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Catalytical nano-immunocomplexes for remote-controlled sono-metabolic checkpoint trimodal cancer therapy

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Checkpoint immunotherapies have been combined with other therapeutic modalities to increase patient response rate and improve therapeutic outcome, which however exacerbates immune-related adverse events and requires to be carefully implemented in a narrowed therapeutic window. Strategies for precisely controlled combinational cancer immunotherapy can tackle this issue but remain lacking. We herein report a catalytical nano-immunocomplex for precise and persistent sono-metabolic checkpoint trimodal cancer therapy, whose full activities are only triggered by sono-irradiation in tumor microenvironment (TME). This nano-immunocomplex comprises three FDA-approved components, wherein checkpoint blockade inhibitor (anti-programmed death-ligand 1 antibody), immunometabolic reprogramming enzyme (adenosine deaminase, ADA), and sonosensitizer (hematoporphyrin) are covalently immobilized into one entity via acid-cleavable and singlet oxygen-activatable linkers. Thus, the activities of the nano-immunocomplex are initially silenced, and only under sono-irradiation in the acidic TME, the sonodynamic, checkpoint blockade, and immunometabolic reprogramming activities are remotely awakened. Due to the enzymatic conversion of adenosine to inosine by ADA, the nano-immunocomplex can reduce levels of intratumoral adenosine and inhibit A2A/A2B adenosine receptors-adenosinergic signaling, leading to efficient activation of immune effector cells and inhibition of immune suppressor cells in vivo. Thus, this study presents a generic and translatable nanoplatform towards precision combinational cancer immunotherapy.
In vitro evaluation of the acidic TME/sono-activation of the nano-immunocomplex a Schematic mechanisms of the acidic TME/sono-activated disassociation of the nano-immunocomplex and the OFF-ON switches of sonodynamic ¹O2 generation, ADA-induced Ade degradation, and aPD-L1-mediated PD-L1 blockade. b The generation of ¹O2 in HNP, PNP, and HPNP in 1× PBS buffer (pH 7.4) ([HP] = 20 μmol/L or [ADA] = 800 U/L) as a function of the sono-irradiation (1.0 MHz, 1.2 W/cm², 50% duty cycle) time (n = 3). HPNP versus PBS: p < 0.0001. c TEM images and d DLS profiles of HPNPs in different conditions (pH 7.4, pH 7.4 with sono-irradiation, pH 6.8, and pH 6.8 with sono-irradiation). The experiments were repeated independently three times with similar results. e HPLC profiles and f quantification of Ade and Ino after 8 h incubation of HPNPs ([ADA] = 40 U/L) in PBS solutions containing Ade (20 mmol/L) with different treatments (pH 7.4, pH 7.4 with sono-irradiation, pH 6.8, and pH 6.8 with sono-irradiation) (n = 3). pH 6.8 with sono-irradiation versus pH 7.4: p < 0.0001. g Ade degradation profiles of HPNPs in the presence of Ade at different conditions (pH 7.4, pH 7.4 with sono-irradiation, pH 6.8, and pH 6.8 with sono-irradiation) (n = 3). pH 6.8 with sono-irradiation versus pH 7.4: p < 0.0001. h PD-L1 blockade efficiency after 12 h incubation of HPNPs at different conditions (pH 7.4, pH 7.4 with sono-irradiation, pH 6.8, and pH 6.8 with sono-irradiation) (n = 3). pH 6.8 with sono-irradiation versus pH 7.4: p < 0.0001. Sono-irradiation: 1.0 MHz, 1.2 W/cm², 50% duty cycle for 6 min. Statistical significance in b and g was calculated via a two-tailed Student’s t-test. Statistical significance in f and h was calculated via one-way ANOVA with a Tukey post-hoc test. ****p < 0.0001. The mean values and SD are presented. Source data are provided as a Source Data file.
… 
In vitro nano-immunocomplex-mediated activatable sono-metabolic checkpoint trimodal cancer therapy a Schematic illustration of experiment implementation for cellular uptake, SDT, ICD induction, and BMDC maturation. b Proposed mechanism of acidic TME/sono-activation of SDT and IMT for antigen presentation and DC maturation. c Confocal fluorescence images of 4T1 cancer cells after 12 h incubation with HNP, PNP, or HPNP ([HP] = 20 μmol/L or [ADA] = 800 U/L). d Confocal fluorescence images of 4T1 cells after 12 h incubation with HNP, PNP, or HPNP ([HP] = 20 μmol/L or [ADA] = 800 U/L), followed by staining with H2DCFDA with or without sono-irradiation (1.0 MHz, 1.2 W/cm², 50% duty cycle) for 6 min. The experiments in c and d were repeated independently three times with similar results. e Relative cell viabilities of 4T1 cells after 24 h incubation with HNP, PNP, or HPNP at different HP or ADA concentrations with or without sono-irradiation for 6 min (n = 3). HPNP + US versus HPNP: p < 0.0001. f Relative Ade content in the cell culture medium after 12 h incubation of 4T1 cells with HNP, PNP, or HPNP ([HP] = 1 μmol/L or [ADA] = 40 U/L) in the presence of additional Ade (10 mmol/L) by HPLC assay (n = 3). HPNP + US versus HPNP: p < 0.0001. g Quantification of HMGB1 expression in 4T1 cell nuclei after 12 h incubation with HNP, PNP, or HPNP ([HP] = 20 μmol/L or [ADA] = 800 U/L) with or without sono-irradiation for 6 min (n = 5). HPNP + US versus HPNP: p < 0.0001; HNP + US versus HNP: p < 0.0001. h Flow cytometry assay and i quantification of the matured DCs (CD80⁺CD86⁺) after 12 h incubation of BMDCs with the 4T1 cell supernatants with different treatments (n = 3). 4T1 cells were incubated with HNP, PNP, or HPNP ([HP] = 20 μmol/L or [ADA] = 800 U/L) with or without Ade addition or sono-irradiation for 6 min. HNP + US with Ade addition versus without Ade addition: p < 0.0001; HPNP + US versus HPNP with Ade addition: p < 0.0001; HPNP + US versus HPNP without Ade addition: p < 0.0001. Statistical significance in e was calculated via two-tailed Student’s t-test. Statistical significance in f, h, and i was calculated via one-way ANOVA with a Tukey post-hoc test. ****p < 0.0001. The mean values and SD are presented. Source data are provided as a Source Data file.
