor immunotherapy efficacies
generated by PTT with Fe3O4-R837 SPs of the primary tumor.
Figure 6a shows the experimental design. 4T1 cells are
orthotopically injected into the right mammary fat pads, and
they can spontaneously metastasize to lungs or liver.59,60 When
the volume of tumor reaches ∼200 mm3, the mice are i.v. injected
with Fe3O4-R837 SPs and attached to a magnet every 3 days
three times (on day 10, 13, and 16). Twenty four hours p.i.,
tumors are exposed to a 808 nm NIR laser at 0.33 W/cm2 for 30
min. After laser irradiation, anti-PD-L1 antibody are i.v. injected
into the mice at a dose of 75 μg/mouse. As shown in Figure S10,
compared with the control group, PTT with Fe3O4-R837 SPs can
eliminate the primary tumor and PD-L1 blockade therapy alone
fails to delay the tumor proliferation. On day 24, tumor-bearing
mice are all sacrificed to evaluate the extent of metastasis by
checking the tumor nodules in the lungs and liver. The
dehydrated tissues are imaged by digital camera and further
sectioned and stained with hematoxylin−eosin (H&E) staining.
For the control group, Figure 6b,f,j,n shows that there are many
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tumor nodules in lungs and large area of edge necrosis in liver.
Anti-PD-L1 blockade therapy alone shows little effect on
preventing lungs/liver metastasis (Figure 6c,g,k,o). Although
the primary tumor elimination by PTT with Fe3O4-R837 SPs can
reduce the possibility of tumor metastasis and does not cause a
local tissue necrosis, there are still some tumor nodules in lungs
and obvious metastasis around the blood vessels in liver (Figure
6d,h,l,p). Only by combining the two treatments can the tumor
metastasis be effectively eliminated (Figure 6e,i,m,q). Such
results are consistent with the previous literature in that the
individual anti-PD-L1 blockade therapy has almost no effect on
4T1 tumor cells and only once the immune system is activated by
other immunogenic therapies, the efficiency of blockade therapy
can be greatly enhanced.35 Therefore, we demonstrate that the
tumor-specific immunity activated by PTT with immune
adjuvants can enhance the effect of checkpoint blockade therapy,
leading to the elimination of primary tumor and the prevention
of lungs/liver metastasis.
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Figure 7. In vivo Fe3O4-R837 SP PTT with PD-L1 blockade eliminates primary 4T1 tumors and inhibits the growth of distant tumors. (a) Schematic
illustration showing the experiment’s design. 4T1 tumors are planted on both sides of the mice. The right tumors are designed as “primary tumors” for
PTT, and the left tumors are designed as “distant tumors” without PTT. (b, c) The primary and distant tumor volume growing trend for different groups.
(d, e) Tumor weights at the end point of different groups. Arrows represent the time of materials injection (black) and irradiation (red). “−” and “+”
mean treatment without or with irradiation, respectively. Error bars are based on standard deviations of five parallel samples.

Fe3O4-R837 SP PTT Plus PD-L1 Blockade To Inhibit the
Growth of Distant Tumors. In practical therapies, the
metastatic tumors may have already existed so the studies on
the inhibition of metastatic tumor growth is necessary. To
facilitate the demonstration and further study that synergistic
therapy can induce immune cells to specifically kill 4T1 tumor
cells and thus prevent metastasis, the larger pre-existing
simulated distant tumors are constructed for the analysis of
immune cell content.
