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Multifunctional Nanoparticle Loaded Injectable
Thermoresponsive Hydrogel as NIR Controlled Release
Platform for Local Photothermal Immunotherapy to Prevent
Breast Cancer Postoperative Recurrence and Metastases
Yan Peng Jia, Kun Shi, Fan Yang, Jin Feng Liao, Ru Xia Han, Li Ping Yuan, Ying Hao,
Meng Pan, Yao Xiao, Zhi Yong Qian,* and Xia Wei Wei*
For breast cancer patients who have undergone breast-conserving surgery,
effective treatments to prevent local recurrences and metastases is very
essential. Here, a local injectable therapeutic platform based on a thermo-
sensitive PLEL hydrogel with near-infrared (NIR)-stimulated drug release is
developed to achieve synergistic photothermal immunotherapy for preven-
tion of breast cancer postoperative relapse. Self-assembled multifunctional
nanoparticles (RIC NPs) are composed of three therapeutic components
including indocyanine green, a photothermal agent; resiquimod (R848), a
TLR-7/8 agonist; and CPG ODNs, a TLR-9 agonist. RIC NPs are physically
incorporated into the thermosensitive PLEL hydrogel. The RIC NPs encap-
sulated PLEL hydrogel (RIC NPs@PLEL) is then locally injected into the
tumor resection cavity for local photothermal therapy to ablate residue tumor
tissues and produce tumor-associated antigens. At the same time, NIR also
triggers the release of immune components CPG ODNs and R848 from ther-
moresponsive hydrogels PLEL. The released immune components, together
with tumor-associated antigens, work as an in situ cancer vaccine for postsur-
gical immunotherapy by inducing effective and sustained antitumor immune
effect. Overall, this work suggests that photothermal immunotherapy based
on local hydrogel delivery system has great potential as a promising tool for
the postsurgical management of breast cancer to prevent recurrences and
metastases.
Y. P. Jia, Dr. K. Shi, F. Yang, R. X. Han, L. P. Yuan, Dr. Y. Hao, M. Pan,
Y. Xiao, Prof. Z. Y. Qian, Prof. X. W. Wei
State Key Laboratory of Biotherapy and Cancer Center
National Clinical Research Center for Geriatrics
West China Hospital
Sichuan University
Chengdu 610041, P. R. China
E-mail: zhiyongqian@scu.edu.cn; xiaweiwei@scu.edu.cn
Dr. J. F. Liao
State Key Laboratory of Oral Diseases
West China School of Stomatology
Sichuan University
Chengdu 610041, P. R. China
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adfm.202001059.
DOI: 10.1002/adfm.202001059
Adv. Funct. Mater. 2020, 2001059 2001059 (1 of 12)
1. Introduction
Breast cancer has been the leading cancer
diagnosed in female globally.[1,2] Surgical
resection, especially breast-conserving sur-
gery (BCS) has been a preferred therapy
method for primary breast cancer with its
advantage of better cosmetic results.[3,4]
Unfortunately, the residue tumor cells
around the resection cavity usually lead
to quick recurrence and metastasis, which
could result in patient death.[5–7] Worse
still, for postoperative patients, severe
local immunosuppression associated
with wound healing and inflammation
further increases the incidence of relapse
and metastases by promoting cancer cell
invasion and suppressing the activity of
antitumor immune cells.[8,9] Although
therapies including chemotherapy and
radiotherapy are often provided after sur-
gery in clinics, they bear disadvantages
such as undesirable side effects, treat-
ment resistance, or radiation-induced
damage.[10,11] Therefore, developing effec-
tive and safe strategies to address relapse
and metastases for patients undergone the
BCS are urgently needed.
Recently, immunotherapy has shown great potential to
prevent tumor regression and metastasis by utilizing the
immune system of the body to kill the tumor cells.[12–14] Alter-
natively, photothermal therapy (PPT) represents a noninva-
sive therapy in which heat generated by photothermal agents
upon near-infrared (NIR) light irradiation is applied to kill
cancer cells. Moreover, the tumor-associated antigens gener-
ated in situ after photothermal tumor ablation could present
immune vaccine-like functions.[15–17] Considering the immu-
nosuppressive microenvironment after surgery, strategies
based on synergistic photothermal immunotherapy thereby
have great potential to control postsurgery local relapse and
metastasis.[18]
Although photothermal immunotherapy shows such advan-
tages, overactive immune system due to the systemic adminis-
tration of therapeutic agents could also lead to either adverse
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Scheme 1. Schematic illustration of a) the preparation process of RIC NPs@PLEL hydrogels and b) photothermal-immune therapy to prevent
postsurgery tumor reoccurrence.
