Reinforcing the Induction of Immunogenic Cell Death Via Artificial Engineered Cascade Bioreactor-Enhanced Chemo-Immunotherapy for Optimizing Cancer Immunotherapy

Article abstract of DOI:10.1002/smll.202101897

Traditional chemo-immunotherapy can elicit T cell immune response by inducing immunogenic cell death (ICD), however, insufficient ICD limits the lasting antitumor immunotherapeutic efficacy. Herein, tadpole–ovoid manganese-doped hollow mesoporous silica coated gold nanoparticles (Au@HMnMSNs) as biodegradable catalytic cascade nanoreactors are constructed to generate intratumoral high-toxic hydroxyl radicals combined with DOX and Aspirin (ASA) for enhancing the induction of ICD and maturation of dendritic cells (DCs). The released Mn²⁺ can catalyze endogenous H₂O₂ to hydroxyl radicals, while internal gold nanoparticles mimetic glucose oxidase (GOx) converted glucose into H₂O₂ to accelerate the generation of hydroxyl radicals. On the other hand, tadpole oval-structured Au@HMnMSNs can avoid the inactivation of gold nanoparticles due to strong protein adsorption. The introduction of ASA is to recruit DCs and cytotoxic T lymphocytes (CTLs) to tumor sites and restrain the intratumoral infiltration of immunosuppressive cells by decreasing the expression of prostaglandin E2 (PGE₂). Accordingly, this work presents a novel insight to introduce GOx-like catalytic cascade ICD nano-inducer into antitumor immunotherapy for synergistic tumor therapy.

Full text of DOI:10.1002/smll.202101897

ReseaRch aRticle
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Reinforcing the Induction of Immunogenic Cell Death Via
Artificial Engineered Cascade Bioreactor-Enhanced Chemo-
Immunotherapy for Optimizing Cancer Immunotherapy
Kai Sun, Jinzhong Hu, Xiangyu Meng, Yunfeng Lei, Xuezhong Zhang, Zhuoxuan Lu,
Liming Zhang,* and Zhifei Wang*
immune attack. Cancer immune cycle is the
Traditional chemo-immunotherapy can elicit T cell immune response by
inducing immunogenic cell death (ICD), however, insufficient ICD limits
the lasting antitumor immunotherapeutic efficacy. Herein, tadpole–ovoid
manganese-doped hollow mesoporous silica coated gold nanoparticles
process of cancer immunotherapy through
a series of steps to initiate antitumor
immune response. Recently, more research
has begun to focus on remodulating the
immunosuppression tumor microenviron-
ment (ITM) to achieve better antitumor
(Au@HMnMSNs) as biodegradable catalytic cascade nanoreactors are
constructed to generate intratumoral high-toxic hydroxyl radicals combined
with DOX and Aspirin (ASA) for enhancing the induction of ICD and maturation
[3,4]
immunotherapy effects.
Immunogenic cell death (ICD) plays
a crucial role in activating the immune
2+
of dendritic cells (DCs). The released Mn can catalyze endogenous H O to
2
2
[5,6]
response.
The dying tumor cells under-
hydroxyl radicals, while internal gold nanoparticles mimetic glucose oxidase
GOx) converted glucose into H O to accelerate the generation of hydroxyl
going ICD can release tumor-associated
antigens (TAAs) and damage-associated
molecular patterns (DAMPs), including the
surface exposure of calreticulin (CRT) and
high mobility group box 1 (HMGB1) release
to potentiate the recruitment and antigen
presentation of antigen presenting cells
(
2
2
radicals. On the other hand, tadpole oval-structured Au@HMnMSNs can
avoid the inactivation of gold nanoparticles due to strong protein adsorption.
The introduction of ASA is to recruit DCs and cytotoxic T lymphocytes (CTLs)
to tumor sites and restrain the intratumoral infiltration of immunosuppressive
cells by decreasing the expression of prostaglandin E2 (PGE ). Accordingly, this
2
(
APCs) and boost the infiltration of cytotoxic
work presents a novel insight to introduce GOx-like catalytic cascade ICD nano-
inducer into antitumor immunotherapy for synergistic tumor therapy.
[7–9]
T lymphocytes (CTLs) into tumor tissue.
The process could induce the maturation of
dendritic cells (DCs), which are one of the
most important APCs, to initiate innate and
adaptive immunity.[10] Therefore, the phenotypic and functional
maturation of DCs determine the therapeutic effect of antitumor
immune response. However, inherent troubles with DCs and the
low level of ICD induction seriously block antitumor efficiency in
1. Introduction
Cancer immunotherapy, which can activate and boost the innate
immune system to generate antitumor immunity to combat tumor
growth and metastasis, is considered to be a promising treat-
ment strategy for the future.[1,2] However, due to the low immune
response rate, the efficacy of immunotherapy is much lower than
expectations. Moreover, tumor microenvironment (TME) can pro-
mote virtue of immune evasion and immunosuppression to avoid
[11–13]
immunosuppressive microenvironment.
Cyclooxygenase-2 (COX-2) has been confirmed to be a key
factor in the immune suppression by upregulating prosta-
[
14,15]
glandin E2 (PGE ) in tumor cells.
High COX-2 expres-
2
sion could cause tumor cells to hinder the migration of DCs,
strengthen the barrier between T cells and tumor cells, and
[
16–18]
K. Sun, J. Hu, X. Meng, Y. Lei, X. Zhang, Prof. Z. Wang
School of Chemistry and Chemical Engineering
Southeast University
Nanjing 211189, People’s Republic of China
E-mail: zfwang@seu.edu.cn
recruit more immunosuppressive cells.
Thus, inhibition of
COX-2 can reverse tumor immunosuppression and effectively
improve the efficiency of tumor cells elimination. As an FDA-
approved anti-inflammatory drug, Aspirin (acetylsalicylic acid,
[
19]
ASA) is one of the COX inhibitors. In previous studies, it
was found that the introduction of ASA would increase tumor
Prof. Z. Lu, Prof. L. Zhang
Key Laboratory of Tropical Translational Medicine of Ministry of
Education & Hainan Provincial Key Laboratory of Tropical Medicine
Hainan Medical University
[
20–22]
infiltrating lymphocytes in chemo-preventive treatment.
Nevertheless, the down-regulation of COX-2/PGE expression
2
Haikou 571199, People’s Republic of China
E-mail: lmzhang1980@163.com
of ASA has not been studied in the process of DCs maturation
induced by ICD to stimulate potential anti-tumor immunity.
Although traditional chemo-drugs kill tumor cells by apop-
tosis or necrosis, and induce ICD to stimulate the maturation
of DCs, the level of ICD produced by chemo-drugs alone is
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/smll.202101897.
DOI: 10.1002/smll.202101897
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Scheme 1. Schematic diagram of PEGylated Au@HMnMSNs ICD nanoinducers for eliciting potent antitumor immunotherapeutic efficacy.
inadequate, which impedes the activation of cytotoxic T lym-
phocytes and infiltration into the tumor tissue.
for achieving GOx-like catalysis enhanced chemodynamic
therapy (CDT), which combined with chemotherapy to induce
sufficient ICD for release of TAA, and then stimulate potent
systemic antitumor immunity. On the other hand, the introduc-
tion of ASA was to remodel ITM by restraining the expression
[23–25]
Herein, we first constructed a tadpole–ovoid manganese-
doped hollow mesoporous silica coated gold nanoparticle (Au@
HMnMSN) to reinforce the induction of ICD and maturation
of DCs after loading doxorubicin (DOX) and ASA (Scheme 1).