… 
In vivo NIR fluorescence imaging and nano-immunocomplex-mediated activatable sono-metabolic checkpoint trimodal cancer therapy a Pharmacokinetic analysis of blood concentration of HP in BALB/c mice at t = 1, 2, 4, 8, 12, or 24 h post-injection of HNP or HPNP (n = 3). b NIR fluorescence imaging of 4T1 tumor-bearing BALB/c mice at t = 0, 2, 4, 6, 8, 12, or 24 h post-injection of HNP or HPNP (injection dose: 200 μL, [HP] = 1 mmol/L, or [ADA] = 40 U/mL). c Biodistribution of HNP or HPNP in 4T1 tumor-bearing mice at 24 h after systemic administration (n = 3). HPNP versus HNP in tumors: p = 0.0035. d Quantitative analysis of SOSG MFIs in tumor tissues from HNP-, PNP-, or HPNP-injected mice with or without sono-irradiation (1.0 MHz, 1.2 W/cm², 50% duty cycle) for 6 min (n = 5). HNP + US versus HNP: p < 0.0001; HPNP + US versus HPNP: p < 0.0001. e Schematic illustration of the schedule for bilateral tumor model implantation and nano-immunocomplex-mediated activatable sono-metabolic checkpoint trimodal cancer therapy. Growth curves of primary tumors (f) and distant tumors (g) after different treatments (n = 5). 6 versus 1 in f: p < 0.0001; 8 versus 1 in f: p < 0.0001; 8 versus 1 in g: p < 0.0001. h Survival curves for the mice after different treatments using the Kaplan–Meier method (n = 5). p < 0.0001. i Quantification of caspase-3 expression (n = 5). 6 versus 1 in primary tumors: p < 0.0001; 8 versus 1 in primary tumors: p < 0.0001; 8 versus 1 in distant tumors: p < 0.0001. j Histological H&E staining of lung in 4T1 tumor-bearing mice. Images are representative of three biologically independent mice. k Schematic illustration of the schedule for tumor rechallenge study. l Growth curves of the reinoculated tumors (n = 5). HPNP versus HNP: p = 0.0014. m Survival curves for the mice with reinoculated tumors using the Kaplan–Meier method (n = 5). p = 0.0003. Cell viability of 3T3 and 4T1 cells as target cells (T) after incubation with effector T cells (E) isolated from spleen of the rechallenged and HNP- (n) or HPNP-injected (o) mice as a function of the E/T ratios (n = 4). 4T1 versus 3T3 in n: p = 0.0027; 4T1 versus 3T3 in o: p < 0.0001. Statistical significance inc, d, i, n, and o was calculated via one-way ANOVA with a Tukey post-hoc test. Statistical significance in f, g, and l was calculated via two-tailed Student’s t-test. Statistical significance in h and m was calculated via the log-rank test. **p < 0.01, ***p < 0.001, and ****p < 0.0001. The mean values and SD are presented. Source data are provided as a Source Data file.
… 
In vivo mechanistic study of nano-immunocomplex-mediated activatable sono-metabolic checkpoint trimodal cancer therapy a Flow cytometry assay of tumor-infiltrating T lymphocytes (CD8⁺ and CD4⁺) and quantification of CD3⁺ T cells (b) and CD3⁺CD8⁺ Teffs (c) in primary tumors (n = 3). 8 versus other groups in b and c: p < 0.0001. d Flow cytometry quantification of CD3⁺CD8⁺ Teffs in blood (n = 3). 8 versus other groups: p < 0.0001. e Schematic illustration of the schedule for implantation and treatment of 4T1 tumor-bearing immunodeficient NSG mice. Growth curves of primary tumors (f) and distant tumors (g) after different treatments (n = 5). HPNP + US versus saline in f: p = 0.0002; HPNP + US versus saline in g: not significant (ns). h Quantification of HMGB1 expression in primary tumors (n = 5). 8 versus 5: p < 0.0001; 6 versus 3: p < 0.0001. i Flow cytometry quantification of matured DCs (CD80⁺CD86⁺) in TDLNs (n = 3). 8 versus 5: p < 0.0001; 6 versus 3: p < 0.0001. Flow cytometry assay (j) and quantification (k) of CD4⁺Foxp3⁺ Tregs in primary tumors (n = 3). 8 versus 1: p < 0.0001. Quantification of Teff/Treg ratio in primary (l) and distant (m) tumors (n = 3). 8 versus other groups in l and m: p < 0.0001. Flow cytometry assay and quantification of F4/80⁺CD206⁺ M2 Macs (n and o) and CD11b⁺Gr-1⁺ MDSCs (p and q) in primary tumors (n = 3). 8 versus 1 in o: p = 0.0004; 8 versus 1 in q: p < 0.0001. r The relative populations of immune effector cells and immune suppressor cells after different treatments (n = 3). s Quantification of granzyme B expression in primary tumors (n = 5). 8 versus 5 in primary and distant tumors: p < 0.0001. t The Ade content in primary tumors after different treatments (n = 3). 8 versus other groups: p < 0.0001. Injection dose: 200 μL, [HP] = 1 mmol/L, or [ADA] = 40 U/mL; sono-irradiation: 1.0 MHz, 1.2 W/cm², 50% duty cycle for 6 min. Statistical significance in b–d, h, i, k–m, o, q, s, and t was calculated via one-way ANOVA with a Tukey post-hoc test. Statistical significance in f and g was calculated via a two-tailed Student’s t-test. ***p < 0.001, and ****p < 0.0001. The mean values and SD are presented. Source data are provided as a Source Data file.
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ARTICLE
Catalytical nano-immunocomplexes for
remote-controlled sono-metabolic checkpoint
trimodal cancer therapy
Chi Zhang 1, Jingsheng Huang1, Ziling Zeng1, Shasha He1, Penghui Cheng1, Jingchao Li1& Kanyi Pu 1,2,3
Checkpoint immunotherapies have been combined with other therapeutic modalities to
increase patient response rate and improve therapeutic outcome, which however exacerbates
immune-related adverse events and requires to be carefully implemented in a narrowed
therapeutic window. Strategies for precisely controlled combinational cancer immunotherapy
can tackle this issue but remain lacking. We herein report a catalytical nano-immunocomplex
for precise and persistent sono-metabolic checkpoint trimodal cancer therapy, whose full
activities are only triggered by sono-irradiation in tumor microenvironment (TME). This
nano-immunocomplex comprises three FDA-approved components, wherein checkpoint
blockade inhibitor (anti-programmed death-ligand 1 antibody), immunometabolic repro-
gramming enzyme (adenosine deaminase, ADA), and sonosensitizer (hematoporphyrin) are
covalently immobilized into one entity via acid-cleavable and singlet oxygen-activatable lin-
kers. Thus, the activities of the nano-immunocomplex are initially silenced, and only under
sono-irradiation in the acidic TME, the sonodynamic, checkpoint blockade, and immuno-
metabolic reprogramming activities are remotely awakened. Due to the enzymatic conversion
of adenosine to inosine by ADA, the nano-immunocomplex can reduce levels of intratumoral
adenosine and inhibit A2A/A2B adenosine receptors-adenosinergic signaling, leading to
efcient activation of immune effector cells and inhibition of immune suppressor cells in vivo.