A bilateral orthotopic 4T1 tumor model is introduced into the
experiments to prove that the synergistic therapy cannot only
prevent the tumor metastasis but also inhibit the pre-existing
metastatic tumors (Figure 7a). The first tumor is inoculated into
the right mammary fat pads as the primary tumor, and the second
tumor is inoculated into the left mammary fat pads as an artificial
mimic of distant metastatic tumor. After the SPs are systemically
injected, the primary tumors are eliminated by PTT or not. Then,
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anti-PD-L1 antibody are i.v. injected into the mice at a dose of 75
μg/mouse three times on day 11, 14, and 17. The sizes of the
primary and distant tumors in different groups are measured by a
digital caliper every other day. Figure 7b,c shows that anti-PD-L1
alone contributes minimally to the inhibition of both the primary
and the distant tumors. Fe3O4 SPs/Fe3O4-R837 SPs injection
alone shows almost no effect on the primary or distant tumor
growth. PTT with Fe3O4 SPs/Fe3O4-R837 SPs in the absence of
anti-PD-L1 three times can eradicate the primary tumor
effectively but does not inhibit the growth of the distant tumor
significantly. The distant tumor growth of Fe3O4-R837 SP PTT
group is only partially delayed because the immune system can be
successfully activated, as mentioned above. Only the combina-
tion of Fe3O4-R837 SP PTT and anti-PD-L1 treatment can
completely eradicate the primary tumor and control the growth
of the distant tumor. In addition, the average weight of mice in all
groups are monitored. As indicated in Figure S11, no significant
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Figure 8. Mechanism study of inhibiting the metastasis. (a) Schematic illustration showing the design of experiments. The distant tumors are collected
for flow analysis; the percentages of CD45+ leukocytes (CD45+PI−) (b), NK cells (CD45+CD3ε−NKp46+PI−) (c), B cells (CD45+CD3ε−B220+PI−)
(d), CD8+ T cells (CD45+CD3ε+CD8+PI−) (e), and CD4+ T cells (CD45+CD3ε+CD4+PI−) (f) in the tumors are measured. “−” and “+” in the figure
mean treatment without or with irradiation, respectively. Error bars are based on standard deviations of five parallel samples.

weight loss is observed in either the PTT treatment, anti-PD-L1
alone, or the synergistic therapy of two methods, indicating the
safety of the therapies. Meanwhile, considering the full activation
of the immune system may be harmful to the organs, serum
biochemistry assay is performed in 4T1 tumor-bearing mice on
day 24 after the synergistic therapy and the healthy mice (Table
S1). All parameters of the treatment group are consistent with
those of the healthy one, indicating that such a full activation of
the immune system by synergistic therapy can be tolerated by the
mice.
Mechanism Study of Synergistic Therapy To Inhibit
the Metastasis. Before studying the mechanism of synergistic
therapy, it must be ensured that the systemic antitumor immune
response has been activated in the body, which makes sense for
the immune cell test. Therefore, we evaluate the change of
proinflammatory cytokines, including IL-6, TNF-α, and IFN-γ,
in sera by ELISA kits within 7 days after a group of treatment
(Figure S12). Although anti-PD-L1 alone or Fe3O4-R837 SP
PTT alone can increase the secretion of proinflammatory
cytokines, the secretions induced by the synergistic therapy are
the highest, favorable for triggering antitumor immune response.
However, on day 7, after PTT treatment, all cytokine levels drop
back to the baseline levels, indicating that the inflammation
induced by different kinds of therapy is merely an acute response.
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To investigate the mechanism of synergistic antitumor
immune responses triggered by Fe3O4-R837 SP PTT plus anti-
PD-L1 therapy, the distant 4T1 tumors are excised to test the
immune cells on day 20 (Figure 8a). Immune cells, which can
also be called leukocytes, include all cells that are involved in or
associated with the immune response.27 Since CD45+ molecules
are expressed on all leukocytes, known as leukocyte common
antigen, we measure the percentage of the infiltrating leukocytes
in the distant tumors. Compared to the control group (14.94 ±
1.84%), the percentage of CD45+ leukocytes increase almost 34
and 46% in anti-PD-L1 therapy alone (20.03 ± 1.29%) and
Fe3O4-R837 SP PTT alone (21.84 ± 2.18%) (Figure 8b). For the
subspecies of leukocytes, such as NK cells, compared with the
control group (6.52 ± 0.58%), the increase in anti-PD-L1 group
is about 19% (7.80 ± 0.63%) and in Fe3O4-R837 SP PTT group,
about 47% (9.62 ± 0.98%) (Figure 8c). Similarly, for B cells,
compared with the control group (3.81 ± 0.44%), the increase in
anti-PD-L1 group is about 14% (4.36 ± 0.25%) and in Fe3O4-
R837 SP PTT group, about 58% (6.04 ± 0.15%) (Figure 8d).