Au@HMnMSNs, as cascade nanoreactors, could be degraded
by consuming antioxidant glutathione (GSH) to release Mn2
for Fenton-like reaction to generate highly toxic hydroxyl radi-
of PGE , which could promote the recruitment and maturation
2
of DCs into TME and decrease the barrier between CTLs and
tumor cells. Synergistic GOx-like catalysis-enhanced CDT com-
bined with chemotherapy could provide a promising strategy
to amplify antitumor immunotherapy effects via inducing suf-
ficient ICD.
+
•
[26–28]
cals ( OH) in TME. In a previous study,
firmed that gold nanoparticles can harness glucose oxidase
GOx) like catalytic activity to decompose glucose into gluconic
acid and H O . Interestingly, Au@HMnMSNs could convert
it has been con-
(
2
2
endogenous H O2 into O2 to improve the GOx-like catalytic
2
2
. Results and Discussion
activity of gold nanoparticles. Meanwhile, tadpole oval-struc-
tured Au@HMnMSNs could avoid the inactivation of gold
nanoparticles due to strong protein adsorption in biological
fluids and promote further GOx-like catalysis.[ As a result,
the regeneration of H O could dedicate to Fenton-like reaction
2.1. Synthesis and Characterization
29]
Tadpole–ovoid Au@HMnMSNs with tumor microenviron-
ment responsive degradation behavior were fabricated via a
2
2
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Figure 1. TEM images of A) Au@MSNs and B) Au@HMnMSNs. Inset: A photograph of Au@HMnMSNs in the PBS soulution. C) SEM images of
Au@HMnMSNs. D) XRD pattern and E,F) XPS spectrum of Au@HMnMSNs. G) UV–vis spectra of PEGylated Au@HMnMSNs before and after encap-
sulation with DOX and ASA. H) The change of TGA curves. I) EDS elemental mapping of Si, O, Mn, and Au of Au@HMnMSNs.
two-step procedure. As shown in Figure 1A, Au@MSNs with
single gold core-mesoporous silica shell were prepared using a
facile one-pot method with an average size of 105 nm. Then,
Au@MSNs doped Mn into mesoporous silicon shell under
high-temperature hydrothermal condition to form hollow
mesoporous Au@HMnMSNs, indicating that Au@HMnMSNs
with a uniform size of 120 ± 5 nm containing ≈18 nm gold core
were successfully prepared (Figure 1B,C). The wide-angle X-ray
diffraction pattern (XRD) of the Au@HMnMSN showed several
sharp peaks (38.2°, 44.4°, 64.7°, 77.7°, and 81.7°) and broadened
peaks (21.3°, 33.2°, and 56.2°), which were respectively assigned
to cubic metal of gold (JCPDS 04–0784) and amorphous state of
Mn (SiO ) (JCPDS 33–0904) (Figure 1D). Furthermore, the ele-
immunogenicity of the host’s immune system.[30] After surface
PEGylation of Au@HMnMSNs through amidation reaction,
the hydrodynamic sizes were measured from 115 to 170 nm
and the zeta potential had changed (Figure S3, Supporting
Information). The prepared PEG–Au@HMnMSNs was also
verified by the Fourier transform infrared (FT-IR) spectroscopy
and thermogravimetric analysis (TGA) curves (Figure S4, Sup-
porting Information; Figure 1H). With the continuous surface
modification of the nanoparticles, the absorption values of gold
nanoparticle at 535 nm were decreased under the same concen-
tration (Figure S5, Supporting Information), which manifested
the formation of PEG–Au@HMnMSNs. Subsequently, the N2
adsorption–desorption isotherm and pore size distribution of
PEG–Au@HMnMSNs were as shown in Figure S6, Supporting
x
4 y
ment mapping and energy dispersive X-ray spectroscopy (EDS)
of Au@HMnMSN were conducted to confirm that Si, O, Mn,
and Au were expectedly distributed within the Au@HMnMSN,
which manifested the successful synthesis of Au@HMnMSN
Information and the surface area and average pore size were
266.63 m2
g
−1
and 2.8 nm, respectively, exhibiting the pres-
ence of mesoporous structure. ASA, which could inhibit the
expression of COX-2 as a representative drug for New Uses of
Old Drugs, and chemotherapy drug DOX were co-loaded into
PEG–Au@HMnMSNs by strong π–π stacking interaction and
physical absorption. As shown in Figure 1G, DOX/ASA-loaded
PEG-Au@HMnMSNs showed two strong absorption peaks at
256 and 278 nm compared with PEG–Au@HMnMSNs, which
were attributed to the presence of DOX and ASA, respectively.
In addition, the drug loading capacities of ASA and DOX
were computed to be 7.6% and 9.5% via physical absorption
and strong π–π stacking interaction according to TGA and
the UV–vis standard curve (Figure 1H; Figure S8, Supporting
Information). DOX/ASA@PEG-Au@HMnMSNs also exhibited
(Figure 1I; Figure S1, Supporting Information). The results also
showed that the mass ratios of Au and Mn were nearly 15.13%
and 13.24%. The X-ray photoelectron spectroscopy (XPS) indi-
cated that the main peak of Mn 2p3/2 was divided into three
characteristic peaks at 641.08, 642.38, and 643.68 eV, each of
2
+
3+
which respectively corresponded to the valence of Mn , Mn ,
4
+
and Mn (Figure 1E,F). Besides, Mn-doped hollow mesoporous
silica nanoparticles (HMnMSNs) were successfully prepared
(Figure S2, Supporting Information).
The PEGylated Au@HMnMSNs (PEG–Au@HMnMSNs)
could increasingly accumulate into tumor tissue by enhanced
permeability and retention (EPR) effect and overcome the
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Figure 2. Biodegradation of PEG–Au@HMnMSNs in SBF solution. A) Schematic description of ASA/DOX-release from PEG–Au@HMnMSNs in the
biodegradation process (inset: chemical composition). B) TEM images of PEG–Au@HMnMSNs after degradation and C) corresponding accumulated
releasing profiles of Mn element from PEG–Au@HMnMSNs under different conditions. n = 3, mean ± SD; **P < 0.01.
excellent blood compatibility by hemolysis assay, which could
remain stable in blood circulation (Figure S9, Supporting
Information).
with 8 mꢀ of GSH and pH 5.0 was significantly higher than
−
1
the others up to 42.7, 72.2, 92.5, and 101.2 µg mL over time,
and gold nanoparticles were completely exposed to the SBF
solution for enhancing endogenous H O concentration
2
2
through the GOx-like catalytic oxidation of glucose to H O
2
2
2
.2. Tumor Microenvironment Responsive
and gluconic acid. In addition, the dialysis method was used to
investigate the drug release of PEG–Au@HMnMSNs. Because
acidic and reducing conditions could accelerate the degrada-
tion of PEG–Au@HMnMSNs to release drugs, the difference
between accumulative release of DOX and ASA was not great.
Besides, the release of DOX and ASA within 24 h was up to
79.85% and 84.85% under PBS solution with 5 mꢀ of GSH and
pH 5.0, which was signally higher than other condition groups
(Figure S10, Supporting Information).