Thus, this study presents a generic and translatable nanoplatform towards precision com-
binational cancer immunotherapy.
https://doi.org/10.1038/s41467-022-31044-6 OPEN
1School of Chemical and Biomedical Engineering, Nanyang Technological University, 70 Nanyang Drive, 637457 Singapore, Singapore. 2School of Physical
and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, 637371 Singapore, Singapore. 3Lee Kong Chian School of Medicine,
Nanyang Technological University, 59 Nanyang Drive, 636921 Singapore, Singapore. email: kypu@ntu.edu.sg
NATURE COMMUNICATIONS | (2022) 13:3468 | https://doi.org/10.1038/s41467-022-31044-6 | www.nature.com/naturecommunications 1
1234567890():,;
Content courtesy of Springer Nature, terms of use apply. Rights reserved
Cancer checkpoint immunotherapies that block negative
immune regulatory pathways have recently been approved
as rst- or second-line therapies for a growing list of
malignancies (e.g., melanoma, lymphoma, lung cancer, and
bladder cancer)1,2. However, the patient response rate to check-
point immunotherapies is generally low (1030%) which is
mainly due to the low tumor immunogenicity and the existence
of immunosuppressive tumor microenvironment (TME)3,4.To
potentiate the tumor for checkpoint immunotherapy, che-
motherapy has been applied to induce immunogenic cell death
(ICD) to enhance tumor immunogenicity1; whereas, targeted
therapy that modulates immune activation signaling (e.g., toll-like
receptor5and stimulator of interferon gene signaling6)or
immunosuppressive pathways (e.g., indoleamine 2,3-dioxygenase
(IDO)7, macrophage-colony-stimulating factor (M-CSF) reg-
ulatory pathway8, etc.) has been used to reprogram immuno-
suppressive TME9. However, clinical data has revealed that
combinational immunotherapies (e.g., programmed death 1 (PD-
1) antibodies combined with chemotherapeutic paclitaxel or IDO
inhibitors) generally exacerbate immune-related adverse events
(irAEs) in patients10. This is attributed to the fact that che-
motherapy and targeted therapy bring in toxicity to normal cells
in addition to excessive activation of self-reactive T cells induced
by immune checkpoint inhibitors11. Thus, despite their promise
for enhanced antitumor efcacy, combinational cancer immu-
notherapies require to be carefully implemented in a narrow
therapeutic window. Strategies for precisely controlled combina-
tional cancer immunotherapy can potentially tackle this issue but
remain lacking. Electromagnetic energy including magnetic
elds12, light13, X-rays14, and ultrasound (US)15 provides a
noninvasive and precise way to ablate tumors via localized irra-
diation, which minimizes the off-target side effects compared to
chemotherapy and targeted therapy16. Electromagnetic therapies
(such as phototherapy, magnetic hyperthermia therapy, and
radiotherapy) have been proved to induce ICD for enhanced
tumor immunogenicity17. Moreover, electromagnetic energy can
serve as an exogenous stimulus to activate the pharmaceutical
action of therapeutic agents. In comparison to the limited tissue-
penetrating light, highly destructive X-ray, and complicatedly
manipulated magnet eld, US possesses properties of great tissue
penetration depth (>10 cm), high spatiotemporal resolution, good
controllability, and high safety18. By virtue of its clinic translation
potential, sonotherapy has also been combined with immune
checkpoint blockade (ICB) to improve the treatment efciency
for cancer immunotherapy19,20. However, the current sono-
activatable strategy generally relies on US-triggered bursts of
drug-loaded micro- or nano-bubbles21,22, which inevitably causes
drug leakage in normal tissues due to the lack of covalent che-
mical bonding. Consequently, developing a cancer-specic sono-
activatable strategy to precisely control drug activity and mini-
mize off-target toxicity is highly desired.
We herein report the development of a catalytical nano-
immunocomplex for remote-controlled sono-metabolic check-
point trimodal cancer therapy (Fig. 1). The nano-
immunocomplex is composed of all Food and Drug Adminis-
tration (FDA)-approved components including hematoporphyrin
(HP), anti-programmed death-ligand 1 (PD-L1) antibody (aPD-
L1), adenosine deaminase (ADA), and bovine serum albumin
(BSA), which are assembled and covalently immobilized into one
nanoparticle entity via acidic TME-cleavable imine and sono-
activatable thioketal bonds. In physiological conditions, HP, aPD-
L1, and ADA are all inert due to the covalent crosslinked
immobilization of the nano-immunocomplex. Only in the con-
currence of the acidic TME and sono-irradiation, the nano-
immunocomplex not only generates singlet oxygen (1O
2
)to
eliminate tumor cells and induce ICD for improved tumor
immunogenicity but also unleashes aPD-L1 and ADA via the
scission of imine and thioketal bonds. In this regard, aPD-L1
specically binds to PD-L1 on the surface of tumor cells to block
the PD-1/PD-L1 checkpoint pathway23, which can reinvigorate
effector T cells (Teffs) activity and inhibit regulatory T cells
(Tregs) function. ADA, a purine metabolic enzyme, can irrever-
sibly deaminate adenosine (Ade) and persistently convert it to
inosine (Ino) via the substitution of the amino group by a ketal
group, leading to the catalytical depletion of the toxic metabolites
Ade for immunometabolic therapy (IMT)24. This further results
in the elimination of adenosinergic signaling (especially A2AR
and A2BR signalling) and reprogramming of immunosuppressive
TME, which nally promotes the activation of antitumorigenic
immune effector cells (including dendritic cells (DCs) and
effector T cells (Teffs)) and inhibition of protumorigenic immune
suppressor cells (including myeloid-derived suppressor cells
(MDSCs), M2-like macrophages (M2 Macs), and Tregs)2528.As
a result, the catalytical nano-immunocomplex exerts the syner-
gistic antitumor effects via the cancer-specic and remote-
controlled sono-metabolic checkpoint trimodal cancer therapy.
Results
Synthesis and characterization. The nano-immunocomplex was
synthesized by the crosslinking of different molecules including
ethylenediamine-modied HP (HP-NH
2
), aPD-L1, ADA, and
BSA via the acidic TME-responsive crosslinker glutaraldehyde
(GA) and sono-activatable bis-N-hydroxy succinimide (NHS)-
conjugated thioketal (thioketal-NHS) crosslinker (Fig. 1a, Sup-
plementary Fig. 1). First, thioketal-NHS and HP-NH
2
were pre-
pared, and their intermediates were characterized by 1H NMR
and ESI-MS. Then, a mixture of HP-NH
2
, aPD-L1, and ADA in a
2 M sodium chloride solution was added with thioketal-NHS and
stirred at 4 °C overnight. BSA and GA were further added into the
solution and stirred at 4 °C for another 12 h. Thereafter, the
obtained nano-immunocomplex (HP-proteins-crosslinking
nanoparticles, abbreviated as HPNPs) was puried to remove the
free crosslinkers and proteins and washed three times with
phosphate buffer saline (PBS) solutions via ultraltration. For
comparison, two control nanoparticles, HP-crosslinking nano-
particles (HNPs) and proteins-crosslinking nanoparticles (PNPs),
were synthesized via similar methods. HNPs without the proteins
(aPD-L1 and ADA) were fabricated by the crosslinking of HP-
NH
2
, thioketal-NHS, BSA, and GA, while PNPs without the
sonosensitizer (HP) were prepared by the crosslinking of aPD-L1,
ADA, BSA, thioketal-NHS, and GA.