Such results indicate that compared with anti-PD-L1 alone, the
percentage of NK cells and B cells is more affected by Fe3O4-
R837 SP PTT group. However, for CD8+ T cells, compared with
the control group (0.28 ± 0.06%), the increase in anti-PD-L1
group is about 239% (0.95 ± 0.06%) and in Fe3O4-R837 SP PTT
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group, about 200% (0.84 ± 0.09%) (Figure 8e). For CD4+ T
cells, compared with the control group (0.77 ± 0.18%), the
increase in anti-PD-L1 group is about 179% (2.15 ± 0.26%) and
in Fe3O4-R837 SP PTT group, about 156% (1.97 ± 0.18%)
(Figure 8f). Such results suggest that anti-PD-L1 may have more
influence on promoting the accumulation of CD8+ and CD4+ T
cells in the distant tumor sites. Although the previous data
demonstrate that Fe3O4-R837 SP PTT can activate the systemic
immune response, the increments of CD8+ and CD4+ T cells are
not significant. The reason is that Fe3O4-R837 SP PTT activates
large numbers of negatively regulatory T cells while activating
systemic immunity, resulting in a small increase in the total
number of T cells. Therefore, we suppose only through the
synergistic therapy, the activation of systemic immunity while
inhibiting negatively regulatory T cells, the treatment efficiency
can be maximized. This consideration is further proved by the
experimental data. Compared to that in the control group (14.94
± 1.84%), the total percentage of CD45+ leukocytes increases
almost 70% in the synergistic therapy group (25.47 ± 2.09%).
Similarly, the percentage of NK cells (11.24 ± 0.70%), B cells
(6.54 ± 0.19%), CD8+ T cells (1.39 ± 0.07%), and CD4+ T cells
(2.76 ± 0.30%) significantly increase in comparison with not
only the control group but also the anti-PD-L1 alone or Fe3O4-
R837 SP PTT alone.
■
CONCLUSIONS
In summary, immunogenic Fe3O4-R837 SPs have been designed
for the effective treatment of metastatic cancer by combining
PTT and PD-L1 checkpoint blockade therapy. With the help of
an external magnetic field, Fe3O4-R837 SPs can directly destroy
the tumors upon NIR irradiation, activating the immune system
by inducing DCs’ maturation and secretion of cytokines. Further
in combination with the PD-L1 checkpoint blockade therapy,
Fe3O4-R837 SPs cannot only eradicate the primary tumors
directly exposed to PTT but also prevent the lungs/liver
metastasis and inhibit the pre-existing distant tumors left after
PTT. Our studies show the great potential of integrating Fe3O4-
based PTT with checkpoint blockade therapy to realize a
remarkable synergistic therapeutic outcome and broaden the
application of cancer immunotherapy in vivo.
■
EXPERIMENTAL SECTION
Materials. Iron acetylacetonate (Fe(acac)3, 99.9+%), benzyl ether
(99%), oleic acid (OA, 90%), oleyamine (OLA, 70%), 1,2-
hexadecanediol (90%), Imiquimod (R837), and fluorescein diacetate
(FD) were purchased from Sigma-Aldrich. Poly(ethylene glycol)-block-
poly(lactic-co-glycolic acid) copolymer (mPEG-PLGA, 50:50 w/w, Mw
∼ 2000:5000 Da) was purchased from Daigang Biomaterial Co., Ltd.
Propidium iodide (PI) was purchased from Invitrogen. Dulbecco’s
modified Eagle’s medium with fetal bovine serum and high glucose was
purchased from Gibco. Chloroform and dimethyl sulfoxide (DMSO)
were of analytical grade. Deionized water and absolute ethanol were
used as received. Antiprogrammed death ligand 1 used in vivo was
obtained from BioXCell (α-PD-L1, clone: 10F.9G2, catalog no.
BE0101). Anti-CD11c (N418), anti-CD80 (16-10A1), anti-CD86
(GL-1), anti-CD16/32 (93), anti-CD45 (30-F11), anti-CD3ε (145-
2C11), anti-CD4 (RM4-5), anti-CD8a (53-6.7), anti-NKp46 (29A1.4),
and anti-B220 (RA3-6B2) antibodies were purchased from Biolegend.
Mouse interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and
interferon γ (IFN-γ) ELISA Kit were purchased from Dakewe biotech.
Preparation of OA-Stabilized Fe3O4 NPs. OA-stabilized Fe3O4
NPs were prepared by thermal decomposition route following the
previous literature.54 Briefly, 2 mmol Fe(acac)3, 6 mmol OLA, 6 mmol
OA, 5 mmol 1,2-hexadecanediol, and 20 mL of benzyl ether were mixed.
The mixture was then cycled between vacuum and nitrogen three times
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and kept at 80 °C for 20 min under vacuum and vigorous stirring.
Afterward, the mixture was raised to 200 °C with the rate of 20 °C/min,
kept for 30 min at this temperature under nitrogen atmosphere, and
then refluxed under 265 °C for another 30 min. Then, the reaction
solution was cooled to room temperature naturally. With the help of a
magnet, Fe3O4 NPs were extracted and washed by adding hexane and
ethanol three times and finally dissolved in chloroform.