effects or limited administration dosage.[19] In this regard, local
drug delivery represents a desirable administration method for
postsurgical therapy.[20–23] Injectable biodegradable hydrogels
are among the best candidates for local drug release with the
following merits: higher dosage at tumor site than the systemic
approach; minimized adverse effects to normal tissues; a sus-
tained and even controlled drug release; moreover, multi­ ple
drugs that act synergistically could be incorporated into the
same hydrogel together for combination therapy.[21,24–27] In
particular, injectable intelligent hydrogels have been widely
explored as locally controlled drug delivery system with the
unique property of sol–gel phase transition when triggered by
stimuli such as pH, temperature, and light.[28–31] Among them,
temperature-responsive hydrogels represent an ideal candi-
date for sustained and controlled local drug release. They are
sol at room temperature, which allow them to load agents and
be injected into the tumor site, then they transformed into gel
upon injection at the physiological temperature. As a result,
loaded drugs can be released from the hydrogel in a sustained
way.[30,32] To be noted, some temperature-responsive hydrogels
can undergo further transformation such as gel to sol again
when triggered by a higher temperature, making it possible to
release drug in a controlled pattern.[32] Recently, except to be
applied in photothermal therapy, NIR responsive photothermal
agents have also been reported as effective heat generators for
controlled drug release.[28,30] Therefore, for temperature-sensi-
tive hydrogels, developing NIR as switches to achieve locally
controlled drug release has great potential in tumor therapy
application.
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As mentioned above, with all the advantages of photo-
thermal immunotherapy, NIR controlled drug release as well
as localized hydrogel delivery system, exploring the feasibility
of designing an all-in-one powerful hydrogel therapeutic plat-
form for effective prevention of postresection recurrence and
metastasis is very necessary. However, to the best of our knowl-
edge, there are almost no previous reports proposed it. Herein,
in this study, we developed a localized photothermal immuno-
therapy platform for postresection prevention of breast cancer
recurrences and metastases as shown in Scheme 1. An inject-
able thermosensitive PDLLA-PEG-PDLLA (PLEL) hydrogel
incorporating three-component nanoparticles RIC NPs was
fabricated as a localized NIR-controlled drug delivery system.
PDLLA-PEG-PDLLA was prepared as described before.[32] RIC
NPs is formed by self-assembly technology with indocyanine
green (ICG), resiquimod (R848), and CPG ODNs. ICG is an
FDA-approved excellent NIR fluorescent dye for PTT with supe-
rior photothermal effect. R848 is a small molecular agonist of
TLR-7/8, CPG ODNs is TLR-9 agonist, they are both used to
activate dendritic cells (DCs) via multiple TLRs and stimulate
proinflammatory cytokines release. However, the three func-
tional components have either poor aqueous stability or easy
degradation issues. Here our hydrogel local delivery system
solved these problems. Upon local injection, RIC NPs@PLEL
systems changed from liquid state at room temperature to the
gel (above 37 °C). Upon further NIR laser irradiation, ICG gen-
erated high temperature (above 45 °C) promote further trans-
formation of the gel to sol, thus triggering on-demand release
of R848 and CPG ODNs, together with the tumor-associated
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Figure 1. A) TEM image and B) particle size distribution of RIC NPs. The RIC NPs were negatively stained with 1% phosphotungstic acid for TEM
imaging. C) UV–vis absorption spectra of RIC NPs. D) Near infrared imaging of blank PLEL hydrogel, free ICG, and ICG@PLEL hydrogel under 808 nm
laser irradiation. E) Temperature changes of (D) under NIR laser irradiation.

antigens generated in situ after PTT of residue tumor, they
play vaccine-like functions by inducing subsequent antitumor
immune response.
2. Results and Discussion
2.1. Preparation and Characterization of RIC NP Loaded
PLEL Hydrogels
RIC NPs are prepared by self-assembly method of three mole-
cules ICG, CPG, and R848. ICG is an NIR dye with strong photo­
thermal effect, CPG and R848 work as important immune
stimulators by activating the TLRs of DCs. To be noted, the
three components we chose are of good biocompatibility, which
make it a promising candidate for further clinical application.
The successful preparation of RIC NPs is demonstrated by
the transmission electron microscope image which showed a
well-defined morphology and uniform size of about 180 nm of
the RIC NPs (Figure 1A). The mean hydrodynamic size of RIC
NPs measured is about 280 nm as shown in Figure 1B. The
UV–vis absorption spectrum of RIC NPs revealed the charac-
teristic absorption peaks of ICG at about 780 nm, CPG at about
260 nm, and R848 at about 340 nm, further suggesting the RIC
NPs were successfully prepared (Figure 1C). The PLEL hydrogel
is synthesized and characterized as described in our previous
studies.[32] The RIC NPs is incorporated into PLEL hydrogels
by physically mixing them with certain weight ratios. As we
intended to apply RIC NP loaded PLEL hydrogels for photo-
thermal enhanced immunotherapy to prevent the postoperative
breast cancer recurrence, the excellent photothermal effect
is expected to achieve photothermal ablation of postsurgical
residue tumor tissues and thermal-induced immunotherapy.
Therefore, we investigated the photothermal effect of free ICG
and ICG loaded PLEL hydrogels to confirm the photothermal
effect of ICG is not weakened when incorporating into the
hydrogel, blank PLEL hydrogel was used as control. As shown
in Figure 1D,E, under 808 nm laser irradiation (1 W cm−2), the
temperature of the hydrogel increased to almost 60 °C within
1 min, and finally reached about 70 °C over time. These results
verified no obvious difference of photothermal ability between
free ICG and ICG@PLEL hydrogels, implying that being
encapsulated into hydrogel did not affect the photothermal
effect of ICG.