The physical and optical characteristics of these crosslinking
NPs (HNP, PNP, and HPNP) were studied. The transmission
electron microscopy (TEM) images indicated the similar particle
sizes and uniform size distribution of HNP (~47 nm), PNP
(~58 nm), and HPNP (~66 nm) (Supplementary Fig. 2a). The
dynamic light scattering (DLS) analysis also veried the similar
hydrodynamic sizes of HNP, PNP, and HPNP (Supplementary
Fig. 2b) and high stability of HPNP in both PBS solutions and cell
culture media (Supplementary Fig. 3). The zeta potential further
conrmed their similar surface potential owing to the cross-
linking of negatively charged BSA on the surface of these NPs
(Supplementary Fig. 2c). HNP and HPNP had similar UV-vis
absorption spectra with characteristic absorption peaks from the
HP unit at 372 nm, 503 nm, 535 nm, 565 nm, and 620 nm
(Supplementary Fig. 2d, e). The uorescence spectra of HNP and
HPNP were also similar with the characteristic emission peaks at
624 nm and 687 nm, which exhibited a slight redshift from the
HP characteristic emission peaks at 613 nm and 668 nm owing to
the existence of a ππinteraction of HP in the crosslinking
nanoparticles29 (Supplementary Fig. 2f). In contrast, PNP did not
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show any characteristic absorption or emission peak because of
the absence of HP. These data validated that these NPs had
similar physical properties, and HNP and HPNP exhibited similar
optical characteristics.
The cancer-specic and remote-controlled activation of the
nano-immunocomplex was studied (Fig. 2a). The generation of
1O
2
of these NPs was rst detected by using singlet oxygen sensor
green (SOSG) as a uorescence indicator30. The frequency
(1.0 MHz), power (1.2 W/cm2), duty cycle (50%), and irradiation
time (8 min) of US were all controlled at a safety range to ensure
the reversible permeabilization into the skin without safety
concerns31. The uorescence intensity of SOSG at 520 nm
gradually increased for HNP and HPNP under sono-irradiation
(1.0 MHz, 1.2 W/cm2, 50% duty cycle), which demonstrated the
generation of 1O
2
(Fig. 2b). The uorescence intensity of HNP
and HPNP exhibited about 2.3-fold increment after sono-
irradiation for 8 min. In contrast, the uorescence intensity of
the control and PNP groups did not show an obvious increment.
These data validated that the generation of 1O
2
for HNP and
HPNP upon sono-irradiation was owing to the existence of HP
and the crosslinking of different proteins (aPD-L1 and ADA) did
not affect the sonodynamic properties of HP.
Afterward, the morphological changes and functional activa-
tion of the smart nano-immunocomplex were studied. The TEM
images of HPNP exhibited a spherical morphology and uniform
size distribution in pH 7.4 solutions (Fig. 2c). After sono-
irradiation or incubation in pH 6.8 solutions, the particle sizes
were enlarged and a small portion of HPNP was disassociated
owing to the scission of the acidic TME-responsive imine bonds
or the sono-activatable thioketal bonds. Notably, the spheric
nanoparticles almost disappeared after the simultaneous treat-
ments of sono-irradiation and incubation in pH 6.8 solutions,
indicating the complete disassociation of HPNP. The DLS
analysis further elucidated the changes of particle sizes after
different treatments, which was consistent with the TEM results
(Fig. 2d). The generation of 1O
2
in acid conditions exhibited an
obvious increase compared to that in pH 7.4 solutions (Supple-
mentary Fig. 4), further indicating the acidic TME-responsive
disassociation of HPNP.
To verify the acidic TME/sono-activation of ADA along with
the disassociation of nanoparticles, high-performance liquid
chromatography (HPLC) analysis was conducted after incubation
of HPNP in PBS solutions containing the substrate Ade (Fig. 2e).
The elution peaks at 8.1 and 10.6 min were ascribed to Ino and
Ade, respectively. The free enzymes ADA exhibited a high
catalytic activity with only 8% residues of Ade in pH 7.4 solutions
after 8 h incubation (Fig. 2f). Meanwhile, the metabolites Ino
relatively increased due to the conversion of Ade to Ino via the
catalysis of ADA. After incubation of HPNP in pH 6.8 solutions
for 8 h and subsequent sono-irradiation for 6 min, the relative
Ade contents greatly decreased to 21%. In contrast, the remaining
Ade contents were 83%, 52%, or 59% after the incubation of
HPNP in the conditions of pH 7.4, pH 6.8, or pH 7.4 with sono-
irradiation. This was owing to the spatially restricted interaction
b
NN
NN
NH
2
NH
2
H
2
N
H
2
N
a
O O
c
OS
O
OH
HN
H
N
O
OH
N
N
NH
H
N
O
N
N
H
O
O
O
S
S
O
N
H
OO
O
S
S
O
N
H
1
O
2
O
SH
O
N
H
O
SH
O
N
H
NH
2
NH
2
N
N
N
N
H
2
N
O
OHOH
HO
O
NH
N
N
N
O
O
OHOH
HO
O
1
O
2
O
2
ADA Ade
Ino
Anti-tumor Pro-tumor
i. Acidic TME-mediated imine scission
pH
BSA GA
Nano-immunocomplex
HP-NH
2
Crosslinking
Thioketal-NHS
ADA
NH
2
NH
2
NH
2
aPD-L1
Acidic TME/
sono-activation
SDT
ICB
IMT
OFF
OFF
OFF
SDT
ICB
IMT
ON
ON
ON
ii. ICB: PD-1/PD-L1 blockade
PD-1PD-L1
Teffs
aPD-L1 Teffs activation
PD-L1 blocking
ii. Sonodynamic thioketal scission
US
iii. IMT: catalytical reprogramming of immunosuppressive TME
Antigen presentation
DCs
M2 polarization
M2
Macs
MDSCs activation
MDSCs Tregs
Tregs activation
Adenosinergic signaling elimination
i. SDT: ICD
SDT
HP
TAAs release
Tumor
cells
US irradiation
Disassociation
O
N
O
OS S O
O
O
OO
N
O
O
OH
HN
NH
O
OH
N
N
NH
H
N
O
H
2
N
H
2
N
Crosslinking
Fig. 1 Schematic illustration of the smart catalytical nano-immunocomplex for remote-controlled sono-metabolic checkpoint trimodal cancer therapy.
aSynthesis and activation of the nano-immunocomplex for synergistic sonodynamic therapy (SDT), immune checkpoint blockade (ICB), and
immunometabolic therapy (IMT). bAcidic TME/sono-activatable molecular scission mechanisms of the nano-immunocomplex. (i) Acidic TME-mediated
scission of imine bond. (ii) Sonodynamic scission of thioketal bond. cAcidic TME/sono-activatable cancer therapeutic mechanisms of the nano-
immunocomplex. (i) Hematoporphyrin (HP)-mediated SDT to induce 1O
2
generation, immunogenic cell death (ICD), and tumor-associated antigens
(TAAs) release. (ii) aPD-L1-mediated ICB for effector T cells (Teffs) activation via blocking PD-1/PD-L1 signaling pathway. (iii) Adenosine deaminase
(ADA)-mediated IMT to promote the antigen presentation of dendritic cells (DCs), reduce the protumorigenic polarization of M2 macrophages (M2
Macs), and inhibit the activation of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) via the elimination of adenosinergic pathway
by catalytical conversion of adenosine (Ade) to inosine (Ino) for immunosuppressive TME reprogramming.
