Modularly Designed Peptide Nanoprodrug Augments Antitumor Immunity of PD-L1 Checkpoint Blockade by Targeting Indoleamine 2,3-Dioxygenase

Article abstract of DOI:10.1021/jacs.9b12232

The limited efficacy of single-agent immune checkpoint inhibitors in treating tumors has prompted investigations on their combination partners. Here, a tumor-homing indoleamine 2,3-dioxygenase (IDO) nanoinhibitor is reported to selectively inhibit immunosuppressive IDO pathway in the tumor microenvironment. It is self-assembled from a modularly designed peptide-drug conjugate containing a hydrophilic targeting motif (arginyl-glycyl-aspartic acid; RGD), two protonatable histidines, and an ester bond-linked hydrophobic IDO inhibitor, which exhibits pH-responsive disassembly and esterase-catalyzed drug release. Markedly, it achieved potent and persistent inhibition of intratumoral IDO activity with a reduced systemic toxicity, which greatly enhanced the therapeutic efficacy of programmed cell death-ligand 1 blockade in vivo. Overall, this study provides a promising paradigm of combinatorial normalization immunotherapy by exploiting a targeted IDO nanoinhibitor to augment the antitumor immunity of checkpoint inhibitors.

Full text of DOI:10.1021/jacs.9b12232

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Article
Modularly Designed Peptide Nanoprodrug Augments Anti-Tumor Immunity
of PD-L1 Checkpoint Blockade by Targeting Indoleamine 2, 3-Dioxygenase
Xuexiang Han, Keman Cheng, Ying Xu, Yazhou Wang, Huan Min, Yinlong Zhang,
Xiao Zhao, Ruifang Zhao, Greg Anderson, Lei Ren, Guangjun Nie, and Yiye Li
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b12232 • Publication Date (Web): 16 Jan 2020
Downloaded from pubs.acs.org on January 17, 2020
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Modularly Designed Peptide Nanoprodrug Augments Anti-Tumor
Immunity of PD-L1 Checkpoint Blockade by Targeting Indoleamine
2, 3-Dioxygenase
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Xuexiang Han†,‡,#, Keman Cheng†,§,#, Ying Xu†,#, Yazhou Wang†, Huan Min†, Yinlong Zhang†,‡, Xiao
Zhao†,‡, Ruifang Zhao†,‡, Gregory J. Anderson^, Lei Ren§, Guangjun Nie*,†,‡ and Yiye Li*,†,‡
†CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience,
National Center for Nanoscience and Technology, Beijing 100190, P.R. China
‡Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, Beijing 100049,
P.R. China
§Department of Biomaterials, Key Laboratory of Biomedical Engineering of Fujian Province, College of Materials, Xiamen
University, Xiamen 361005, P.R. China
^QIMR Berghofer Medical Research Institute, Royal Brisbane Hospital, Brisbane, Queensland 4029, Australia.
ABSTRACT: The limited efficacy of single-agent immune checkpoint inhibitors in treating tumors has prompted investigations on
their combination partners. Here, a tumor-homing indoleamine 2, 3-dioxygenase (IDO) nanoinhibitor is reported to selectively
inhibit immunosuppressive IDO pathway in the tumor microenvironment. It is self-assembled from a modularly designed peptide-
drug conjugate containing a hydrophilic targeting motif (arginyl-glycyl-aspartic acid; RGD), two protonatable histidines and an
ester bond-linked hydrophobic IDO inhibitor, which exhibits pH-responsive disassembly and esterase-catalyzed drug release.
Markedly, it achieved potent and persistent inhibition of intratumoral IDO activity with reduced systemic toxicity, which greatly
enhanced the therapeutic efficacy of programmed cell death-ligand 1 blockade in vivo. Overall, this study provides a promising
paradigm of combinatorial normalization immunotherapy by exploiting a targeted IDO nanoinhibitor to augment the anti-tumor
immunity of checkpoint inhibitors.