Biodegradation Behavior
Since MnO bonds were sensitive to acidic and reducing
tumor microenvironment, PEGAu@HMnMSNs with manga-
nese embedded into the silicon shell could accelerate the pro-
cess of biodegradation.[
31,32]
To imitate acidic endo/lysosomal
(pH 5.0–6.5/4.5–5.0) and overexpressed GSH of tumor micro-
[33]
environment, simulated body fluid (SBF) solutions with dif-
ferent GSH concentrations (5 and 8 mꢀ) and pH values (6.8
and 5.0) were used to investigate the degradation of PEG–Au@
HMnMSNs at various time intervals (Figure 2). The mor-
phology of PEG–Au@HMnMSNs was visually observed by
TEM, and the amounts of released Mn were determined by
ICP-MS analysis. The morphology of PEG–Au@HMnMSNs
stepwise cracked with the increase of time, GSH concentra-
tion, and acidity via TEM. The release rate of Mn in the group
•
2.3. GSH Depletion-Enhanced OH Generation
High expression of GSH in tumor cells could weaken the
efficacy of CDT-based treatments. In view of the sensitive
property of PEG–Au@HMnMSNs to acidic and reducing envi-
ronment, GSH consumption detection assay was investigated
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Figure 3. A) Degradation of DTNB by remanent GSH after treating with different concentration of PEG–Au@HMnMSNs in the PBS solutions. B) MB
•
consumption by H O plus GSH-treated PEG–Au@HMnMSNs in the NaHCO /CO buffer solutions. C) ESR spectra of demonstrating OH generation of
2
2
3
2
PEG–Au@HMnMSNs. D) Schematic graph describing the PEG–Au@HMnMSNs as GOx-like mimetics. E) UV–vis spectra of PEG–Au@HMnMSNs after
incubation. Inset: Corresponding actual photographs. F) MB degradation by H O plus GSH-treated PEG–Au@HMnMSNs after addition of glucose.
2
2
using an indicator 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB),
which could react with GSH to GSSH and TNB. As shown
in Figure 3A, GSH could react with different concentration
of PEG–Au@HMnMSNs (50, 100, 200, and 400 µg mL ). As
the concentration of PEG–Au@HMnMSNs increased, more
GSH could be consumed. As a result, remanent GSH reacting
with DTNB could produce less TNB with an absorption peak at
2.4. GOx-Like Catalytic Performance Assessments
of PEG–Au@HMnMSNs
−
1
Before degradation, PEG–Au@HMnMSNs could protect the
surface of gold nanoparticles from passivation and impact fur-
ther catalysis in TME. A horseradish peroxidase (HRP)-based
colorimetric measurement was utilized to investigate the GOx-
like catalytic oxidation of glucose to H O (Figure 3D,E). The
4
12 nm in acidic PBS solution, revealing the consumption of
2
2
2+
GSH by PEG–Au@HMnMSNs to release Mn for Fenton-like
catalysis.
appearance of characteristic green color of oxABTS (maximum
absorbance at 405 nm) verified the enzymatic activity of Au
nanozyme. As shown in Figure 3C, the characteristic signals
Another major feature was the overexpression of H O in
2
2
2+
•
tumor cells, and released Mn could convert endogenous
of OH (1:2:2:1) were remarkably enlarged. In view of overex-
•
−
H O into highly toxic OH with the assistance of HCO /CO
pression of in tumor cells, when 20 and 40 mꢀ concentrations
of glucose along with low concentration of H O were respec-
2
2
3
2
[
34,35]
physiological buffer.
As shown in Figure 3B, methylene
blue (MB) was degraded by OH as an indicator, which could
be used to detect the production of OH. As the concentration
2
2
•
tively added, the MB was degraded more, indicating that more
hydroxyl radicals were generated due to the increased level of
H O by catalytic oxidation of glucose (Figure 3F).
•
of GSH increased from 0 to 5 mꢀ, the MB was degraded in
the presence of GSH-response PEG–Au@HMnMSNs and
H O . However, the degradation of MB was reduced at 10 mꢀ
2
2
2
2
of GSH, indicating GSH could restrict the CDT efficiency by
eliminating the generated OH. As the concentration of GSH
2.5. GOx-Like Catalysis-Enhanced Chemodynamic Activity
•
increased from 0 to 10 mꢀ, the absorbance wavelength of MB at
Inspired by the excellent properties of PEG–Au@HMnMSNs
to generate OH, GOx-like catalysis-enhanced chemodynamic
•
6
65 nm barely changed, to confirm that GSH and H O did not
2
2
cause the reduction of MB signal (Figure S11, Supporting Infor-
activity was analyzed in vitro. The cascade nanozyme was
mation). Besides, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), as
a trapping agent, was used to directly capture short-lived OH
expected to catalyze glucose to produce H O2 and gluconic
2
•
acid, consuming oxygen. The intracellular pH and O variation
2
by electron spin resonance measurement (ESR). The character-
were investigated to uncover the GOx-like catalytic reaction of
PEG–Au@HMnMSNs. The fluorescence intensity of BCECF
AM could be quenched at acidic environment. As shown in
Figure 4A,B, 4T1 cells treated with PEG–Au@HMnMSNs qual-
itatively and quantitatively indicated the weakest fluorescence
•
istic signals of OH (1:2:2:1) could be obviously observed after
2+
treating with H O and released Mn under different condi-
2
2
tions (Figure 3C), further proving the GSH depletion-reinforced
•
OH production.
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Figure 4. A) The fluorescent images of 4T1 cells incubated with PBS, HMnMSNs, and PEG–Au@HMnMSNs after BCECF-AM staining for pH measure-
ments. Scale bar: 40 µm. B) Flow cytometry assays of pH levels in 4T1 cells. C) The fluorescent images of 4T1 cells incubated with PBS, HMnMSNs,
and PEG–Au@HMnMSNs after RDPP staining. Scale bar: 40 µm. D) Flow cytometry assays of ROS levels in 4T1 cells, and E) corresponding mean
fluorescence intensity of PBS (a), PEG-Au@HMnMSNs (b), DOX/ASA@PEG–Au@HMnMSNs (c), and DOX/ASA@PEG–Au@HMnMSNs plus glu-
cose (d). F) CLSM images of 4T1 cells after incubation with PEG–Au@HMnMSNs. DAPI (nuclei, blue) and DOX fluorescence (red). Scale bar: 40 µm.
G) CLSM and corresponding surface plot images of ROS levels induced by PEG–Au@HMnMSNs. Scale bar: 40 µm. H) Relative cell viability of 4T1
cells treated with different formulations via CCK-8 assay. n = 3, mean ± SD; *P < 0.05; **P < 0.01; ***P < 0.001.
signal compared with PBS and HMnMSNs groups without
function of Au nanozyme, displaying the formation of glu-
conic acid to cause a decrease in the intracellular pH. RDPP,
and Mn (Fenton catalyst) in cancer therapy, we also measured
the experiment with intracellular ROS enhanced by treatment
with PEG–Au@MSNs, PEG–HMnMSNs, and PEG–Au@
HMnMSNs in 4T1 cells (Figure S13, Supporting Information).
DCF fluorescence induced by PEG–Au@MSNs and PEG–
HMnMSNs in 4T1 cells was both stronger than the control
group, which respectively attributed to the GOx-like catalyzed
H O production in the presence of AuNPs, and the generation
of OH by Mn catalyst. However, DCF fluorescence intensity
induced by combined PEG–Au@HMnMSNs in 4T1cells was
the strongest, indicating the function of catalytic cascade nano-
reactors. Flow cytometry was used to quantitatively analyze the
effects of loading drugs and glucose on intracellular ROS levels,
and the clear shift could be observed in Figure 4D,E, further
suggesting GOx-like catalysis-enhanced chemodynamic activity.
Furthermore, Figure S14, Supporting Information clarified the
as O probe, could increase the signal of red fluorescence in the
2
absence of O . Compared with PBS and HMnMSNs groups,
2
4
T1 cells treated with PEG–Au@HMnMSNs showed stronger
red fluorescence, reflecting the GOx-like catalytic reaction that
consumed the glucose and O (Figure 4C).