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of Ade with ADA and the crosslinking-mediated shielding of the
catalytic sites of ADA in the nano-immunocomplex. The Ade
degradation proles further validated the acidic TME/sono-
activation of ADA (Fig. 2g, Supplementary Fig. 5). To further
study the acidic TME/sono-activation of aPD-L1, the PD-L1
blockade efciency was detected by enzyme-linked immunosor-
bent assay (ELISA). The ELISA results exhibited the highest PD-
L1 blockade efciency (99%) after incubation of HPNP in pH
6.8 solutions and sono-irradiation (Fig. 2h). In contrast, the PD-
L1 blockade efciency was only 45%, 60%, or 66% after
incubation of HPNP in the conditions of pH 7.4, pH 6.8, or
pH 7.4 with sono-irradiation. This is due to the incomplete
disassociation of HPNP and partial release of aPD-L1 in the
conditions of pH 7.4, pH 6.8, or pH 7.4 with sono-irradiation.
These results further validated the acidic TME/sono-activation of
the nano-immunocomplex for ADA-induced Ade degradation
and aPD-L1-mediated PD-L1 blockade.
In vitro studies of sono-metabolic checkpoint trimodal cancer
therapy. To evaluate the cellular uptake of NPs, 4T1 murine
breast cancer cells were rst incubated with HNP, PNP, or HPNP
for 12 h and imaged by confocal uorescence microscopy (Fig. 3a,
b). The red uorescence signals could be detected in HNP- or
HPNP-incubated cells (Fig. 3c). The mean uorescence intensities
(MFIs) of HNP- or HPNP-incubated cells were 79.8 or 69.4,
indicating their similar cellular uptake by 4T1 cells (Supple-
mentary Fig. 6a). In contrast, the PNP-incubated cells did not
show obvious red uorescence, because the red uorescence
012345678
1.0
1.5
2.0
2.5
F/F
0
Time (min)
PBS
HNP
PNP
HPNP
****
92%
17%
48% 41%
79%
8%
83%
52% 59%
21%
ADA
pH 7.4
pH 6.8
pH 7.4
pH 6.8
0
50
100
150
Ade degradation efficiency (%)
Ade Ino
****
+ US- US
024681012
0
50
100
150
Ade content (%)
Time (h)
ADA
pH 7.4 pH 7.4 + US
pH 6.8 pH 6.8 + US
****
1 10 100 1000 10000
0
10
20
30
40
50
60
Number (%)
Diameter (nm)
pH 7.4 pH 7.4 + US
pH 6.8 pH 6.8 + US
6 8 10 12
Time (min)
ADA
pH 7.4
pH 6.8
pH 7.4 + US
pH 6.8 + US
Inosine Adenosine
pH 7.4
pH 6.8
pH 7.4
pH 6.8
0
50
100
150
PD-L1 blockade efficiency (%)
****
+ US
- US
b
cde
fgh
a
pH/US
ADA
Ade
Ino
OFF
aPD-L1 PD-L1
OFFOFF
HP
O2
1O2
Ade
Ino
ON
PD-L1 blockade
ONON
O2
1O2
0.2 μm
pH 7.4 pH 7.4 + US
pH 6.8 pH 6.8 + US
Fig. 2 In vitro evaluation of the acidic TME/sono-activation of the nano-immunocomplex. a Schematic mechanisms of the acidic TME/sono-activated
disassociation of the nano-immunocomplex and the OFF-ON switches of sonodynamic 1O
2
generation, ADA-induced Ade degradation, and aPD-L1-
mediated PD-L1 blockade. bThe generation of 1O
2
in HNP, PNP, and HPNP in PBS buffer (pH 7.4) ([HP] =20 μmol/L or [ADA] =800 U/L) as a
function of the sono-irradiation (1.0 MHz, 1.2 W/cm2, 50% duty cycle) time (n=3). HPNP versus PBS: p< 0.0001. cTEM images and dDLS proles of
HPNPs in different conditions (pH 7.4, pH 7.4 with sono-irradiation, pH 6.8, and pH 6.8 with sono-irradiation). The experiments were repeated
independently three times with similar results. eHPLC proles and fquantication of Ade and Ino after 8 h incubation of HPNPs ([ADA] =40 U/L) in PBS
solutions containing Ade (20 mmol/L) with different treatments (pH 7.4, pH 7.4 with sono-irradiation, pH 6.8, and pH 6.8 with sono-irradiation) (n=3).
pH 6.8 with sono-irradiation versus pH 7.4: p< 0.0001. gAde degradation proles of HPNPs in the presence of Ade at different conditions (pH 7.4, pH 7.4
with sono-irradiation, pH 6.8, and pH 6.8 with sono-irradiation) (n=3). pH 6.8 with sono-irradiation versus pH 7.4: p< 0.0001. hPD-L1 blockade efciency
after 12 h incubation of HPNPs at different conditions (pH 7.4, pH 7.4 with sono-irradiation, pH 6.8, and pH 6.8 with sono-irradiation) (n=3). pH 6.8 with
sono-irradiationversus pH 7.4: p< 0.0001. Sono-irradiation: 1.0 MHz, 1.2 W/cm2, 50% duty cycle for 6 min. Statistical signicance in band gwas
calculated via a two-tailed Studentst-test. Statistical signicance in fand hwas calculated via one-way ANOVA with a Tukey post-hoc test.
****p< 0.0001. The mean values and SD are presented. Source data are provided as a Source Data le.
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signals came from the sonosensitizer HP. The intracellular lyso-
some colocalization analysis further indicated the effective
endosomal escape of NPs after incubation for 6 h (Supplementary
Fig. 7).