Next, the viscoelastic behavior of RIC NP loaded PLEL
hydrogels and blank PLEL hydrogels were evaluated. The
results confirmed a similar temperature response (10–60 °C)
between them (Figure 2A,B). For both RIC NP loaded PLEL
hydrogels and blank PLEL hydrogels, the storage modulus
(G′) was higher than loss modulus (G″) at a physical tempera-
ture of 37 °C, indicating the gel status of the hydrogel. When
the temperature reached approximately 45 °C, the hydrogel
remained a higher loss modulus (G″) than storage modulus
(G′), which revealed that the hydrogel returned to the flowing
sol state. These findings demonstrated our hydrogel system
exhibited optimal photothermal effect and thermosensitive
property. We next observed the appearance changes of RIC NP
loaded PLEL hydrogels at different temperatures. As presented
in Figure 2C, the hydrogel has good liquidity at 4 °C. With
the increase of temperature, the hydrogel underwent sol–gel

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Figure 2. A,B) Temperature dependence of storage (G′) and loss modulus (G″) for blank PLEL and RIC NPs@PLEL as a function of temperature.
C) Reversible sol–gel transformation photos of PLEL hydrogels and RIC NPs@PLEL hydrogels.

transformation at 37 °C, and swelling behavior was observed.
When the temperature came to 55 °C, hydrogel shrinkage and
gel–sol transformation occurred. To be noted, the whole trans-
formation process from 4 to 55 °C was reversible. This result
confirmed the thermally responsive property of RIC NP loaded
PLEL hydrogels. Furthermore, Figure 2C also implied that laser
irradiation could directly control the rheological status of RIC
NP loaded PLEL hydrogel by photothermal effect. Therefore, it
is concluded that the RIC NPs@PLEL hydrogels is a promising
candidate for topical photothermal cancer therapy and it may
serve as a local drug delivery system capable of NIR-triggered
drug release.
2.2. In Vitro and In Vivo Biocompatibility Assay
Although PLEL has been applied with good biocompatibility
before, it is still necessary to evaluate its biosafety as a synthe-
sized material. As for the three components in RIC NPs, ICG
as a photothermal agent should undergo safety test to con-
firm its safety, while for R848 and CPG ODNs, there is usu-
ally no need since they could function with small dose and
they work by stimulating immune cells such as DCs. The cyto-
toxicity of ICG@PLEL hydrogel was thus investigated via the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
(MTT) assay. As shown in Figure 3A,B, 4T1 and RAW246.7 cell

Figure 3. In vitro cell viability of A) DC 2.4 cells and B) RAW 246.7 cells incubated with ICG@PLEL hydrogels at various concentrations. C–E) Blood
biochemistry data of mice in the PLEL group and ICG@PLEL group (ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline
phosphatase; TP: total protein; BUN: blood urea nitrogen).

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lines were respectively cocultured with the nanocomplexes at
various concentrations for 24 h. The assays showed that ICG@
PLEL exhibited little cytotoxicity in the given concentration
range with the cell viability all higher than 80%. For in vivo
application, we estimated the biosafety of our PLEL hydrogel
system. 14 days after injection of PLEL hydrogel or ICG@PLEL.
The blood of mice in each group was collected and subjected to
a blood chemical analysis. There were no detectable changes in
the concentration of the biomarkers of liver enzymes including
alanine aminotransferase and aspartate aminotransferase,
kidney function (blood urea nitrogen) in the blood across treat-
ment groups (Figure 3C–E). Besides, the injection site was also
collected for histopathological analysis and no inflammation
was observed (Figure S2, Supporting Information).
2.3. Extended and Controlled Release Behavior of RIC NP
Loaded PLEL Hydrogels
In recent years, smart drug release behavior triggered by multi-
stimuli such as NIR laser, pH, temperature, and enzymes have
showed enormous advantages in drug delivery application.[32]
Since our PLEL hydrogel has thermal sensitive property, we
then studied the NIR laser triggered drug release behaviors
of PLEL hydrogel to see if it could serve as an ideal scaffold
for NIR controlled drug release. As well known, hydrogel has
the advantage of sustained drug release property. First, we
confirmed that our hydrogel PLEL could extend the release of
its payloads in vivo. As a model drug, ICG was used as free
ICG solution or ICG@PLEL hydrogel and both were injected
locally to the side of healthy female BALB/c mice. Then, the
mice were monitored at different time points through fluores-
cence in vivo imaging system (IVIS) imaging (Figure 4A), and
Figure 4. A) In vivo extended release of ICG from ICG@PLEL hydrogel recorded by IVIS and B) quantitative analysis. C) In vivo controlled release of
CPG from PLEL hydrogel recorded by IVIS and D) quantitative analysis.
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the results were further quantified as shown in Figure 4B. For
free ICG, the fluorescence intensity weakened rapidly within
24 h revealing that the free ICG diffuses away or even degraded
very rapidly. In contrast, fluorescence intensity of ICG encapsu-
lated in PLEL hydrogel remained strong all over the monitoring
period. These findings proved that the PLEL hydrogel could
efficiently extend the local release of its payloads. Such drug
sustainable release property of PLEL hydrogel is promising in
keeping relatively stable and long-term drug concentrations in
vivo for perdurable therapy.