malignancies.8 However, only a small fraction of patients can
benefit from this and the overall clinical efficacy of aPD-L1
INTRODUCTION
Immune escape is a fundamental characteristic during tumor
monotherapy is still limited due to the existence of other
growth and progression. Tumor cells exploit multiple
immunosuppressive molecules in the TME.9,10 Therefore,
mechanisms to evade host immune surveillance,1 such as
combinatorial normalization immunotherapy by concurrently
immune checkpoints, which refer to co-inhibitory receptors
blocking multiple pathways in the TME to boost the anti-
expressed on immune effector cells or immunosuppressive
tumor immune response of checkpoint inhibitor-based
ligands expressed on tumor cells.2,3 One of the best
backbone therapy is widely regarded as the future of immune-
characterized immune checkpoint molecules is the
oncology.11-14 Among various combination partners,
programmed cell death-ligand 1 (PD-L1), which is up-
indoleamine 2, 3-dioxygenase (IDO) inhibitors have great
regulated by malignant cells.4 Ligation of PD-L1 to
potential to enhance the efficacy of checkpoint blockade and
programmed cell death receptor (PD-1) expressed on T cells in
are under active investigation in clinic.15-17
the tumor microenvironment (TME) suppresses T-cell
activation and proliferation.5
IDO is a cytosolic tryptophan-catabolizing enzyme that
converts the essential amino acid L-tryptophan (Trp) to
kynurenine (Kyn).18 It is constitutively expressed in many
tissues where it induces immune tolerance and prevents
normal tissue injury.17 Over-expression of IDO in tumor cells
suppresses T-cell responses and recruits regulatory T (Treg)
cells via Trp depletion and Kyn accumulation in the TME,
leading to immune evasion and tumor growth.19,20 Since IDO
is an important target for immunotherapeutic intervention,
several small molecule inhibitors are under clinical
development,15 including NLG919, a highly potent inhibitor.
Correcting tumor-induced immune deficiency selectively in
the TME to restore a natural anti-tumor immune capacity
without severe systemic immune disorders is termed as
normalization immunotherapy,6 which has revolutionized the
methodology of tumor therapy. In particular, immune
normalization by targeting the PD-1/PD-L1 pathway holds
great promise for regaining a lost anti-tumor immunity by
restoring T cell activity.6,7 In clinical trials, anti-PD-L1
antibody (aPD-L1) treatment induced durable tumor
regression and prolonged stabilization of disease in various
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However, as an imidazoleisoindole derivative, NLG919
successful preparation of NLG-RGD was confirmed by
analytical high performance liquid chromatography (HPLC,
Figure 1C), matrix-assisted laser desorption ionization with
time of flight mass spectrometry (MALDI-TOF-MS, Figure
1D) and Fourier transform infrared spectrometry (FT-IR,
Figure S2).
suffers from poor water solubility and demands frequently
high-dose administration to maintain IDO inhibition.17
Furthermore, the nonspecific distribution of NLG919 and its
strong potency can break IDO-mediated systemic immune
balance and lead to severe adverse effects.16
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Scheme 1. Schematic illustration of the modular design and self-
assembly of NLG-RGD and the proposed mechanism of
combinatorial normalization immunotherapy by concurrent
blocking IDO and PD-L1 using NLG-RGD NI and aPD-L1.
NLG-RGD NI is effectively internalized by cancer cells via αvβ3
integrin receptor-mediated endocytosis (i) followed by acidic pH-
triggered disassembly (ii) and esterase-catalyzed NLG919 release
(iii).
Figure 1. Synthesis and characterization of NLG-RGD. (A)
Synthesis of NLG-SA. (B) SPPS synthsis of NLG-RGD. (C)
HPLC trace of NLG-RGD. The purity of NLG-RGD is above
95%. (D) MALDI-TOF-MS analysis of NLG-RGD.
To specifically normalize the Trp pathway in tumors and to
avoid the shortcomings aforementioned, we develop a targeted
IDO nanoinhibitor (denoted as NLG-RGD NI) based on a
modularly designed, self-assembled peptide-drug conjugate
NLG-RGD (Scheme 1), which shows the perfect integrity of
structure and function: 1) RGD, a hydrophilic peptide, can
achieve tumor targeting by binding to αvβ3 integrin receptor; 2)
Preparation and Characterization of NLG-RGD NI. Due
to the π-π stacking and hydrophobic interactions, the
amphiphilic NLG-RGD can self-assemble into NLG-RGD NI
with a low critical micelle concentration (CMC, 2.63 μM)
(Figure S3). Transmission electron microscope (TEM) image
revealed that NLG-RGD NI had a spherical morphology with
a particle size of ~50 nm (Figure 2A), and its hydrodynamic
diameter was ~70.2 nm with a surface charge of ca. -5.1 mV
in neutral phosphate buffered saline (PBS, Figure 2B,C and
Table S2). When pH was lowered to 6.5, NLG-RGD NI still
maintained its spherical nanostructure (Figure S4). However,
after pH dropped to 5.0, NLG-RGD NI quickly lost its
structural integrity and become monomers or small aggregates
in 2 h due to the disassembly driven by the protonated
imidazole groups (Figure S5).25 Meanwhile, its hydrodynamic
size dropped to ~2.9 nm and surface charge increased to ca.