The sonodynamic activity and cytotoxicity of NPs were studied
in 4T1 cells. The 1O
2
uorescent turn-on probe 2,7-dichlor-
odihydrouorescein diacetate (H
2
DCFDA) was used to evaluate
the generation of 1O
2
in cells after sono-irradiation30. The
obvious green uorescence signals from the 1O
2
-oxidized 2,7-
dichlorouorescein (DCF) could only be detected in HNP- or
HPNP-incubated and sono-irradiated cells (Fig. 3d). The MFIs of
HNP- or HPNP-incubated and sono-irradiated cells were 92.7 or
93.5, both of which were about 25-fold higher than that of the
unirradiated cells (Supplementary Fig. 6b). Afterward, the
cytotoxicity of NPs was studied by using the 5-(3-carboxy-
methoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl)-tet-
razolium (MTS) assay. Before sono-irradiation, the HNP-, PNP-,
or HPNP-incubated cells exhibited relatively low cytotoxicity
with the cell viability of above 80% even at a high concentration
of NPs (Fig. 3e). This demonstrated the low cytotoxicity of
these NPs without sono-irradiation. After sono-irradiation, the
HNP- or HPNP-incubated cells showed the increased cytotoxicity
in a concentration-dependent manner. The cell viabilities of
HNP- or HPNP-incubated and sono-irradiated cells decreased to
~25% at the HP concentration of 8 μmol/L. These data validated
the good sonodynamic therapeutic properties of the nano-
immunocomplex.
The acidic TME/sono-activated Ade degradation was further
studied in 4T1 cells by using HPLC analysis of the Ade contents
in the cell supernatant. The relative Ade content was 82.02%
without any treatments owing to the autologous metabolism of
Ade by 4T1 cells (Fig. 3f). After incubation with HPNP and
subsequent sono-irradiation for 6 min, the relative Ade content
exhibited a remarkable decrease to 0.02%. However, PNP-
incubated cells, HPNP-incubated cells, or PNP-incubated and
sono-irradiated cells did not show obvious decrease of the Ade
contents (76.40%, 71.27%, or 62.02%). These data validated that
sono-irradiation could remotely control the activation of ADA,
leading to the degradation of Ade in cell levels after incubation
with the nano-immunocomplex.
To investigate the antitumor immunity after the acidic TME/
sono-activated degradation of Ade, ICD and DC maturation
were further studied (Fig. 3a, b). The release of high-mobility
group protein B1 (HMGB1) from the cell nuclei to extracellular
Control
PNP
HPNP
PNP
HPNP
0.00
0.05
50
60
70
80
90
100
Ade content (%)
****
+ US
- US
20
40
60
80
100
120
HP(ADA) concentration (μM(U/L))
Cell viability (%)
HNP HNP + US
PNP PNP + US
HPNP HPNP + US
0(0)
2(80)
4(160)
6(240)
8(320)
****
a b
cd
Control
HNPPNPHPNP
Control
HNPPNP HPNP
0
25
50
75
100
HMGB1 MFI (a.u.)
- US
+ US
**** ****
gh i
ef
Control
HNPPNPHPNP
Control
HNPPNPHPNP
0
10
20
30
40
50 - Ade
+ Ade
CD80
+
CD86
+
DCs (%)
- US
+ US
+ US
- US- US
****
**** ****
HNP PNP HPNP HNP + US PNP + US HPNP + US
-Ade
+ Ade
CD80
CD86
20 μm
Control HNP
PNP HPNP
Control HNP PNP HPNP
20 μm
-US
+ US
Fig. 3 In vitro nano-immunocomplex-mediated activatable sono-metabolic checkpoint trimodal cancer therapy. a Schematic illustration of experiment
implementation for cellular uptake, SDT, ICD induction, and BMDC maturation. bProposed mechanism of acidic TME/sono-activation of SDT and IMT for
antigen presentation and DC maturation. cConfocal uorescence images of 4T1 cancer cells after 12 h incubation with HNP, PNP, or HPNP
([HP] =20 μmol/L or [ADA] =800 U/L). dConfocal uorescence images of 4T1 cells after 12 h incubation with HNP, PNP, or HPNP ([HP] =20 μmol/L
or [ADA] =800 U/L), followed by staining with H
2
DCFDA with or without sono-irradiation (1.0 MHz, 1.2 W/cm2, 50% duty cycle) for 6 min. The
experiments in cand dwere repeated independently three times with similar results. eRelative cell viabilities of 4T1 cells after 24 h incubation with HNP,
PNP, or HPNP at different HP or ADA concentrations with or without sono-irradiation for 6 min (n=3). HPNP +US versus HPNP: p< 0.0001. fRelative
Ade content in the cell culture medium after 12 h incubation of 4T1 cells with HNP, PNP, or HPNP ([HP] =1μmol/L or [ADA] =40 U/L) in the presence of
additional Ade (10 mmol/L) by HPLC assay (n=3). HPNP +US versus HPNP: p< 0.0001. gQuantication of HMGB1 expression in 4T1 cell nuclei after
12 h incubation with HNP, PNP, or HPNP ([HP] =20 μmol/L or [ADA] =800 U/L) with or without sono-irradiation for 6 min (n=5). HPNP +US versus
HPNP: p< 0.0001; HNP +US versus HNP: p< 0.0001. hFlow cytometry assay and iquantication of the matured DCs (CD80+CD86+) after 12 h
incubation of BMDCs with the 4T1 cell supernatants with different treatments (n=3). 4T1 cells were incubated with HNP, PNP, or HPNP
([HP] =20 μmol/L or [ADA] =800 U/L) with or without Ade addition or sono-irradiation for 6 min. HNP +US with Ade addition versus without Ade
addition: p< 0.0001; HPNP +US versus HPNP with Ade addition: p< 0.0001; HPNP +US versus HPNP without Ade addition: p< 0.0001. Statistical
signicance in ewas calculated via two-tailed Studentst-test. Statistical signicance in f,h, and iwas calculated via one-way ANOVA with a Tukey post-
hoc test. ****p< 0.0001. The mean values and SD are presented. Source data are provided as a Source Data le.
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supernatants was rst detected to evaluate ICD32.TheHNP-or
HPNP-incubated and sono-irradiated 4T1 cells exhibited
obvious decrease of green uorescence signals from FITC-
labeled anti-HMGB1 antibodies in the cell nuclei (Supplemen-
tary Fig. 8). The MFIs of HNP- or HPNP-incubated and sono-
irradiated cells were 16.3 or 18.2 (Fig. 3g), which exhibited 4.1-
or 3.5-fold decreases compared to the unirradiated cells.