For NIR controlled release test, BALB/c mice bearing 4T1
tumor were injected with cy-3 labeled CPG@PLEL intratumorally
and divided into two groups of CPG@PLEL hydrogel and CPG@
PLEL hydrogel/NIR. For the CPG@PLEL hydrogel/NIR group,
NIR irradiation was performed 1 h before IVIS imaging. Mice
in both groups were then monitored at different time points by
fluorescence IVIS imaging (Figure 4C,D). It is found that the flu-
orescence signal of CPG@PLEL group without laser treatment
still retained intense fluorescence signal for 48 h. In contrast, for
the CPG@PLEL hydrogel/NIR group, the fluorescence intensity
decreased a lot compared with CPG@PLEL without NIR. Similar
results were observed when the laser treatment was repeated for
another two cycles, suggesting that the release rate of CPG could
be regulated intelligently by switching on or off of NIR laser.
Thus, these data demonstrated that PLEL hydrogel can serve as a
stable, biodegradable depot with the release of anticancer drugs
controlled by the stimuli of NIR irradiation.
2.4. DC Maturation Assay In Vitro
DCs, the most important antigen-presenting cells (APCs), play
crucial roles in antitumor immunity. Immature DCs (iDCs)
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collect antigens from the surrounding fluid and then process
them into peptides during migration to nearby draining lymph
nodes, where they present the peptide major histocompatibility
complex (MHC) to T cell receptors (TCR) for T cell activation.
The maturation of DCs could be confirmed by co-stimulatory
molecules CD80 and CD86, the typical markers indicating
maturation of DCs. After bone marrow-derived dendritic cells
(BMDCs) were treated in various formulations for 24 h, the
percentage of DCs maturation (CD11c+CD80+CD86+) was
then analyzed by flow cytometry (Figure 5A–E). It turned out
that DCs maturation in the RIC NPs@PLEL/NIR group was up
to 32%, the highest among all groups. In contrast, the same
amount of RIC NPs@PLEL without NIR induced a little less
DC maturation of 25%. To be noted, ICG@PLEL/NIR group
alone also promoted DC maturation three times higher than
the blank group suggesting the photothermal effect did trigger
immune response. Since matured DC secretes immune-related
cytokines such as tumor necrosis factor α (TNF-α), we meas-
ured the concentration of cytokines in the medium supernatant
by ELISA kit. It was found that the secretion levels of TNF-α
and IL-6 totally enhanced after RIC NPs@PLEL/NIR treatment
(Figure 5F–G). These results implied that tumor-associated
antigens liberated from PPT-triggered tumor cell lysis, together
with TLR agonists R848 and CPG ODNs, they induced DC
maturation in vitro efficiently.
2.5. In Vivo Inhibition of Breast Cancer Recurrence
by Photothermal Immunotherapy
The superior photothermal effect, relatively precise NIR con-
trolled local drug release, as well as strong immune effect of
RIC NPs@PLEL hydrogel motivated us to further explore its
potential application in anticancer photothermal immuno-
therapy. Considering hydrogel’s great advantages in local drug
delivery especially for postresection model, we expected that
our RIC@PLEL hydrogel could prevent local recurrence and
metastases of breast cancer after surgery. The design of our in
vivo experiment was shown in Figure 6A. Female BALB/c mice
with orthotopic 4T1-Luc breast cancer was used. Half an hour
after resection of primary tumor as shown in Figure S1 in the
Supporting Information, the hydrogel-based therapeutic formu-
lations were injected into the surgical bed. For NIR laser irra-
diation groups, the temperature of injection site rose to about
60 °C rapidly under 808 nm laser irradiation, it is high enough
to kill the residue tumor tissues (Figure 6B). This result also
demonstrated the excellent photothermal effect of ICG@PLEL
hydrogel for in vivo application. The growth of the postresec-
tion residue tumors in each group was monitored every other
day for one mouth. As shown in Figure 6C, the survival rates
in all the therapeutic groups are encouraging 100%, except 80%
in ICG@PLEL/NIR group, suggesting that PTT alone could
destroy the tumor residues and induce antitumor immune
response to some extent. However, when talking about recur-
rence rate, we found that only in RIC NPs@PLEL/NIR group,
80% of the mice were completely cured without recurrence
(Figure 6D). This could be attributed to the antitumor immune
response since NIR laser irradiation triggering tumor antigen
generation as well as R848 and CPG ODN release, which
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promoting intratumoral infiltration and proliferation of effector
CTLs. Although in all the rest groups, tumor recurrence hap-
pened on the whole mice, there are still differences in the delay
of recurrence. The quickest recurrence occurred in the PLEL
group. Figure 6E analyzed the tumor volume among time after
therapy, the results further confirmed the most effective pre-
vention of recurrence in RIC NPs@PLEL/NIR group. However,
due to the sensitivity problems of luciferase during biolumines-
cence images, the IVIS results in Figure 7A existed deviations
from the actual results. Even so, the IVIS results still revealed
the recurrence rate varies among different groups. In the PLEL
group, recurrence happened at an earlier time among all the
mice when compared with other therapeutic groups. On the
whole, RIC NPs@PLEL/NIR group presented the best result.