2
2
•
2
2+
GOx-like
catalysis-enhanced
intracellular
reactive
oxygen species (ROS) levels were visualized by 2′,7′-
dichlorodihydrofluorescein diacetate (DCF-DA), which could be
oxidized to green fluorescent 2′,7′-dichlorodihydrofluorescein
36]
(
DCF) by the presence of ROS.[ DCF fluorescence induced
PEG–Au@HMnMSNs in 4T1 cells was gradually stronger with
the prolonged incubation time (1, 6, 12 h) (Figure 4G). To fur-
ther investigate the function of AuNP (GOx-mimic enzyme)
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changes of H O level in 4T1 cells for 24 h of incubation. ASA
with the cytotoxicity assay, indicating that GOx-like catalysis-
enhanced CDT combined with chemotherapy could induce
ICD. HMGB1 distributes in the nucleus of the normal cells,
but it can migrate outside of the tumor cells undergoing ICD.
The amounts of HMGB1 in the supernatants were detected
by a HMGB1 ELISA Kit. The HMGB1 release of DOX/ASA@
PEG–Au@HMnMSNs group was 402 pg mL , which was
1.37-fold,1.67-fold, 2.98-fold, and 4.63-fold higher than that of
the PEG–Au@HMnMSNs, DOX/ASA, HMnMSNs, and con-
trol groups, respectively (Figure S20, Supporting Information).
Furthermore, the expression level of HMGB1 was upregulated
with DOX/ASA@PEG–Au@HMnMSNs treatment by western
blot (Figure 5C).
2
2
and DOX could promote the generation of endogenous H O .
2
2
However, after the introduction of PEG–Au@HMnMSNs, the
level of H O was decreased, which was due to the release of
2
2
2
+
•
Mn catalyzing H O to produce highly toxic OH.
2
2
−
1
2
.6. Cellular Uptake and Cytotoxicity Evaluation in Vitro
To deliver agents into tumor cells for efficient tumor treatment,
the cellular uptake behavior of PEGylated Au@HMnMSNs
was designed to detect on 4T1 cells. As displayed in Figure 4F,
the red fluorescence from DOX-loaded PEG–Au@HMnMSNs
and the blue fluorescence DAPI staining cell nucleus revealed
prominent overlay with the extension of incubation time. Flow
cytometry was also utilized to quantify the overlay of fluores-
cence (Figure S15, Supporting Information), both verifying the
internalization of PEG–Au@HMnMSNs via endocytosis.
In vitro GOx-like catalysis-enhanced CDT combined with
chemotherapy of 4T1 cells was investigated via CCK-8 assay
to evaluate the inherent CDT enhanced effect of PEG–Au@
HMnMSNs. As shown in Figure 4H, all formulations except
the ASA group displayed a dose-dependent cytotoxicity to 4T1
cells. PEG–Au@HMnMSNs showed a lower cell inhibition rate
compared with PEG–HMnMSNs and PEG–Au@MSNs due to
the lack of gold nanozyme and Mn catalyst, respectively. How-
ever, it showed no obvious cytotoxicity to normal liver cells L-02
under different concentration (Figure S16, Supporting Informa-
tion). It’s worth noting that the administered ASA and DOX
doses were calculated according to the drug loading capacity of
PEG–Au@HMnMSNs. After introduction of DOX and ASA,
DOX/ASA@PEG–Au@HMnMSNs exhibited a distinct inhibi-
tion rate. The flow cytometry and live (green)/dead (red) cells
staining assay further confirmed potential enhancement effect
of DOX/ASA@PEG–Au@HMnMSNs in inducing apoptosis of
Given the satisfied ICD-induction results, the immuno-
genicity of the tumor cells was evaluated by acting on DCs
maturation after various treatments in vitro. DCs matura-
tion could be verified by costimulatory molecules CD80 and
CD86, which were considered as markers to elicit T cell-
[
39]
mediated immune responses.
The DCs maturation fre-
quency (CD11c+CD80+CD86+) of the control group was 8.64%,
which sharply increased to 57.1% after DOX/ASA@PEG–Au@
HMnMSNs-treated BMDCs by flow cytometry (Figure 5D).
During DCs maturation, other types of immune cells were
regulated by releasing cytokines, including interleukin 6 (IL-6,
an important marker of humoral immunity), interleukin 12p70
(IL-12p70, important marker of innate immunity), interferon
γ (IFN-γ, positive role in the cytotoxic functions of CTLs), and
tumor necrosis factor α (TNF-α, important marker of cellular
immunity). As displayed in Figure 5E, the highest secretion
levels of IL-6, IL-12p70, IFN-γ, and TNF-α could be examined
in DOX/ASA@PEG-Au@HMnMSNs-treated supernatant by
using mouse inflammation kit. In contrast, the level of inter-
leukin 10 (immunosuppression cytokine) was reduced.
Although the maturation of DCs were crucial initiators of
innate and adaptive immune responses, the overexpressed
4
T1 cells to cause a higher cell mortality than single CDT (Fig-
PGE impeded the migration of DCs, strengthened the barrier
2
ures S17 and S18, Supporting Information).
between T cells and tumor cells, and recruited more immuno-
suppressive cells. A well-known anti-inflammatory drug ASA
improved the immune microenvironment by the inhibition of
[
14,15,19]
2
.7. ICD-Induced Immune Activation
COX-2 expression and secretion of PGE2.
As shown in
Figure 5F,G, the prohibitive expression level of ASA on COX-2
Such synergistic GOx-like catalysis-enhanced CDT combined
with chemotherapy not only kills cancer cells, but more impor-
tantly, it could activate antitumor immune response as an ICD
nano-inducer to remodulate the ITM and achieve better anti-
tumor immunotherapy effects. CRT, as a dominant biomarker
during ICD, acts as an “eat me” signal to guide DCs to take
up the dying tumor cells. And HMGB1, another surrogate
and PGE was detected by western blot and ELISA in tumor
2
cells (4T1) and tumor-associated macrophages (RAW264.7).
The expression level of COX-2 was observably decreased in 4T1
and RAW264.7 as the concentration of ASA increased. More-
over, the level of COX-2 was down-regulated caused by DOX/
ASA@PEG–Au@HMnMSNs greater than that induced by con-
trol, ASA, DOX, and PEG–Au@HMnMSNs in 4T1 cells. The
biomarker for ICD-associated immunogenicity, can stimulate
secretions of PGE in the supernatants of 4T1 cells were quan-
2
DCs maturation and antigen presentation.[
37,38]
As displayed in
titatively calculated by ELISA, which was consistent with the
results of COX-2 protein expression. It indicated that ASA could
effectively inhibit the expression level of COX-2 and PGE2.
Figure 5, the CRT exposure and HMGB1 release in 4T1 cells
were assessed. DOX/ASA@PEG–Au@HMnMSNs-treated 4T1
cells could more observably augment the expression level of
CRT (higher green fluorescence intensity), compared with PBS,
HMnMSNs, DOX/ASA, and PEG–Au@HMnMSNs treatments
2.8. In Vivo Biodistribution and Infiltration of CD8+
and CD4+ T Lymphocytes
(Figure 5A). Flow cytometry was further utilized to confirm the
high expression of CRT induced by DOX/ASA@PEG–Au@
HMnMSNs, quantitatively (Figure 5B; Figure S19, Supporting
Information). The increasing expression of CRT was consistent
We further evaluated the in vivo biodistribution of PEG–Au@
HMnMSNs in the 4T1 tumor-bearing female BALB/c mice, via
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Figure 5. A) CLSM images and B) flow cytometric detection of CRT exposure on the surface of 4T1 cells treated with PBS (a), HMnMSNs (b), DOX/
ASA (c), PEG–Au@HMnMSNs (d), and DOX/ASA-loaded PEG-Au@HMnMSNs (e). Scale bar: 20 µm. C) Western blot analysis of released HMGB1
from the 4T1 cells into the extracellular supernatant. D) Expression levels of surface molecules (CD80/CD86) on BMDCs after treatments by flow
cytometry analysis (gated on CD11c+). E) Quantification of cytokine production via using mouse inflammation kit for IL-10, IL-12p70, IL-6, IFN-γ, and
TNF-α analysis. F,G) Levels of COX-2 expression and PGE secretion in 4T1 cells after different treatments. n = 3; *P < 0.05; **P < 0.01; ***P < 0.001.