13.2 mV (Figure 2C).
Two histidines,
a pH-recognizable moiety, can drive
disassembly in the lysosome; 3) Ester bond, a hydrolysable
linker, can achieve controlled drug release upon esterase
treatment; 4) NLG919, a hydrophobic unit, can inhibit IDO
activity. Compared to previously reported polymer-NLG919
nanoconjugates,10,21-24 NLG-RGD NI consists of
a
biodegradable peptide backbone and a targeting motif with a
drug loading content as high as 27% (Table S1). Morever, the
NLG-RGD NI greatly improves the bioavailability of NLG919
and exerts persistent inhibition of IDO activity with reduced
systemic toxicity, which significantly boosts aPD-L1 based
immunotherapy by reversing immune tolerance in both
subcutaneous and orthotopic pancreatic tumor models.
The leakage of NLG919 from NLG-RGD NI was very slow
without esterase (Figure S6A). However, upon esterase
treatment, NLG-RGD NI displayed a sustained NLG919
release behavior at pH 7.4 or 6.5 and the release was
obviously accelerated at pH 5.0 (Figure S6B-D and Figure 2D).
This phenomenon can be explained by the increased
accessibility between NLG-RGD monomer and esterase after
the acidic pH-triggered structural dissociation. Such a
RESULTS AND DISCUSSION
Synthesis and Characterization of NLG-RGD. NLG-
RGD was synthesized in two steps. First, a succinic acid-
modified NLG919 (NLG-SA) was prepared (Figure 1A and
Figure S1). Then, NLG-RGD was synthesized by a standard
solid-phase peptide synthesis (SPPS) method using Fmoc
protected amino acids and NLG-SA (Figure 1B). The
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cooperatively responsive nanoinhibitor is supposed to prevent
To demonstrate that inhibition of IDO by NLG-RGD NI
leads to enhanced T-cell proliferation, an in vitro T cells and
Pan02 cells co-culture experiment was carried out.21,26
NLG919 treatment led to a significant increase of EdU+ T cells
(Figure 2F and Figure S9), confirming the effectiveness of
IDO blockade in stimulating T-cell proliferation. Notably, the
proportion of EdU+ T cells was also markedly elevated
following NLG-RGD NI treatment. However, in line with its
inferior inhibition of IDO activity, NLG-RAD NI exhibited
moderate promotion of T-cell replication.
the premature release of NLG919 during its blood circulation,
and enable the controlled release of NLG919 after engulfed
into acidic lysosomes.
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Next, we studied the cellular uptake behavior of
nanoinhibitors. In order to track them, protoporphyrin IX-
labeled peptides, PpIX-RGD and PpIX-RAD, were
synthesized (Figure S10 and S11), and then were co-
assemblied with NLG-RGD and NLG-RAD to prepare
PpIXNLG-RGD NI and PpIXNLG-RAD NI (Table S2),
respectively. After incubation with Pan02 cells for 0.5 h,
PpIXNLG-RGD NI exhibited a stronger fluorescent signal than
PpIXNLG-RAD NI, confirming its more efficient cellular
uptake (Figure S12A). Moreover, a highly co-localized
fluorescence of PpIX and Lysotracker was observed in both
groups, suggesting that these internalized nanoinhibitors were
further transported to lysosomes. It is worth mentioning that
lysosome is an acidic organelle (pH ~5) with abundant
esterases,27 which is in favor of cooperatively responsive
NLG919 release. At 6 h post-treatment, a higher PpIX signal
was still observed in PpIXNLG-RGD NI treated cells and the
fluorescence was mainly distributed in the cytoplasm, where
the remaining prodrug molecules can be further hydrolyzed
with the help of cytosolic esterases and the uncaged NLG919
can interact with IDO. The much higher cellular uptake of
PpIXNLG-RGD NI was also confirmed by flow cytometry
(Figure S12B, C). It is identified that PpIXNLG-RAD NI is
preferentially taken up by clathrin-mediated endocytosis and
the internalization of PpIXNLG-RGD NI is mainly dependent
on caveolae-mediated endocytosis (Figure S13), which are
consistent with previous reported nanoparticles involved in
RGD modification.28 Together, these data suggest that RGD-
mediated active targeting results in increased cellular uptake
of NLG-RGD NI compared to NLG-RAD NI, finally leading
to enhanced IDO inhibition.