Besides, representative mice photos of tumor site were also
recorded to show the tumor recurrence in Figure 7B. Hema-
toxylin and eosin (H&E) staining of tissues including heart,
liver, spleen, and kidney in various groups indicated that there
is no visible tissue damage (Figure 7C). In addition, except for
the modest decrease after surgery, no obvious body weight
loss was observed across all groups during the whole therapy
period (Figure 7D), implying the high biocompatibility of our
hydrogel system. Lung metastasis is another malignant clin-
ical problem for breast cancer patients undergone operation.
Evaluation of lung metastasis is also a very important factor
for therapy effectiveness assessments. As shown in Figure 8A,
the photos of lung tissues clearly revealed that for lung tissue
in the untreated group severe metastases occurred with the
most lung nodules found. For the RIC@PLEL hydrogels/NIR
group, the lung tissue kept health with no metastasis nodules
found. HE analysis of lung tissues further confirmed these
results. Besides, the lung weight and the number of pulmonary
nodules among all the groups were also analyzed. Compared
with the untreated group, the lung weight and nodules both
decreased in different therapy groups. (Figure 8B,C). Taken
together, these results proved that during the in vivo therapy
process, the RIC NPs@PLEL/NIR hydrogels mediated photo-
thermal ablation, as well as photothermally triggered release of
RIC NPs in resection site, effectively elicits systemic immunity
response against both tumors recurrence and metastasis.
To understand the fundamental immune mechanisms behind
the tumor inhibition effects of RIC@PLEL hydrogels/NIR, we
determined the DCs and T cells proliferation and activation by
flow cytometry. For in vivo DC maturation analysis, 4T1-bearing
mice were intratumorally administrated with various PLEL-based
formulations, then sacrificed 3 days later to harvest the draining
LNs for flow cytometry analysis of DCs maturation. As shown
in Figure 9A,C, RIC NPs@PLEL/NIR remarkably activated 24%
DC maturation while for RIC NPs@PLEL without NIR irradia-
tion the percentage was a little lower. In comparison, bare NIR
irradiation in ICG@PLEL group also promoted DC maturation
slightly, which could be attributed to NIR-triggered liberation
of tumor antigens. Furthermore, the serum levels of TNF-α
also showed significant up-regulation in mice after RIC NPs@
PLEL/NIR treatments (Figure 9E). Overall, the in vivo findings
verified that our RIC NPs@PLEL/NIR could efficiently induce
DC maturation and may act as an in situ vaccine to initiate
the immune responses. As well known, CD8+T cells, called
cytotoxic T lymphocytes (CTL), could directly recognize and
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Figure 5. A) Expression levels of surface molecules (CD80 and CD86) on BMDCs after various treatments in transwell system. B) Schematic illustra-
tion of transwell system for DCs/4T1 tumor cells coculture. C–E) Quantification of expression levels of CD80 and CD86 on the surface of BMDCs.
F,G) TNF-α and IL-6 secreted in the BMDC cell culture supernatant after different treatments. **p < 0.01 and ***p < 0.001.

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Figure 6. A) Schematic diagram of the postsurgery breast cancer model and postoperative therapy of the tumor. B) IR thermal images of postsurgery
4T1 residue tumor-bearing mice with 808 nm laser irradiation. C) Survival curves of mice after various treatments. D) Recurrence-free rate of mice after
various treatments. E) Tumor volume curves after various treatments.

Figure 7. A) In vivo bioluminescence images to evaluate the recurrence of 4T1-Luc tumor after various treatments. B) Typical photographs of mice to
evaluate the recurrence of 4T1-Luc tumor after various treatments. C) H&E staining of mice tissues after various treatments. D) Body weight curves
after various treatments. The scale bar indicated 100 µm.

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Figure 8. A) Typical photographs and H&E staining of 4T1 metastatic foci for mice after different treatments. The scale bar indicated 100 µm. B) Lung
weight at the end of different treatments. C) Lung metastatic nodules at the end of different treatments. **p < 0.01 and ***p < 0.001.
kill cancer cells. On the other hand, CD4+T cells play impor-
tant roles in the regulation of adaptive immunities. There-
fore, we collected spleens of mice in all groups after different
treatments to test the CD8+ (CD3+CD4−CD8+) and CD4+
(CD3+CD4+CD8−) T cells by flow cytometry (Figure 9B,D). The
percentage of CD8+T cells in RIC NPs@PLEL/NIR group was
dramatically increased by about twofold compared to the con-
trol. This result confirmed that the combination of RIC NPs@
PLEL vaccination and laser irradiation significantly activated
the antitumor immune response. In contrast, no significant
enhancement of CD4+T cell was observed in therapy groups.