Preparation of mPEG-PLGA-Capped Fe3O4 SPs. mPEG-PLGA-
capped Fe3O4 SPs were synthesized by an emulsification method.51−53
OA-stabilized Fe3O4 NPs and mPEG-PLGA were dissolved in
chloroform with the concentration of 20 and 200 mg/mL, respectively.
Under a nitrogen atmosphere and mechanical stirring at room
temperature, 0.5 mL of OA-stabilized Fe3O4 NPs, 50 μL of mPEG-
PLGA, and 0.5 mL of DMSO were injected dropwise into 10 mL of
deionized water. After the ultrasonic treatment, the emulsion was further
stirred to evaporate organic solvent for 4 h. The SPs were washed by
centrifugation (6000 rpm, 10 min) three times and dialyzed (molecular
weight cut-off (MWCO): 7 kDa) against deionized water for 24 h, which
were defined as Fe3O4 SPs.
Preparation of mPEG-PLGA-Capped Fe3O4-R837 SPs. The
synthesis of mPEG-PLGA-capped Fe3O4-R837 SPs was similar to that of
mPEG-PLGA-capped Fe3O4 SPs. R837 was dissolved in DMSO with
the concentration of 4 mg/mL. Under a nitrogen atmosphere and
mechanical stirring at room temperature, 0.5 mL of OA-stabilized Fe3O4
NPs, 50 μL of mPEG-PLGA, and 0.5 mL of R837 were injected
dropwise into 10 mL of deionized water. After the ultrasonic treatment,
the emulsion was further stirred to evaporate the organic solvent for 4 h.
The products were washed by centrifugation (6000 rpm, 10 min) three
times and dialyzed (MWCO: 7 kDa) against deionized water for 24 h,
which were defined as Fe3O4-R837 SPs.
R837 Loading Standard Curve and Releasing Experiments.
R837 solution was prepared in DMSO with the concentration of 1 mg/
mL and then diluted into 2.5, 5, 7.5, and 10 μg/mL standard solution by
water. The absorbance of R837 solution with different concentrations at
322 nm was measured by UV−vis absorption spectra. According to the
absorbance, a standard curve was obtained. The encapsulation efficiency
and loading efficiency of Fe3O4-R837 SPs were calculated from the UV−
vis absorption spectra against the standard curve. For R837 release, the
SPs were irradiated under NIR laser and centrifuged to get the
supernatant. The released concentration and percentage can also be
calculated from the absorbance at 322 nm according to the standard
curve.
Cellular Uptake and Efflux. 4T1 cells seeded in six-well plates (2 ×
105 cells/well) were incubated with Fe3O4 SPs/Fe3O4-R837 SPs at a
concentration of 150 μg/mL for 2, 4, 12, and 24 h. Then, cells were
collected and washed three times with PBS, counted with a
hemocytometer, and lysed with aqua regia (HCl/HNO3 = 3:1) to test
the Fe3+ concentration with ICP-OES. The uptake level was expressed as
the amount of Fe3O4 SPs (nmol) per 104 cells.
To investigate the efflux of SPs, 4T1 cells were incubated with Fe3O4
SPs/Fe3O4-R837 SPs at a concentration of 150 μg/mL for 4 h. Then, the
culture medium was discarded and the 4T1 cells were washed with PBS
three times. Two milliliters of fresh culture medium was added to each
well, and cells were further cultured for 2, 4, 12, and 24 h. Thereafter, the
cells were collected and washed three times with PBS, counted with a
hemocytometer, and lysed with aqua regia to test the Fe3+ concentration
with ICP-OES. Results were expressed as the percent of the difference
value of SPs being retained to the 4 h cellular uptake amount.
The bright field images of internalization were also observed under a
fluorescence microscope. Fe3O4-R837 SPs were incubated with 4T1
cells for 2, 4, 12, and 24 h and washed with PBS three times, fixed with
4% paraformaldehyde, and observed under the fluorescence microscope.
Cytotoxicity Assay in Vitro. 4T1 cells were seeded in 96-well plates
at a density of 4 × 103 cells per well. Different concentrations of Fe3O4
SPs/Fe3O4-R837 SPs were incubated with cells for 24 h. The cell
viability was detected by standard MTT assay according to the
manufacturer’s instructions. The experiment was repeated five times.