Figure 2. Preparation and characterization of NLG-RGD NI. (A)
TEM image of NLG-RGD NI at pH 7.4. Scale bar, 200 nm. (B)
The size distribution of NLG-RGD NI at pH 7.4. (C)
Hydrodynamic size and zeta potential of NLG-RGD NI after
equilibrium at pH 7.4, 6.5 or 5.0 for 2 h. (D) NLG919 release
profile upon esterase treatment at different pHs. (E) IDO
inhibition in Pan02 cells. (F) T-cell proliferation assay. Mean ±
SD., n = 3. **p < 0.01, ***p < 0.001, one-way ANOVA.
IDO Inhibition in Vitro. We then examined the
cytotoxicity of NLG-RGD NI towards Pan02 cells. The result
demonstrated that it had no influence on cell viability at
concentration up to 100 μM (Figure S7). Next, its capacity to
block the conversion of Trp to Kyn was evaluated. For
comparison, a non-targeted control nanoinhibitor (NLG-RAD
NI) with similar physical properties was prepared by replacing
glycine with alanine (Figure S8 and Table S2). NLG919 was a
potent inhibitor of IDO activity, with a half maximum effect
concentration (EC50) of 1.5 μM (Figure 2E and Table S3). Its
Pharmacokinetics and Pharmacodynamics. Self-
assembling peptide-drug conjugates are suggested to prolong
the blood circulation of their hydrophobic cargo.29 To prove
this, a model hydrophobic molecule PpIX and PpIXNLG-RGD
NI were injected intravenously into Pan02 tumor-bearing mice
and their blood clearance behaviors were investigated. As
expected, PpIX underwent rapid blood clearance and there was
little circulating PpIX at 0.5 h post-injection (Figure S14).
However, PpIXNLG-RGD NI was cleared much more slowly
and a strong signal was still observed even after 6 h,
suggesting its favorable pharmacokinetics.
inhibition was dose-dependent in
a narrow range of
concentrations. NLG-RAD NI also showed dose-dependent
inhibition, but over a wider range of concentrations and with a
much higher EC50 (7.3 μM) than NLG919. The reduced
potency of NLG-RAD NI can be explained by requiring
additional disassembly and hydrolysis steps to become its
pharmacological active form. Notably, although NLG-RGD
NI was less effective (EC50 = 3.9 μM) than NLG919, it had a
stronger inhibitory effect on IDO activity than NLG-RAD NI
presumably due to its enhanced cellular uptake.
To further confirm RGD-mediated active tumor targeting in
vivo, the bio-distribution of PpIXNLG-RAD NI and PpIXNLG-
RGD NI in tumors and major organs was revealed by
fluorescence imaging (Figure S15). At 24 h post-injection, the
tumor from PpIXNLG-RAD NI treated mouse exhibited a
higher fluorescence intensity than any other organ, suggesting
its preferential tumor accumulation via the enhanced
permeability and retention (EPR) effect.30-32 Notably,
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PpIXNLG-RGD NI treatment led to
a much stronger
fluorescence signal in tumor than its counterpart, proving its
more effective tumor accumulation via active targeting.
Moreover, the tumor still maintained a strong fluorescent
signal even at 48 h post-injection of PpIXNLG-RGD NI,
indicating its long-term tumor retention. In sharp contrast,
PpIX showed a weak fluorescent signal in tumor after 24 h
and a negligible fluorescent signal after 48 h due to the rapid
metabolism and poor tumor accumulation of small molecules.