To be noted, with the thermal sensitive property, our PLEL
hydrogel is injectable at room temperature. Once injected into
the surgical bed where the temperature is 37 °C, the hydrogel
transformed into gel condition with no mobility as shown in
Figure S2 in the Supporting Information, which is advanta-
geous for local administration and extended release of thera-
peutic components. The efficient inhibition of local breast
cancer recurrence by the RIC NPs@PLEL hydrogel was due
to the synergetic effects of photothermal therapy and immu-
notherapy. First, the in situ injection of hydrogel system can
effectively eliminate residual tumor tissues by photothermal
ablation. Second, PPT itself can initiate moderate immunolog-
ical responses by triggering tumor associated antigens release
from tumor lysis and some “danger signals” such as HSP
70.[33] Third, RIC NPs, containing typically used immunoadju-
vants R848 and CPG ODNs, were released from hydrogel upon
NIR irradiation to further enhance immune response. Fourth,
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unlike free CpG ODNs or R848, nanotechnology has further
improved the in vivo stability and DCS uptake efficiency of
these adjuvants. Collectively, the basic mechanism of hydrogel
system’s therapeutic effect is the activation of DCs, and sub-
sequent exposure of the activated DCs to tumor antigens in
vivo so that a tumor-specific T cell response is induced. RIC
NPs@PLEL/NIR hydrogel could thus be considered an in situ
autologous cancer vaccine that utilizes whole tumor cells as the
source of tumor antigens.
It is reported that in the process of photothermal ablation
of cancer, the increased temperature, as an exogenous cellular
stress, killed tumor cell mostly through necrosis. Necrotic cells
released tumor associated antigens as well as damage asso-
ciated molecular pattern molecules (DAMPs). DAMPs are
inflammatory mediators including ATP, nucleic acids and intra-
cellular proteins such as heat shock proteins (Hsp70) and high-
mobility group box 1 (HMGB-1). [33] DAMPs working as danger
signals have attracted much attention in recent years in cancer
research. These inflammation molecules with immunostimu-
latory properties are normally existed within live cells. Once
secreted by damaged tumor cells, they are found to improve
adaptive antitumor immunity by promoting maturation of
antigen-presenting cells and production of cytokines. [34] These
DAMPs are reported to bind specific pattern-recognition recep-
tors (PRRs) such as Toll-like receptors (TLRs) and trigger their
activation. [35] As well known, PRRs are important costimula-
tory molecules expressed by natural immune cells and play an
important role in regulating innate immune response. Notably,
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Figure 9. Representative flow cytometry plots showing A) matured DCs in lymph node and B) cytotoxic CD3 + CD8 + T cells and CD3 + CD4 + T cells
in spleens after various treatments. C,D) Quantitation of A) the matured DCs and B) CD8 + T cells and CD4+T cells in the spleen. E) TNF-α levels
detected by ELISA assay in the serum of mice after different treatments. *p < 0.05 and **p < 0.01.

the immune effect of photothermal ablation during tumor
treatment deserves more exploration.
3. Conclusion
In summary, we developed a local injectable therapeutic platform
by loading multifunctional nanoparticles RIC NPs (composed
of photothermal agent ICG, immunoadjuvants R848, and CPG
ODNs) into thermoresponsive hydrogel PLEL. The hybrid RIC
NPs@PLEL hydrogel system exhibited excellent biocompat-
ibility both in vitro and in vivo. In the orthotopic breast cancer
model undergone breast-conserving surgery, the RIC NPs@
PLEL system successfully ablated residue tumors through NIR-
induced photothermal ablation. Furthermore, the photothermal
ablation, together with the loaded immunoadjuvant, triggered a
strong immune response to inhibit lung metastasis and induced
a strong antitumor immunity to prevent locoregional tumor
recurrence. Taken together, our strategy demonstrated that the
simple fabrication procedure of RIC NPs@PLEL hydrogel,
as well as its ability to simultaneously induce a photothermal
enhanced immune response, holds great potential for clinical
prevention of postsurgical recurrence of breast cancer.
4. Experimental Section
Materials: Poly (ethylene glycol) (PEG, Mn = 1500, 1000, and 2000,
respectively), stannous octoate (Sn (Oct)2, 95%), ε-caprolactone
(ε-CL), and MTT were obtained from Sigma-Aldrich (USA). Penicillin–
streptomycin, DMEM medium, RMPI 1640 medium, and fetal bovine
serum (FBS) were all purchased from Gibco (USA). CpG ODNs
(5′-TCC ATG ACG TTC CTG ATG C-3′) and cy3-labeled CpG ODNs were
provided by Sangon Biotech (Shanghai, China). R848 was purchased
from InvivoGen (France). ICG was purchased from melonepharma
(Dalian, China). TNF-α, IL-6 ELISA kits were purchased from Invivogen.
Fluorochrome-labeled antibodies were all purchased from eBioscience
(USA). Recombinant mouse GM-CSF and IL-4 were purchased from
Peprotech. All other chemical reagents used in this study were of
analytical grade and used without further purification.