2
intravenous injection of Cy5.5-labeled PEG–Au@HMnMSNs
and observation by IVIS small animal imaging system at prede-
termined time intervals. As shown in Figure 6A, Cy5.5-labeled
nanoparticles exhibited increasing fluorescence in tumor sites
with the prolonged injection time (0.5, 3, 12, and 24 h), and
the fluorescence intensity reached a maximum within 12 h,
indicating high accumulation and long retention time around
tumor sites. The mice were sacrificed to obtain main organs
and tumors for ex vivo fluorescence imaging after 24 h of injec-
tion. The fluorescence intensity at the tumor site was higher
than other main organs.
manifesting the reinforcement of innate antitumor immune
responses. In addition, the immunofluorescent examinations
of the tumor sections with CD8+ and CD4+ staining further
verified the previous results (Figure 6D). Meanwhile, the
immunohistochemical and immunofluorescent analysis of the
sections confirmed that DOX/ASA@PEG–Au@HMnMSNs
significantly decreased the expression level of COX-2 and pro-
moted CRT exposure compared with other treatments, which
activated antitumor immune response to achieve better poten-
tial antitumor immunotherapy effects. To further study the
effect of independent loading of DOX or ASA in PEG–Au@
HMnMSNs on the COX-2 suppression and antitumor immune
response, the immunohistochemical and immunofluorescent
analyses of the sections were employed in Figure S21, Sup-
porting Information. When ASA was introduced into PEG–
Au@HMnMSNs, DOX/ASA-loaded PEG–Au@HMnMSNs
and ASA-loaded PEG–Au@HMnMSNs significantly decreased
the expression level of COX-2, compared with DOX-loaded
PEG–Au@HMnMSNs, which indicated COX-2 suppression
induced potential anti-cancer immunotherapy. In addition, the
immunofluorescent examinations of the tumor sections with
To evaluate GOx-like catalysis-enhanced CDT combined
with chemotherapy for synergistic antitumor effect, the infil-
tration of CD8+/CD4+ cytotoxic T lymphocytes (CTLs) were
analyzed by flow cytometry in tumor. CD8+ T cells were one of
most significant immune cells to limit tumor progression and
induce CTLs killing cancer cells. CD4+ T cells played a vital
role in the regulation of adaptive immunities.[ As shown
in Figure 6B, the population of cytotoxic CD8+ and helper
CD4+ T cells were remarkably increased in the DOX/ASA@
PEG–Au@HMnMSNs group compared with monotherapy,
40]
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Figure 6. A) In vivo fluorescent images of 4T1 tumor-bearing BALB/c mice after the intravenous injection, and ex vivo fluorescent images of major
organs and tumor after 24 h of injection. B) The flow cytometric analysis of the populations of cytotoxic CD8+ and helper CD4+ T cells in 4T1 tumor-
bearing BALB/c mice after different treatments. C) Growth curves of tumor volume in tumor-bearing mice with various treatments. D) H&E, TUNEL,
and COX-2 staining of tumor slices in different groups. The level of increasing CRT exposure on the tumor sections and the infiltration of CD8+ T cells
and CD4+ T cells by immunofluorescence staining assay. Scale bar: 50 µm. n = 3, mean ± SD; ***P < 0.001.
CD8+ and CD4+ staining further verified that DOX/ASA@
PEG–Au@HMnMSNs could induce more CD8+/CD4+ cells
to activate antitumor immune response, compared with ASA-
loaded PEG–Au@HMnMSNs and DOX-loaded PEG–Au@
HMnMSNs.
2.9. In Vivo Antitumor Efficacy and Biosafety Evaluation
To reach desirable antitumor efficacy, 4T1 tumor-bearing female
BALB/c mice were intravenously injected with different for-
mulations at day 0, 3, 6, and 9. The tumor growth profiles in
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Figure 6C; Figure S22, Supporting Information demonstrate
that DOX/ASA@PEG–Au@HMnMSNs conduced to robust
potential antitumor effect with reduction in tumor volume
compared with PBS group, ASA group, HMnMSNs group,
DOX groups, and PEG–Au@HMnMSNs group, suggesting the
limitation of monotherapy failed to completely suppress the
tumor growth. Synergistic GOx-like catalysis-enhanced CDT
combined with chemotherapy was further verified by hematox-
ylin and eosin (H&E) and terminal deoxynucleotidyl transferase
dUTP nick-end labeling (TUNEL) staining (Figure 6D). H&E
staining images showed bulk necrosis and acellular regions
of tumor cells after treatment with DOX/ASA@PEG–Au@
HMnMSNs. The TUNEL staining assay exhibited higher cell
apoptosis induced by DOX/ASA@PEG–Au@HMnMSNs than
other treatments.
In addition, the body weights of the 4T1 tumor-bearing
BALB/c mice remained stable after treatments, indicating
negligible systemic toxicity (Figure S23, Supporting Informa-
tion). H&E staining results displayed no obvious physiological
morphology changes in the main organs (heart, liver, spleen,
lung, and kidney) between control and treatment groups,
showing the safety of the formulations (Figure S24, Supporting
Information).
Sigma–Aldrich Chemical Company. DCFH–DA, H2O2, and GSH assay
kit, 4′,6-diamidino-2-phenylindole (DAPI), calcein acetoxymethyl
ester/propidium iodide (Calcein-AM/PI), 2″,7″-bis-(2-carboxyethyl)-
5
-(and-6) carboxy fluorescein, acetoxymethyl ester (BCECF-AM), Cell
Counting Kit-8 (CCK-8), and cyclooxygenase 2 monoclonal antibody
were supplied from Beyotime Institute of Biotechnology. Antibodies for
calreticulin (CRT) were purchased from Bioss Biotech Co., Ltd. Mouse
inflammation kit for IL-10, IL-12p70, IL-6, MCP-1, IFN-γ, and TNF-α
analysis was purchased from BD Biosciences Co., Ltd. Anti-mouse
CD80-PE, anti-mouse CD86-PE-Cyanine5, anti-mouse CD11c-FITC, anti-
CD3-PE- Cyanine5, anti-CD4-FITC, and anti-CD8-FITC were obtained
from Invitrogen. All other chemicals were of analytical grade and used
without further purification.
Preparation of Au@MSNs: Typically, 100 mg of CTAB and 1.2 mL of
NaOH (0.5 ꢀ) were dissolved in 50 mL of deionized water, and then
the solution was heated to 80 °C for 15 min under stirring. 2 mL of
formaldehyde solution (3.7 wt%) was added, followed by 1.6 mL of
HAuCl (0.05 mL) aqueous solution. After stirring for 15 min, an ethanol
4
(
1 g) solution of TEOS (0.5 g) was added and the reaction mixture was
kept at 80 °C for 1.5 h under stirring. The precipitate was collected via
centrifugation at 13 000 rpm for 15 min, washed with water and once with
ethanol to remove residual reactants, and finally dried under vacuum. To
remove CTAB, the products were stirred for 12 h in a methanol solution
of NaCl (1 wt%) for three times. The purple-red products were collected
by centrifugation, and then washed with deionized water and ethanol
several times, followed by vacuum drying.