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To prove that the admiring pharmacokinetics of NLG-RGD
NI leads to the favorable pharmacological inhibition of IDO,
we measured Kyn and Trp content in tumor after different
treatment (Figure 3A). The ratio of Kyn to Trp decreased
slightly at 24 h post-injection of NLG919 and then recovered
at 48 h, suggesting a weak and transient inhibitory effect. In
contrast, NLG-RGD NI exerted more potent suppression of
Kyn conversion than NLG919 and exhibited sustained
inhibition of intratumoral IDO activity over 48 h. Consistent
with its inferior tumor accumulation and cellular uptake, the
inhibitory effect of NLG-RAD NI was much weaker than
NLG-RGD NI. We also examined the systemic IDO activity
by measuring Kyn and Trp content in blood (Figure 3B).
NLG919 dramatically reduced the ratio of Kyn to Trp in 24 h,
indicating its potent inhibition of systemic IDO activity.
Thanks to the controlled release of NLG919, both NLG-RAD
NI and NLG-RGD NI showed moderate systemic IDO
suppression in 48 h, implying the reduced risk of arousing
immune disorders.33 Together, these results suggest that NLG-
RGD NI can significantly improve the bioavailability of
NLG919 and preferentially inhibit intratumoral IDO activity
in a sustained manner. It is worth mentioning that while
NLG919 is orally administrated twice a day to maintain
systemic IDO inhibition in clinical settings,17 our improved
IDO formulation may reduce the dose as well as frequency
and still provide superior intratumoral IDO suppression.
Figure 3. Combinatorial normalization immunotherapy using
NLG-RGD NI and aPD-L1 in the subcutaneous Pan02 tumor
model. (A) Kyn/Trp ratio in tumor. (B) Kyn/Trp ratio in blood.
The dash lines indicate the range of Kyn/Trp ratio in healthy mice.
(C) Scheme of combinatorial normalization immunotherapy. (D)
Tumor growth curves. (E) Photograph of excised tumors. Scale
bar, 1 cm. (F) Tumor weights. (G) Tumor inhibition rates. Mean ±
SD., n = 4. *p < 0.05, **p < 0.01, ***p < 0.001, one-way
ANOVA.
Dual-Blockade of IDO and PD-L1 for Combinatorial
Normalization Immunotherapy. Since IDO inhibition as a
standalone therapeutic intervention has limited efficacy in
preventing tumor progression,34 considerable efforts have been
made to investigate the combination therapy of immune
checkpoint blockade and IDO inhibition.35 So we first
evaluated the ability of NLG-RGD NI to augment the efficacy
of PD-L1 blockade immunotherapy in the subcutaneous Pan02
tumor model (Figure 3C). Single-agent aPD-L1 demonstrated
a mild anti-tumor activity with an undesirable tumor inhibition
rate (TIR) of 28.4% (Figure 3D-G). Surprisingly, when
combined with NLG919, aPD-L1 did not further delay tumor
growth (TIR = 27.2%). The insufficient IDO inhibition within
tumor should be blamed for this, which could be the same
reason for the recent failure of phase III ECHO-301 trial.36
A
similar result was observed when aPD-L1 was combined with
NLG-RAD NI (TIR = 33.7%). However, NLG-RGD NI in
combination with aPD-L1 dramatically boosted the therapeutic
efficacy with an improved TIR of 57.4%, which is
approximately two-fold higher than that of aPD-L1
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monotherapy and other combinations. The superior anti-tumor
efficacy of this combinatorial normalization immunotherapy
was further confirmed in the orthotopic Pan02 tumor model
with the highest TIR of 60.8% (Figure S16). Collectively,
these results indicate that the robust and persistent inhibition
of intratumoral IDO activity, which is only provided by NLG-
RGD NI, is a prerequisite for greatly improving the anti-tumor
response of PD-L1 blockade.