Adv. Funct. Mater. 2020, 2001059 2001059 (10 of 12) © 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
Preparation and Characterization of RIC NPs@PLEL Hydrogel:
RIC nanoparticles were prepared by the self-assembly method of
CpG ODNs, R848, and ICG solution. In brief, a certain amount of
CpG ODNs, R848, and ICG was weighted and dissolved by deionized
(DI) water, and then mixed and stirred for half an hour to form RIC
nanoparticles. The dynamic nanoparticle size and zeta potential of
resultant RIC NPs were determined by Nano ZS (Malvern Instruments,
UK), and the UV–vis absorption spectra were measured by the UV–vis
absorption spectrometry (PE, USA). Transmission electron microscope
(TEM) (JEM-2100F, JEOL Ltd., Japan) was also used to confirm the visual
morphology of RIC NPs. PLEL thermal-sensitive hydrogel was prepared
and characterized by the group as described previously. [32] RIC NPs
incorporated PLEL hydrogel (RIC NPs@PLEL) was prepared by simple
physical mixing with a certain ratio. The rheology of PLEL hydrogel and
RIC NPs@PLEL hydrogel were measured by using an HAAKE Rheostress
6000 rheometer (Thermo Scientific, USA). The storage modulus (G′) and
loss modulus (G″) were measured as functions of temperature between
10 to 60 °C. In addition, the appearance changes of RIC NP loaded PLEL
hydrogels at different temperatures were recorded by taking photos.
In Vitro Photothermal Effect: For in vitro evaluation of photothermal
property of ICG, free ICG, PLEL hydrogel, and ICG@PLEL hydrogel
were added into EP tubes and irradiated with an 808 nm NIR laser at a
power density of 1.0 W cm−2 for 5 min. This process was simultaneously
recorded by a Fluke Ti32 Infrared (IR) thermal camera (Fluke Ti32, USA)
In Vitro Cytotoxicity Test: Mouse breast cell line 4T1 and luciferase
labeled 4T1 were originally from ATCC and cultured in complete RMPI
1640 (supplemented with 10% fetal bovine serum, 1% penicillin, and
1% streptomycin). Macrophage cell line RAW246.7 and DC2.4 were
maintained in complete Dulbecco’s Modified Eagle Medium (DMEM).
Cells were all incubated in a humidified incubator at 37 °C with 5% CO2.
MTT assays were used to detect cellular viability of ICG@PLEL with
RAW246.7 and DC2.4 cell lines. Briefly, RAW246.7 and DC2.4 cells were
seeded in transwell plates and incubated for 24 h. Then, ICG@PLEL of
various concentrations were added and incubated for another 24 h. MTT
solution was used to determine cell viabilities.
Systemic Toxicity Evaluation: Female wide-type BALB/c (6–8 weeks
old) were purchased from Beijing HFK Bioscience Co., Ltd. They were
housed in an SPF environment with a temperature of 25 ± 2 °C and a
relative humidity of 50–60% with 12 h light–dark cycles. Free access to
food and water was allowed. The animal experiments were all approved
by the Animal Care and Use Committee of Sichuan University (Chengdu,
China) and were carried out according to the approved guidelines. All
mice were treated humanely. Female BALB/c mice (3 mice per group)
were injected with the PLEL hydrogel and ICG@PLEL hydrogel via sc
injection. Two weeks later, serum samples were collected to evaluate the
liver/kidney functions.
In Vivo Extended and Controlled Drug Release: To evaluate the in
vivo sustained release behaviors of PLEL hydrogel, ICG incorporated
hydrogels were injected subcutaneously into nontumor-bearing mice. As
control, free ICG solutions were used. Fluorescence was then acquired
at different time point using the IVIS Spectrum (Perkin Elmer).
To further monitor in vivo NIR controlled release of CpG ODNs,
BALB/c mice were subcutaneously inoculated with 1 × 106 4T1 cells.
When the tumor size reached 100 mm3, tumor-bearing mice were
divided into two groups of cy3-CPG ODNs@PLEL and cy3-CPG
ODNs@PLEL/NIR. For mice in cy3-CPG ODNs@PLEL group, 50 µL
of cy3-CPG ODNs@PLEL were intratumorally injected, and for mice
in the cy3-CPG ODNs@PLEL/NIR group, cy3-CPG ODNs/ICG@PLEL
were intratumorally injected. One hour later, mice in the NIR group were
irradiated upon 808 nm NIR laser (1.50 W cm−2, 5 min). Afterward, mice
in the two groups were imaged by the IVIS Spectrum (Perkin Elmer). At
24 and 48 h after injection, the same process was repeated.
Generation of Immature BMDCs: BMDCs were isolated from 6 week
old C57BL/6 mice according to the established protocol.[13] Briefly, mice
were sacrificed and the femurs and tibiae were collected to harvest cells
from bone marrow. The obtained cells were resuspended in red blood
cell lysis buffer (Beyotime) to deplete erythrocytes and then counted
and resuspended to culture dish with RPMI-1640 complete medium
Adv. Funct. Mater. 2020, 2001059 2001059 (11 of 12)
www.afm-journal.de
supplemented with 10 ng mL−1 mouse granulocyte macrophage colony
stimulating factor (GM-CSF) and 1 ng mL−1 mouse IL-4. Culture media
were refreshed every 2 days. After 7 days incubation, the nonadherent
and loosely adherent cells were harvested as dendritic cells. DC
purity was evaluated by flow cytometry (ACEA Biosciences, Inc.). The
percentage of CD11c positive cells in the obtained BMDC was more than
70%.