However, PNP-incubated cells with or without sono-irradiation
did not exhibit obvious decrease of the green uorescence signals
from FITC-labeled anti-HMGB1 antibodies. This conrmed that
ICD was induced by HP-mediated sonodynamic therapeutic
activity. Afterward, DC maturation was detected via ow
cytometry analysis. Bone marrow-derived dendritic cells
(BMDCs) were rst extracted from the mice and further cultured
in the RPMI 1640 cell culture medium33. Then the puried
BMDCs were incubated with the 4T1 cell supernatants after
different treatments for 12 h. The proportion of matured DCs
(CD80+CD86+) was 23.7%, 17.1%, or 28.1% after treatment with
HNP-, PNP-, or HPNP-incubated cell supernatants without
sono-irradiation (Fig. 3h, i). After sono-irradiation, HNP- or
HPNP-incubated groups exhibited 1.6-fold or 1.4-fold incre-
ments relative to that without sono-irradiation. This was owing
to the HP-mediated SDT and ICD for enhanced maturation of
DCs. Ade acts as an immunosuppressive metabolite, which can
impair the antigen presentation function of DCs with decreased
expression of CD80 and CD86 via the adenosinergic A2BR
signaling pathway25,34,35. Thus, DC maturation was further
evaluated with the addition of Ade in the culture medium. The
proportion of matured DCs greatly decreased by 1.4- and 1.3-
fold in HNP- and PNP-incubated and sono-irradiated groups in
the presence of additional Ade, respectively. However, the
HPNP-incubated and sono-irradiated groups did not show
obvious decrease of the matured DCs, which was ascribed to the
acidic TME/sono-activated Ade degradation. As a result, these
data demonstrated that the nano-immunocomplex could effec-
tively induce ICD and DC maturation for enhanced antigen
presentation.
In vivo sono-metabolic checkpoint trimodal cancer therapy.
Nano-immunocomplex-mediated activatable sono-metabolic
checkpoint trimodal cancer therapy was studied in 4T1 tumor-
bearing BALB/c mice. The pharmacokinetics of HNP and HPNP
were rst studied by detecting the uorescence signals from HP
via collecting the blood at different timepoints. HNP- or HPNP-
injected mice exhibited similar pharmacokinetic proles owing to
the similar particle sizes and structures (Fig. 4a). To conrm the
optimal timepoint for sono-irradiation, the accumulation of NPs
in tumor tissues was then evaluated using in vivo NIR uores-
cence imaging in 4T1 tumor-bearing mice after intravenous
injection of HNP or HPNP. The uorescence intensities at tumor
sites of HNP- or HPNP-injected mice gradually increased and
reached the maximum values at 12 h post-injection, which were
4.0- and 5.6-fold higher than that of the background signals,
respectively (Fig. 4b, Supplementary Fig. 9a). Meanwhile, the
ex vivo uorescence imaging of major organs and tissue biodis-
tribution analysis of NPs further demonstrated their effective
tumor accumulation abilities (Fig. 4c, Supplementary Fig. 9b, c).
Notably, the tumor accumulation effect of HPNP was stronger
than HNP, which was owing to the enhanced tumor-targeting
ability of the activated aPD-L1. The immunouorescence staining
images of the tumor tissues in HNP- or HPNP-treated mice also
exhibited obvious red uorescence signals, which came from the
sonosensitizer HP (Supplementary Fig. 10). These data validated
that HNP and HPNP could passively accumulate at tumor sites
due to their small size and the existence of the hydrophilic corona
BSA, and the stronger tumor-targeting ability of HPNP than
HNP was owing to the existence of aPD-L1.
To verify the sonodynamic activity of these NPs, the 1O
2
generation was studied by detecting the green uorescence signals
from SOSG in tumor tissues. The sono-irradiation was conducted
on the tumors at the maximum accumulation timepoint (12 h
post-injection of NPs). The SOSG green uorescence signals were
detected in tumor tissues of HNP- or HPNP-injected and sono-
irradiated mice (Supplementary Fig. 10). Their MFIs exhibited
7.5-fold and 10.5-fold increments relative to the unirradiated
mice, respectively (Fig. 4d). These data conrmed the effective
generation of 1O
2
in tumor tissues of HNP- or HPNP-injected
and sono-irradiated mice.
Afterward, the bilateral 4T1 tumor model was established to
study the nano-immunocomplex-mediated sono-metabolic
checkpoint trimodal cancer therapy (Fig. 4e). 4T1 cells were
subcutaneously inoculated in the right ank of BALB/c mice as
the primary tumor; 5 days later, the same amount of cells was
subcutaneously inoculated in the left ank of mice as the distant
tumor. After 2 days, 4T1 tumor-bearing mice were injected with
mixed proteins (aPD-L1 and ADA), HNP, PNP, or HPNP and
treated with or without sono-irradiation. Then, the growths of the
primary and distant tumors and the survival of mice with
different treatments were monitored. The primary and distant
tumors in HPNP-injected and sono-irradiated mice were
completely inhibited (Fig. 4f, g). The HNP-injected and sono-
irradiated mice exhibited a short-term inhibition (14-day
monitoring period) and an obvious tumor recurrence of the
primary tumors with the prolonged monitoring period to 21 days.
No obvious regression or inhibition of the primary or distant
tumors was observed in the other treatment groups. Moreover,
the HPNP-injected and sono-irradiated mice exhibited prolonged
survivals relative to the mice with other treatments (Fig. 4h). The
therapeutic effects were further conrmed by caspase-3 immuno-
uorescence and hematoxylin and eosin (H&E) staining analysis.
The green uorescence signals from the FITC-labelled anti-
caspase-3 antibodies were detected in primary and distant tumors
of HPNP-injected and sono-irradiated mice at 14 days post-
injection (Supplementary Fig. 11). The MFIs were 31.7-fold and
37.9-fold higher than that of the unirradiated mice, respectively
(Fig. 4i). In contrast, MFIs in primary tumors of HNP-injected
and sono-irradiated mice exhibited a 32.9-fold increment relative
to the unirradiated mice, while the distant tumors did not show
obvious green uorescence signals. Moreover, obvious dead cells
were observed in both the primary and distant tumors of HPNP-
injected and sono-irradiated mice in contrast to the other groups
according to the H&E staining images (Supplementary Fig. 12).
These were also consistent with the caspase-3 immunouores-
cence staining results. To further conrm the inhibition of tumor
metastasis by nano-immunocomplex-mediated therapy, the H&E
staining of lung tissues from the mice with different treatments
was conducted. The obvious metastatic niches were found in all
groups except for the HPNP-injected and sono-irradiated mice at
22 days post-injection (Fig. 4j and Supplementary Fig. 13). As a
result, these data validated that nano-immunocomplex-mediated
therapy effectively inhibited tumor growth and metastasis,
indicating its strong antitumor effects relative to the other
treatments.
The bilateral CT26 tumor model was further established to
study the nano-immunocomplex-mediated sono-metabolic
checkpoint trimodal cancer therapy (Supplementary Fig. 14a).
The experiment details were similar with the bilateral 4T1 tumor
model. First, CT26 tumor cells suspended in RPMI 1640 cell
culture medium were subcutaneously inoculated into the right
ank (primary tumors) of each mouse (1×106cells/mouse).
5 days later, the same amounts of the cells were subcutaneously
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inoculated in the left ank (distant tumors) of the same mouse.