Synthesis of PEG–Au@HMnMSNs: To dope manganese in
mesoporous silicon, the prepared Au@MSNs were first dispersed into
1
0 mL of deionized water. Then, 80 mg of MnSO ·H O and 100 mg of
4 2
disodium maleate were dissolved into 10 mL of deionized water. The
mixture solution was dropwise added into the Au@MSNs solution and
kept for another 30 min under stirring. The final mixture was treated
under hydrothermal condition at 180 °C for 13 h. To remove residual
reactants, the products were collected by centrifugation, and then rinsed
with water and ethanol several times, followed by vacuum drying. The
prepared Au@HMnMSNs were further aminated through APTES. Au@
HMnMSNs (80 mg) and 450 µL of APTES were refluxed in anhydrous
ethanol (30 mL) under magnetic stirring for 24 h. Amine group-modified
Au@HMnMSNs were obtained via centrifugation and washed with
3
. Conclusion
In summary, we developed biodegradable manganese-doped
hollow mesoporous silica coated gold nanoparticles by loading
ASA and DOX, as TME-responsive catalytic cascade nano-
reactors for enhanced CDT synergistic antitumor chemo-
2
+
immunotherapy. PEG–Au@HMnMSNs could release Mn
•
for Fenton-like reaction to produce OH through the degrada-
tion of GSH in TME, and protect the GOx-like activity of Au
to regenerate H O for enhanced CDT effect. Such an inherent
−
2
ethanol. Subsequently, mPEG2 COOH (2.16 × 10 mmol), EDCl
000
2
2
−1
−1
(
5
1.08 × 10 mmol), and NHS (1.08 × 10 mmol) were dissolved in
GOx-like catalysis-enhanced CDT combined with DOX treat-
ment could induce sufficient ICD to promote DC maturation
and enhance the infiltration of CTLs for systemic antitumor
immunity compared with monotherapy. Meanwhile, ASA could
recruit DCs and CTLs to tumor sites, restrain the immunosup-
pressive cells, and remodel ITM by decreasing the expression
mL of anhydrous DMSO and stirred for 4 h to activate the carboxyl
groups. 50 mg of aminated Au@HMnMSNs was dispersed in 5 mL of
anhydrous DMSO, which was added into the previous activated solution
to react for 48 h. The prepared PEG

Au@HMnMSNs were collected via
centrifugation, and then washed with deionized water and ethanol.
Preparation of HMnMSNs: Briefly, 200 mg of CTAB and 300 mg of
ammonium fluoride were dissolved in 50 mL of deionized water at 80 °C
for 15 min under magnetic stirring. Then, 900 µL of TEOS was slowly
added dropwise, and the reaction mixture was kept at 80 °C for 2 h. The
removal of template was the same as the previous steps, and MSNs
were collected via centrifugation. To obtain manganese-doped hollow
mesoporous silicon nanoparticles (HMnMSNs), 80 mg of MnSO ·H O
of PGE . This work provided an innovative insight to introduce
2
GOx-like catalytic cascade ICD nano-inducer into potential anti-
tumor immunotherapy with valid remodulation of TME for
synergistic tumor therapy.
4
2
and 100 mg of disodium maleate were dissolved in 10 mL of deionized
water, and then the mixture solution was added into MSNs solution,
which was treated under hydrothermal condition at 180 °C for 12 h.
The synthesized HMnMSNs were collected via centrifugation, and then
rinsed with deionized water several times.
Characterization: Transmission electron microscopy (TEM) and
scanning electron microscopy (SEM) images were respectively observed
using JEM-2100 transmission electron microscope and field-emission
scanning electron microscopy (Zeiss, Ultra Plus). Energy dispersive X-ray
spectroscopy (EDS) was carried out on JEM-2100 transmission electron
microscope. The valence states of different elements were obtained by
X-ray photoelectron spectroscopy (XPS, Thermo Fisher Nexsa). The
crystal structure analysis was measured via X-ray diffraction (Ultima
IV). The sizes and surface charges of nanoparticles were detected via
Brookhaven BI-200SM apparatus. The FTIR and UV–vis adsorption
4
. Experimental Section
Materials: Manganese sulfate monohydrate (MnSO ·H O),
4
2
cetyltrimethylammonium bromide (CTAB), tetraethyl orthosilicate
TEOS), HAuCl ·4H O, doxorubicin hydrochloride (DOX·HCl),
(
4
2
acetylsalicylic acid (ASA), 3-aminopropyltriethoxysilane (APTES),
N-hydroxysuccinimide (NHS), N-ethylcarbodiimide hydrochloride
(
EDCl), mPEG2000–COOH, methylene blue (MB), glutathione (GSH),
hydrogen peroxide (H O ), and glucose were obtained from Sinopharm
2
2
Chemical Reagent Co., Ltd. 2,2′-Azino-bis(3-ethylbenzothiazoline-
-sulfonic acid) diammonium salt (ABTS), 5,5-dimethyl-1-pyrroline-
N-oxide (DMPO), peroxidase from horseradish (HRP) (type VI),
,5′-dithiobis (2-nitrobenzoic acid) (DTNB), and ruthenium dichloride
4, 7-biphenyl-1, 10-phenanthroline) (RDPP) were purchased from
6
5
(
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properties were measured by Thermo Scientific Nicolet 6700 FTIR
spectrometer and UV-2600 spectrometer, respectively. The generation
of hydroxyl radicals was quantified by an electron spin resonance (ESR)
spectrometer using Bruker EMX plus. Thermogravimetric analysis (TGA)
was conducted on a TG209 F3 thermogravimetric analyzer. The amount
of Mn element was measured by using a TG209 F3 thermogravimetric
analyzer. The surface area and pore size distribution of Au@HMnMSNs
were further performed on Brunauer–Emmett–Teller Micromeritics
Tristar 3000 analyzer.
Drug Loading and Release: In brief, 20 mg of PEG–Au@HMnMSNs
and 4 mg of ASA were dispersed in 10 mL of anhydrous DMSO and
kept under stirring for 24 h at 25 °C. ASA-loaded PEG–Au@HMnMSNs
were collected via centrifugation and washed several times. The
as-synthesized ASA-loaded PEG–Au@HMnMSNs were re-dispersed
in phosphate buffered saline (PBS) solution containing DOX (4 mg),
and the mixture solution stirred for 24 h under dark conditions. ASA/
DOX-loaded PEG–Au@HMnMSNs were collected by centrifugation
and washed several times. The amounts of ASA and DOX in PEG–Au@
HMnMSNs were respectively quantified by TGA and a standard curve via
UV–vis spectra. The drug release based on ASA and DOX experiments
was measured using a dialysis method in PBS. Equal amounts of ASA/
DOX-loaded PEG–Au@HMnMSNs were transferred into dialysis bag
pH = 7.4 and 5.0, respectively. To investigate the GOx-like catalytic
activity of gold nanoparticles, glucose was added to a sample solution.
After 4 h of stirring, 8 mꢀ of H O and 200 mꢀ of DMPO were added
to sample solutions. The OH characteristic peak signals were measured
by ESR spectrometer.
2
2
•
GOx-Like Activity of PEG–Au@HMnMSNs: A horseradish peroxidase
(HRP)-based colorimetric measurement was utilized to investigate
the enzymatic activity of Au nanozyme. Briefly, a mixed solution of
−
1
100 µg mL of PEG–Au@HMnMSNs solution in 10 mꢀ NaCl and
10 mꢀ Tris-HCl buffer in 100 mꢀ NaCl was incubated with 100 mꢀ
glucose for 30 min. Then, the supernatant was collected by centrifugation
to avoid the color interference. Subsequently, the supernatant was added
−
1
to 50 mꢀ Tris-HCl buffer solution containing HRP (5 µg mL ) and ABTS
(1 mꢀ). The resulting solution continued to incubate for 10 min before
measurement of absorbance at 405 nm by UV–vis spectra.