BMDC Maturation Assay: For in vitro DC stimulation experiments,
a transwell system was used in which the upper chamber was saline,
PLEL hydrogel, ICG@PLEL/NIR, RIC NPs@PLEL, and RIC NPs@PLEL/
NIR, respectively, while the lower chamber was seeded with immature
DCs. After various treatments for 24 h, DCs in all groups were collected,
washed, and stained with antibodies of PE antimouse CD11c, APC
antimouse CD86 and FITC antimouse CD80, and then matured DCs
were analyzed by flow cytometry (ACEA Biosciences, Inc.). Meanwhile,
the culture medium in the transwell system was also collected for
measurement of proinflammatory cytokines TNF-α and IL-6 with
cytokine ELISA assay.
In Vivo DC Maturation Assay: For in vivo DC maturation experiment,
when 4T1 tumors inoculated on BALB/c mice reached 100 mm3, saline,
PLEL hydrogel, ICG@PLEL/NIR, RIC NPs@PLEL, and RIC NPs@PLEL/
NIR were injected intratumorally. For NIR treated group, mice were
irradiated with 808 nm NIR laser at 1.5 W cm−2 for 5 min. Three days
later, mice were then sacrificed to harvest draining lymph nodes to
examine DC maturation by flow cytometry. For cytokine detection, serum
samples in all groups were collected from mice at 72 h after various
treatments. TNF-α in the serum were analyzed with ELISA kits.
In Vivo Inhibition of Breast Cancer Recurrence: Female BALB/c mice
were inoculated orthotopically with 1 × 105 4T1-Luc cells in the second
mammary fat pad. When tumor volumes reached around 100 mm3,
mice were randomly divided into five groups. Before surgery, mice
were anesthetized by intraperitoneal injection 4% chloralhydrate.
Then, local resection was performed with a layer of surrounding skin
left to construct the local postoperative recurrence model, and then
wound was sutured with medical clips and threads. Half an hour later,
mice in each group were injected with the following formulations in
the resultant cavity: PLEL hydrogel, ICG@PLEL hydrogel/NIR, ICG-
R848-CPG NPs@PLEL hydrogel, and ICG-R848-CPG NPs@PLEL
hydrogel/NIR. Untreated group was used as control. For the groups
subjected to NIR treatment, 808 nm laser irradiation (1.5 W cm−2,
5 min) was performed 24 h postsurgery. Thermal imaging was
captured using an infrared thermal imaging camera (Ti27, Fluke, USA)
simultaneously. The NIR laser irradiation was repeatedly performed on
days 3 and 5 after surgery. Body weight and tumor recurrence were
monitored every 2 days.
In Vivo Bioluminescence Imaging: After treatments, mice were
inspected by bioluminescent IVIS imaging weekly for local tumor
recurrence detection. In brief, substrate of Luc2 called d-luciferin was
intraperitoneally injected (150 mg kg−1). 10 min later, the mice were
anesthetized under 2% isoflurane and imaged by the IVIS (Perkin
Elmer).
At the end of the experiment, mice were sacrificed and lung and other
tissues of each group were harvested. Furthermore, the lung weight and
pulmonary nodule of each group were counted and recorded. Then, the
samples were fixed for H&E histological analysis.
Analysis of Immunocell In Vivo: To analyze the immune effect induced
by these treatments, when 4T1 tumors inoculated on BALB/c mice
reached 100 mm3, saline, PLEL hydrogel, ICG@PLEL/NIR, RIC NPs@
PLEL, and RIC NPs@PLEL/NIR were injected intratumorally. For NIR
treated group, mice were irradiated with 808 nm NIR laser at 1.5 W cm−2
for 5 min. Six days later, the same formulations were administrated
repeatedly. Mice were sacrificed on day 12 and spleens were harvested
and prepared as single cell suspensions, then stained using antimouse
antibodies of anti-CD3-FITC, anti-CD8a-APC, and anti-CD4-PerCP, and
analyzed by flow cytometry.
Statistical Analysis: All data in the study were indicated as mean result ±
standard deviation (SD). The statistically difference analysis was
evaluated by one-way ANOVA using Origin 2016 software.
© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
The authors greatly appreciate the help of Dr. Shanling Wang from
Analytical & Testing Center in Sichuan University for TEM analysis
of the nanoparticles. This work was financially supported by the
National Natural Science Foundation of China (Nos. 31930067,
31525009, 31800797, and 31771096), the National Key Research
and Development Program of China (Nos. 2017YFC1103502 and
2016YFA0201402), the China Postdoctoral Science Foundation funded
project (No. 2018M631094), the Postdoctoral Innovation Talents
Support Program (No. BX20180207), and 1·3·5 project for disciplines of
excellence, West China Hospital, Sichuan University (ZYGD18002).
Conflict of Interest
The authors declare no conflict of interest.
Keywords
cancer recurrence, controlled drug release, immunotherapy,
photothermal, thermal-responsive hydrogels
Received: February 4, 2020
Revised: March 25, 2020
Published online:
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