Apoptosis Analysis in Vitro. 4T1 cells seeded in six-well plates (2
× 105 cells/well) were treated with Fe3O4-R837 SPs at a concentration
of 150 μg/mL for 4 h and then irradiated with 808 nm NIR laser at
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different power densities for different times. The cell viability was
evaluated by MTT assay. For fluorescence imaging, the treated cells
were fixed with 4% paraformaldehyde and stained with 1 μg/mL PI and
FD to visualize living cells (green fluorescence) and cell apoptosis (red
fluorescence). For flow cytometric analysis, treated cells were harvested,
washed with ice-cold PBS three times, stained with Alexa Fluor 488-
Annexin V and PI in the dark for 20 min at room temperature, and then
analyzed by flow cytometry.
In Vitro DC Stimulation Transwell Experiments. DCs were
isolated from the bone marrow of ∼8 week-old BALB/c mice. The mode
of transwell is the cocultivation system, and the pore diameter is 0.4 μm.
First, 1 × 105 4T1 cells were incubated with R837, Fe3O4 SPs, or Fe3O4-
R837 SPs in the upper compartment and irradiated with 808 nm NIR
laser or not. The residues of 4T1 cells were added into the 5 × 105 DC
culture in the lower compartment using the transwell system. After
various treatments, DCs stained with anti-CD11c, anti-CD80, and anti-
CD86 antibody were analyzed by flow cytometry.
The proinflammatory cytokines in DC medium suspensions,
including IL-6 and TNF-α, were determined by ELISA kits following
standard protocols.
Magnetic Attraction-Enhanced Pharmacokinetics, Biodistri-
bution, and MRI. The 4T1 tumor-bearing mice were intravenously
(i.v.) administrated with Fe3O4-R837 SPs at a dose of 6 mg/kg and
attached to the magnets. Animals were sacrificed (n = 3) at the indicated
time points to collect the same dose of blood from each mouse. Then,
the blood was immediately centrifuged at 3000 rpm for 5 min to harvest
plasma samples and dissolved in aqua regia to analyze the total amount
of Fe3+ with ICP-OES. Major organs and tumors were also collected
after 24 h, and the concentration of Fe3+ was measured with ICP-OES.
Before collecting the organs, the mice need cardiac perfusion with
physiological saline and paraformaldehyde to remove the effects of
blood in organs. The T2-weighted MRI images were acquired after 24 h
p.i. using a 1.5 T human clinical scanner. The organs’ and tumors’
retention rates were calculated by the formula
ID%/g = (drug content in organs
/total amount of drug injections)/organ weight
Apoptosis, DC Stimulation, and Cytokines Release in Vivo.
Female BALB/c mice (6−8 weeks) were divided into groups randomly.
The first 4T1 tumor model was planted by an orthotopic injection of 1 ×
106 4T1 cells into the right mammary fat pads of the mice, and tumors
were allowed to grow until ∼200 mm3 before experiments. Then, the
mice were i.v. injected with Fe3O4 SPs/Fe3O4-R837 SPs at a dose of 6
mg/kg and attached to the magnets. According to the previous
literature,21,27 the laser irradiation after 24 h of injection of NPs is an
ideal condition that has been optimized and verified. In addition,
according to the blood circulation kinetics of Fe3O4-R837 SPs in vivo
and 24 h tumor retention rate, it can be seen that the high drug
accumulation in the tumor area after 24 h is beneficial to maximize the
efficiency of photothermal therapy. Therefore, 24 h after i.v. injection is
chosen as the time point for further experiments. After 24 h, mice were
anesthetized and tumors were irradiated with an 808 nm NIR laser at
0.33 W/cm2 for 30 min. Three days later, lymph nodes were excised and
analyzed by flow cytometry. At the same time, blood was collected and
the serum IL-6, TNF-α, and IFN-γ were determined by using ELISA kits
to evaluate the acute inflammation evoked by the treatment.
Antitumor and Anti-metastatic Activity on Orthotopic 4T1
Model. Tumor-bearing mice were randomly divided into four groups (n
= 5): control, anti-PD-L1, Fe3O4-R837 SP PTT, and Fe3O4-R837 SP
PTT plus anti-PD-L1. The mice were i.v. injected with the materials and
attached to the magnets every 3 days three times. After 24 h of each
injection, mice were anesthetized and irradiated with NIR laser, followed
by i.v. injection with PD-L1 antibody at a dose of 75 μg/mouse. Tumor
volumes were monitored and recorded over 24 days. The volume of
tumor was calculated by the equation
1
V = L × D2
2 Hongchen Sun: 0000-0002-5572-508X
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where L and D (mm) represented the tumor lengths of long and short
axes, respectively. At the end of the experiments, mice were sacrificed
and tumors were excised and weighed. Lungs and livers were harvested,
observed for the tumor nodules, or sectioned and stained with H&E.