Analysis of anti-tumor immune responses. To elucidate
the immunological mechanisms underlying the enhanced anti-
tumor effect of this combinatorial therapy, we next analyzed
tumor infiltrating immune cell populations using flow
cytometry. Among all treatment groups, the combination of
aPD-L1 and NLG-RGD NI resulted in the highest proportion
of CD4+ and CD8+ T cells and interferon-γ (IFN-γ)-producing
CD4+ and CD8+ T cells (Figure 4A-D and Figure S17 and
S18). Moreover, compared to aPD-L1 monotherapy or other
therapeutic combinations, the percentage of Treg (CD4+CD25+)
cells dramatically decreased in aPD-L1 plus NLG-RGD NI
group (Figure 4E and Figure S19). Interestingly, aPD-L1 plus
NLG-RGD NI treatment also significantly increased the
percentage of NK (CD49b+CD69+) cells (Figure 4F and Figure
S20). NK cells also express PD-1 and are sensitive to
tryptophan deficiency,37,38 which can explain the enhanced NK
cell response after this combinatorial therapy. Finally,
intratumoral IFN-γ and interleukin-2 (IL-2), two important
indicators of T-cell activity and proliferation, were analyzed
by enzyme-linked immunosorbent assay (ELISA). Both of
them were found to be the highest in aPD-L1 plus NLG-RGD
NI group (Figure 4G, H). These findings support that aPD-L1
plus NLG-RGD NI efficiently elicits anti-tumor immunity by
enhancing the activation and survival of immune effector cells,
and attenuating the accumulation immunosuppressive Treg
cells. Similar anti-tumor immune responses were also revealed
by flow cytometry analysis in the orthotopic Pan02 tumor
mode (Figure S21).
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Figure 4. Analysis of anti-tumor immune responses in the
subcutaneous Pan02 tumor model. (A) Percentage of CD4+ T cells.
(B) Percentage of CD8+ T cells. (C) Percentage of IFN-γ-
producing CD4+ T cells. (D) Percentage of IFN-γ-producing
CD8+ T cells. (E) Percentage of Treg cells in CD4+ T cells. (F)
Percentage of NK cells in tumor. (G) Intratumoral IFN-γ. (H)
Intratumoral IL-2. Mean ± SD., n = 4. *p < 0.05, **p < 0.01, ***p
< 0.001, one-way ANOVA.
During the treatment period, no obvious changes in mouse
body weight were observed in any group (Figure S22).
Hematoxylin and eosin (H&E) staining results revealed
normal histological morphologies in major organs of all
treated groups (Figure S23). However, serum biochemical
analysis indicated that aPD-L1 plus NLG919 affected liver
function of the treated mice (Figure S24), which is a typical
adverse effect of NLG919.16 In contrast, other therapeutic
combinations demonstrated no noticeable changes of serum
biomarkers. Together, these results suggest the good tolerance
of NLG-RGD NI in combination with aPD-L1.
CONCLUSION
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Marshall, L.; Vahanian, N. N.; Jay Ramsey, W.; Kennedy, E.;
Mautino, M. R.; Link, C. J.; Lin, R. S.; Royer-Joo, S.; Liang, X.;
Salphati, L.; Morrissey, K. M.; Mahrus, S.; McCall, B.; Pirzkall,
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In summary, we report a modularly designed and self-
assembled IDO nanoinhibitor. In vivo studies demonstrate that
it achieves potent and persistent inhibition of intratumoral IDO
pathway due to its preferential tumor accumulation and
sustained drug release. Significantly, this nanoinhibitor boosts
the anti-tumor immune response of PD-L1 blockade by
increasing
immune
effector
cells
and
reducing
immunosuppressive cells. Therefore, it holds great promise to
serve as a powerful combination partner to enhance the
efficacy of current immune checkpoint blockade-based
immunotherapies by combinatorial normalization of the
immunosuppressive TME.
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ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
Materials, methods, 1H-NMR analysis, HPLC analysis,
MALDI-TOF-MS analysis, CMC measurement, acidic pH-
triggered disassembly, cell viability, cellular uptake,
pharmacokinetics, bio-distribution, flow cytometry analysis and
serum biochemical analysis. (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: niegj@nanoctr.cn
*E-mail: liyy@nanoctr.cn
Author Contributions
#These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This work was supported by the grants from National Basic
Research Plan of China (2018YFA0208900, 2016YFA0201600),
National Natural Science Foundation of China (31571021),
Innovation Group of the National Natural Science Foundation of
China (11621505), Frontier Research Program of the Chinese
Academy of Sciences (QYZDJ-SSW-SLH022), and the Key
Laboratory of Biomedical Effects of Nanomaterials and
Nanosafety, CAS.
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