Cell Culture: In this study, Murine breast cancer (4T1) cells were
incubated in RPMI 1640 complete medium containing 10% FBS and 1%
penicillin–streptomycin solution, and all cell lines were cultured under a
humidified atmosphere containing 5% CO at 37 °C.
2
Cellular Uptake Study: 4T1 cells were seeded into confocal petri dishes
and 6-well plates at a density of 1 × 105 cells and incubated for 24 h.
Then, the cells were cultured with DOX-loaded PEG–Au@HMnMSNs at
−
1
(
MWCO: 3500 Da) and then incubated into 25 mL of PBS under different
2 µg mL of DOX. The cellular uptake was obtained by CLSM and flow
cytometry at predetermined time intervals. DAPI was employed to locate
cell nucleus before observation by CLSM.
conditions (pH 7.4, pH 5.0, pH 5.0 + GSH 5 mꢀ). At predetermined time
intervals, the released ASA and DOX were measured by UV–vis spectra.
Degradation Study In Vitro: For the degradation study of PEG–Au@
HMnMSNs, certain mass of PEG–Au@HMnMSNs was dispersed in
the different GSH concentrations (5 and 8 mꢀ) and pH values (6.8 and
Chemodynamic Activity In Vitro: DCFH–DA assay was employed to
2+
qualitatively and quantitatively detect the ROS levels mediated by Mn -
based GOx-catalysis enhanced chemodynamic activity. 4T1 cells were
seeded into confocal petri dishes and 6-well plates at a density of 1 × 105
cells and incubated for 24 h. Then, the medium was replaced with PEG–
5
.0) of simulated body fluid (SBF) solutions, and then stirred gently at
37 °C for scheduled time intervals (6, 12, 24, and 48 h). The mass of
−
1
released manganese ion and the morphology of PEG–Au@HMnMSNs
was respectively determined by ICP-MS analysis and TEM in degradation
solutions.
Hemolysis Assay: To obtain standard erythrocyte solution, the blood
with EDTA from mouse was collected by centrifugation and resuspended
in normal saline. Then, 0.5 mL of erythrocyte suspension was added
to 2 mL of normal saline with dispersed ASA/DOX-loaded PEG–Au@
HMnMSNs. The mixed solution was centrifuged after 4 h of incubation at
Au@HMnMSNs (60 µg mL ) for different incubation times (1, 6, and
12 h). In addition, the medium was also replaced with PEG–Au@MSNs,
PEG–-HMnMSNs, and PEG–Au@HMnMSNs for 6 h of incubation. The
cells were washed with PBS and incubated with DCFH–DA solution for
15 min. Dyed cells were observed with CLSM. To quantitatively examine
the level of ROS, the cells were incubated with PEG–Au@HMnMSNs,
ASA/DOX-loaded PEG–Au@HMnMSNs, and ASA/DOX-loaded PEG–
Au@HMnMSNs plus glucose (50 mꢀ) at 37 °C for another 8 h. The
cells were collected and analyzed by flow cytometry.
3
7 °C. Normal saline and deionized water were respectively dissolved in
erythrocyte suspension as negative and positive controls. The absorbance
A) value of supernatant was determined at 570 nm by UV–vis spectra.
Intracellular pH Detection: Cascade nanozyme was expected to
(
catalyze glucose to produce H O2 and gluconic acid, and consume
2
The percentage of hemolysis was calculated as Hemolysis rate (%) =
oxygen. BCECF AM could be quenched at acidic environment. After 4T1
cells incubation with medium, HMnMSNs, and PEG–Au@HMnMSNs
in confocal petri dishes and 6-well plates, fresh medium containing
5 µꢀ of BCECF AM was added and cultured for another 1 h. The dyed
4T1 cells were observed by CLSM and resuspended in PBS solution for
flow cytometry analysis.
(Asample − Anegative control)/(Apositive control − Anegative control) × 100%.
GSH Consumption Detection: An indicator 5,5′-dithiobis
2-nitrobenzoic acid) (DTNB) was utilized to determine GSH depletion.
(
Different concentrations of PEG–Au@HMnMSNs (50,100, 200, and
−
1
4
00 µg mL ) were added to PBS solutions with GSH (pH = 5.0, 8 mꢀ),
respectively. Then, the mixtures were kept stirring at 37 °C for 2 h, and
supernatants were collected by centrifugation. DTNB solutions were
added and the absorbance of supernatants was measured by UV–vis
spectra.
Intracellular O2 Assay: RDPP, as oxygen probe, was employed to
detect the level of intracellular O . 4T1 cells were seeded into confocal
2
5
petri dishes at a density of 1 × 10 cells and incubated for 24 h. The
medium was replaced with fresh medium, HMnMSNs, and PEG–Au@
HMnMSNs for 8 h. After removal of medium, fresh medium containing
RDPP was added and cultured for another 4 h. Finally, the cells were
observed by CLSM.
Hydroxyl Radical (•OH) Generation by Mn -Mediated Fenton-
2+
•
Like Reaction: Methylene blue (MB) was degraded by OH, which
•
could be used to detect the amount of OH. PEG–Au@HMnMSNs
−
1
(
80 µg mL ) were dispersed in NaHCO /CO buffer solution containing
Intracellular H O Measurement: To detect the level of H O in vitro,
3
2
2
2
2
2
GSH with a series of concentrations (0, 0.5, 1, 5, and 10 mꢀ), and stirred
at 37 °C for 6 h. Similarly, PEG–Au@HMnMSNs and 0.5 mꢀ of GSH
were dissolved in NaHCO /CO buffer solution containing glucose
4T1 cells were seeded into 96-well plates overnight at a density of 5 ×
3
10 per well and then treated with different formulations (ASA, DOX,
3
2
PEG–Au@HMnMSNs, and DOX/ASA-loaded PEG-Au@HMnMSNs).
The level of H O2 was tested via H O2 assay kit according to the
with different concentrations (20 and 40 mꢀ). Then, supernatants were
collected by centrifugation. 8 mꢀ of H O and MB were respectively
2
2
2
2
manufacturer’s instructions after incubation for 24 h.
3
added supernatants, and further incubated for 15 min. Meanwhile, H O2
Cytotoxicity Evaluation in Vitro: In brief, 4T1 cells (6 × 10 per well)
were seeded into 96-well plates and cultured for 24 h. Subsequently,
the medium was replaced with fresh medium containing various
concentration of PEG–HMnMSNs, PEG–Au@MSNs, PEG–Au@
HMnMSNs, ASA, DOX, and ASA/DOX-loaded PEG–Au@HMnMSNs
2
and MB with different concentrations of GSH were used as the control
•
groups. The generation of OH was monitored by the absorbance change
of MB at 665 nm and naked eyes.
ESR Measurements: 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), as
•
−1
a trapping agent, was employed to determine production of OH.
(MassNanoparticle: 12.5, 25, 50, 100, and 200 µg mL ). After 24 h of
−
1
PEG–Au@HMnMSNs (100 µg mL ) were dispersed in buffer solution
incubation, 10 µL CCK-8 was added and treated for another 1 h. A
containing GSH with different concentrations (1 and
5
mꢀ) at
microplate reader was used for measurements at 450 nm.
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5
Cell Apoptosis: 4T1 cells (1 × 10 cells per well) seeded into 6-well plates
In Vivo Antitumor Efficacy: 4T1 tumor-bearing female BALB/c mice
were randomly divided into six groups (n = 3) when the volume of tumor
were cultured for 24 h. Then, 4T1 cells were washed by PBS solutions
and treated with PEG–Au@HMnMSNs (25, 50, and 100 µg mL ) for
4 h. The 4T1 cells were digested, washed, and resuspended with 500 µL
buffer solution. After staining with Annexin-V-FITC and PI for 15 min in
the dark, cell apoptosis was analyzed by flow cytometry.