Abscopal Effect on Bilateral 4T1 Model. BALB/c mice were
injected with 1 × 106 4T1 cells into the right mammary fat pads (primary
tumor) and 2 × 105 4T1 cells into the left mammary fat pads (distant
tumor). When the primary tumor reached ∼200 mm3, mice were
randomly divided into eight groups (n = 5): control, anti-PD-L1, Fe3O4
SPs, Fe3O4 SPs PTT, Fe3O4-R837 SPs, Fe3O4-R837 SPs PTT, Fe3O4-
R837 SPs plus anti-PD-L1, and Fe3O4-R837 SPs PTT plus anti-PD-L1.
The mice were i.v. injected with the materials and attached to the
magnets every 3 days for a total of three injections. After 24 h of each
injection, mice were anesthetized and irradiated with NIR laser, followed
by i.v. injection with PD-L1 antibody at a dose of 75 μg/mouse. The
primary and distant tumor volumes and body weights were monitored
over 24 days. After 24 days, all mice were sacrificed and both the primary
and distant tumors were excised and weighed. The distant tumors were
harvested, treated with 1 mg/mL collagenase I for 1 h, and ground with
the end of a syringe rubber. Cells were filtered, incubated in red blood
cell lysis buffer on ice for 5 min, and washed with PBS three times. To
reduce nonspecific binding to fragment crystallizable receptors (FcRs),
the single-cell suspension was incubated with anti-CD16/32.27 Cells
were further stained with fluorochrome-conjugated antibodies: anti-
CD45, anti-CD3ε, anti-CD4, anti-CD8a, anti-NKp46, anti-B220, and PI
and analyzed by flow cytometry. Blood was also collected, and the serum
IL-6, TNF-α, and IFN-γ were determined by using ELISA kits to
evaluate the acute inflammation evoked by the treatment.
Characterization. Transmission electron microscopy (TEM)
images were measured by a Hitachi H-800 electron microscope.
Scanning electron microscopy (SEM) images were acquired with a
SU8020 Hitachi Company scanning electron microscope. Dynamic
light scattering (DLS) measurements were measured by Zetasizer
NanoZS, Malvern Instruments. UV−vis absorption spectra were
performed with Shimadzu 3600 UV−vis−IR spectrophotometer.
Fourier-transform infrared (FTIR) spectra were obtained by Bruker
IFS66 V instrument. T2 relaxation time was measured by a Bruker
AVANCE III 500 NMR spectroscope. An 808 nm diode laser (LEO
photonics Co. Ltd.) with tunable output power densities was employed
to study the photothermal effect. Fluorescent images of 4T1 cells were
obtained by an Olympus IX71 inverted fluorescence microscope. The
Fe3+ concentration was assessed by PerkinElmer Optima 3300DV
inductively coupled plasma optical emission (ICP-OES) measurements.
Infrared thermal images were acquired by a Fluke infrared (IR) thermal
camera. Flow cytometric analysis was implemented on the BD
Biosciences FACSCalibur flow cytometer. T2-weighted MRI images
were performed by GE Signa 1.5 T unit, General Electric, Milwaukee,
WI. The photographs of mice and lungs/liver were taken by
macroscopic digital imaging workstation for histopathology.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsami.8b05876.
Additional structural characterizations, the encapsulation
and loading efficiency, cellular uptake and efflux, the
release of cytokine, the trend body weight of mice and
serum biochemistry (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*E-mail: daqizhang@yeah.net (D.Z.).
*E-mail: hou63@163.com (Y.H.).
*E-mail: hao_zhang@jlu.edu.cn. Fax: +86 431 85193423 (H.Z.).
ORCID
Yi Liu: 0000-0003-0548-6073
DOI: 10.1021/acsami.8b05876
ACS Appl. Mater. Interfaces 2018, 10, 20342−20355
ACS Applied Materials & Interfaces
Hao Zhang: 0000-0002-2373-1100
Bai Yang: 0000-0002-3873-075X
Author Contributions
H.Z. proposed and supervised the project. H.Z., R.G., X.Z., Y.L.,
H.S., D.Z., Y.H., and B.Y. designed and performed the
experiments and co-wrote the manuscript. C.L., B.L., J.L., and
W.W. participated in most experiments. All authors discussed the
results and commented on the manuscript.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by NSFC (51603084 and 51425303),
JLU Science and Technology Innovative Research Team
2017TD-06, and the Special Project from MOST of China.
■
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