Cell Live–Dead Staining Experiments: To further investigate the
survival and death of cells, 4T1 cells (1 × 10 cells per well) seeded into
-well plates were cultured for 24 h. Then, the cells were incubated
−
1
3
reached 60 mm . The mice were intravenously injected with PBS, ASA
−
1
−1
−1
2
(0.32 mg kg ), DOX (0.5 mg kg ), HMnMSNs (4 mg kg ), PEG–Au@
−
1
HMnMSNs (4 mg kg ), and DOX/ASA-loaded PEG–Au@HMnMSNs
(4 mg kg ) at day 0, 3, 6, and 9. The tumor size (length: L and width W)
and body weight were recorded every 2 days. Tumor volume (V) and
−
1
5
2
relative tumor were calculated as V = (L × W )/2 and Vt/V0 (0: day 0,
6
t: day t), respectively. The treated mice were sacrificed to harvest main
organs for H&E staining, and the obtained tumors from different
groups of mice were sliced for H&E staining, terminal deoxynucleotidyl
TUNEL staining, immunohistochemical staining of COX-2, and
immunofluorescence staining.
with PEG–Au@HMnMSNs, DOX/ASA, and DOX/ASA-loaded PEG–
−
1
Au@HMnMSNs at 60 µg mL of nanoparticles. After staining with
Calcein-AM and PI for 30 min, the treated cells were observed by CLSM.
Western Blotting for the COX-2 Protein: 4T1 and RAW264.7 cells were
seeded into 6-well plates and cultivated for 24 h. After RAW264.7 cells
stimulation with LPS, the 4T1 and RAW264.7 cells were incubated with
various concentrations of ASA. Then, 4T1 cells were treated with fresh
medium, PEG–Au@HMnMSNs, DOX, ASA, and DOX/ASA-loaded PEG–
Au@HMnMSNs for 12 h. The cells were collected by trypsin digestion
and washed with PBS. Total proteins were extracted by RIPA lysis buffer,
and the level of COX-2 protein was detected via western blot analysis.
PGE Level Analysis In Vitro: To further measure the mass of PGE in
The Detection of CD4+ and CD8+ T Lymphocytes in Vivo: To examine the
infiltration of T cells in tumor, saline, ASA, DOX, PEG–Au@HMnMSNs,
and DOX/ASA-loaded PEG–Au@HMnMSNs were intratumorally
injected into 4T1 tumor-bearing BALB/c mice at day 0, 3, and 6. After
the final treatments, the tumors were harvested, cut into several small
pieces, and homogenized to obtain a single-cell suspension. Then, the
cells were washed with PBS and stained with anti-CD3-PE-Cyanine5, anti-
CD4-FITC, and anti-CD8-FITC followed by flow cytometry analysis.
Statistical Analysis: All the data are expressed as the mean ± standard
deviation (SD). Analysis of variance (ANOVA) within GraphPad Prism
8.0 software (San Diego, CA, USA) was used to confirm statistically
significant differences between groups. Statistical significances were
determined using Student’s t test, with the following P values considered
significant: *P < 0.05, **P < 0.01, and ***P < 0.001.
2
2
vitro, 4T1 and RAW264.7 cells were incubated overnight, and then treated
with fresh medium, PEG–Au@HMnMSNs, DOX, ASA, and DOX/ASA-
loaded PEG–Au@HMnMSNs. The mass of PGE in the supernatants were
2
performed on a PGE ELISA Kit according to the manufacturer’s instructions.
2
Induced DAMP Release In Vitro: To detect GOx-like catalysis enhanced
chemodynamic therapy combined with chemotherapy-induced ICD of
the 4T1 cells, CRT exposure and expression of HMGB1 were investigated
in vitro. 4T1 cells were seeded into 6-well plates and incubated with fresh
medium, HMnMSNs, DOX/ASA, PEG–Au@HMnMSNs, and DOX/
−
1
ASA-loaded PEG–Au@HMnMSNs at 50 µg mL of nanoparticles, Supporting Information
respectively. The cells were collected, washed, and cultured with Alexa
Supporting Information is available from the Wiley Online Library or
from the author.
Fluor 488-conjugated anti-CRT antibody for 20 min for flow cytometry
analysis. For CLSM observation, 4T1 cells were incubated with fresh
medium, HMnMSNs, DOX/ASA, PEG–Au@HMnMSNs, and DOX/ASA-
loaded PEG–Au@HMnMSNs. The cells were stained with Alexa Fluor
4
88-conjugated anti-CRT antibody before observation.
To detect the release of HMGB1 in the supernatants corresponding
Acknowledgements
to the samples tested by the above CRT experiments, the amounts of
HMGB1 in the supernatants were performed on a HMGB1 ELISA Kit
according to the manufacturer’s instructions. In addition, 4T1 cells were
collected to measure the level of HMGB1 protein using HMGB1 rabbit
monoclonal antibody by western blot after incubation with DOX/ASA-
This work was financially supported by the National Natural Science
Foundation of China (81771976), the National Key Research and
Development Program of China (Grant 2018YFC1901202), the State Key
Laboratory of Pathogen and Biosecurity (Academy of Military Medical
Science SKLPBS2134), the Fundamental Research Funds for the Central
Universities, and the joint fund of Southeast University and Nanjing
Medical University.
−
1
loaded PEG–Au@HMnMSNs (25 and 50 µg mL ).
ICD-Induced Immune Activation In Vitro: Briefly, bone marrow-derived
dendritic cells (BMDCs) were obtained from primary bone marrow cells
of female BALB/c mice, and BMDCs were seeded into 24-well plates. 4T1
cells were seeded into 24-well plates and cultured for 24 h. Then, the cells
were treated with fresh medium, ASA, DOX, PEG–Au@HMnMSNs, and
Conflict of Interest
The authors declare no conflict of interest.
−1
DOX/ASA-loaded PEG–Au@HMnMSNs at 50 µg mL of nanoparticles
for 12 h. Each group-conditioned supernatant was added into BMDCs
and incubated for 24 h. The stimulated BMDCs were detected using anti-
mouse CD80-PE, anti-mouse CD86-PE-Cyanine5, and anti-mouse CD11c-
FITC by flow cytometry. Each supernatant was used to determine the level
of IL-10, IL-12p70, IL-6, IFN-γ, and TNF-α using mouse inflammation kit Data Availability Statement
for IL-10, IL-12p70, IL-6, IFN-γ, and TNF-α analysis.
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
In Vivo Biodistribution and Imaging Studies: 4T1 tumor-bearing female
BALB/c mice were intravenously administrated with Cy5.5-labeled PEG–
Au@HMnMSNs. The biodistribution and fluorescent intensity were
observed by IVIS small animal imaging system at predetermined time
intervals. Moreover, the fluorescent intensity major organs (heart, liver,
spleen, lung, and kidney) were obtained at a certain time interval. All
animal experimental procedures were approved by the Experimental
Animal Ethics Committee of Southeast University guidelines
Keywords
cascade reaction, chemodynamic therapy, dendritic cells, immune
system activation, immunogenic cell death
(
China). All animal experiments followed the institutional guidelines
of the Experimental Animal Ethics Committee of Southeast University
China) and were performed in compliance with the regulations for the
Received: March 31, 2021
Revised: June 9, 2021
Published online:
(
Administration of Affairs Concerning Experimental Animals of China.
Small 2021, 2101897
2101897 (12 of 13)
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