Then CT26 tumor-bearing BALB/c mice were randomly divided
into three groups (n=5). The mice in each group were
intravenously injected with 200 μL saline or PBS solutions
containing HNP or HPNP. After 12 h post-injection, the primary
tumor of each mouse was treated with sono-irradiation (1.0 MHz,
1.2 W/cm2, 50% duty cycle) for 6 min. The sizes of primary and
distant tumors and the body weights of mice were measured every
2 days for 16 days. The primary and distant tumors in HPNP-
injected and sono-irradiated mice were completely inhibited
(Supplementary Figs. 14b, c and 15). However, both the primary
and distant tumors of HNP-injected and sono-irradiated mice
exhibited moderate inhibition effects. In addition, the body
weights of mice with these treatments did not show obvious
changes (Supplementary Fig. 14d). The H&E staining images of
the major organs including heart, liver, lung, and kidney also did
not show obvious damages at 16 days post-injection (Supple-
mentary Fig. 16). This indicated the good biosafety and
biocompatibility of HNP and HPNP on CT26 tumor-bearing
mice. The caspase-3 immunouorescence and H&E staining
images further veried the antitumor effects of the nano-
immunocomplex-mediated therapy (Supplementary Fig. 14eg),
which were consistent with the results on 4T1 tumor models.
The tumor rechallenge was further studied to evaluate the
long-term antitumor and anti-recurrence effects of nano-
immunocomplex-mediated sono-metabolic checkpoint trimodal
cancer therapy (Fig. 4k). The 4T1 tumor-bearing mice that
survived after multiple HNP or HPNP injections and sono-
irradiation on day 40 were challenged with 4T1 cells on the other
side of anks (Supplementary Fig. 17). The tumor growths in
HPNP-injected mice were greatly inhibited compared to that of
the HNP-injected mice (Fig. 4l). The HPNP-injected mice also
Lung
1-Saline 2-Proteins 3-HNP 6-HNP + US 7-PNP + US 8-HPNP + US
100 μm
4-PNP 5-HPNP
HPNP HNP
6.5
5.5
4.5
4.0
5.0
6.0
7.0
×107 p/s/cm2/sr
0246812
24
Post-injection time (h)
0 1 10 50
0
20
40
60
80
100
120
Cell viability (%)
E/T ratio
3T3 4T1
HNP-injected mice
**
0 1 10 50
0
20
40
60
80
100
120
Cell viability (%)
E/T ratio
3T3 4T1
****
HPNP-injected mice
Heart
LiverSpleen
Lung
Kidney
Tumor
0
2
4
6
8 HNP
HPNP
%ID/g
**
0 102030405060
0
20
40
60
80
100
1 2
3 4
5 6
7 8
Survival (%)
Time (d)
****
12345678
0
50
100
150
Caspase-3 MFI (a.u.)
Primary tumor
Distant tumor
****
****
****
0 5 10 15 20 25
0
50
100
150
Concentration (μM)
Time (h)
HNP
HPNP
048121620
0
10
20
30
40
Relative primary tumor volume
Time (d)
1 2
3 4
5 6
7 8
****
****
048121620
0
50
100
Relative distant tumor volume
Time (d)
1 2
3 4
5 6
7 8
****
ab c
j
efgh
Saline
HNP
PNP
HPNP
Saline
HNP
PNP
HPNP
0
20
40
60
SOSG MFI (a.u.)
- US
+ US
****
****
10 15 20 25 30
0
500
1000
1500
Tumor volume (mm
3
)
Time post tumor rechallenge (d)
HNP
HPNP
**
01020304050
0
20
40
60
80
100
HNP
HPNP
Survival (%)
Time post tumor rechallenge (d)
***
d
i
klmno
Bilateral tumor model
Tumor rechallenge
-40 0-33 -32 -31 -30 -29 -28 -27 -26
Tumor implantation
i.v. injection of nanoparticles
Time (d )
US treatment
Rechallenge
-7 -2 0 1 2 3 4 5 6 7
Primary tumor implantation
Distant tumor implantation
Time (d )
i.v. injection of nanoparticles
US treatment
Fig. 4 In vivo NIR uorescence imaging and nano-immunocomplex-mediated activatable sono-metabolic checkpoint trimodal cancer therapy.
aPharmacokinetic analysis of blood concentration of HP in BALB/c mice at t =1, 2, 4, 8, 12, or 24 h post-injection of HNP or HPNP (n=3). bNIR
uorescence imaging of 4T1 tumor-bearing BALB/c mice at t =0, 2, 4, 6, 8, 12, or 24 h post-injection of HNP or HPNP (injection dose: 200μL,
[HP] =1 mmol/L, or [ADA] =40 U/mL). cBiodistribution of HNP or HPNP in 4T1 tumor-bearing mice at 24 h after systemic administration (n=3). HPNP
versus HNP in tumors: p=0.0035. dQuantitative analysis of SOSG MFIs in tumor tissues from HNP-, PNP-, or HPNP-injected mice with or without sono-
irradiation (1.0 MHz, 1.2 W/cm2, 50% duty cycle) for 6 min (n=5). HNP +US versus HNP: p< 0.0001; HPNP +US versus HPNP: p< 0.0001. eSchematic
illustration of the schedule for bilateral tumor model implantation and nano-immunocomplex-mediated activatable sono-metabolic checkpoint trimodal
cancer therapy. Growth curves of primary tumors (f) and distant tumors (g) after different treatments (n=5). 6 versus 1 in f:p< 0.0001; 8 versus 1 in
f:p< 0.0001; 8 versus 1 in g:p< 0.0001. hSurvival curves for the mice after different treatments using the KaplanMeier method (n=5). p< 0.0001.
iQuantication of caspase-3 expression (n=5). 6 versus 1 in primary tumors: p< 0.0001; 8 versus 1 in primary tumors: p< 0.0001; 8 versus 1 in distant
tumors: p< 0.0001. jHistological H&E staining of lung in 4T1 tumor-bearing mice. Images are representative of three biologically independent mice.
kSchematic illustration of the schedule for tumor rechallenge study. lGrowth curves of the reinoculated tumors (n=5). HPNP versus HNP: p=0.0014.
mSurvival curves for the mice with reinoculated tumors using the KaplanMeier method (n=5). p=0.0003. Cell viability of 3T3 and 4T1 cells as target
cells (T) after incubation with effector T cells (E) isolated from spleen of the rechallenged and HNP- (n) or HPNP-injected (o) mice as a function of the E/T
ratios (n=4). 4T1 versus 3T3 in n:p=0.0027; 4T1 versus 3T3 in o:p< 0.0001. Statistical signicance inc,d,i,n, and owas calculated via one-way
ANOVA with a Tukey post-hoc test. Statistical signicance in f,g, and lwas calculated via two-tailed Studentst-test. Statistical signicance in hand mwas
calculated via the log-rank test. **p< 0.01, ***p< 0.001, and ****p< 0.0001. The mean values and SD are presented. Source data are provided as a Source
Data le.
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exhibited a prolonged survival relative to the HNP-injected mice
(Fig. 4m). Moreover, the cancer-specic T-cell immunity was
evaluated via the co-incubation of tumor (antigen-specic) or
normal (antigen-nonspecic) cells with the isolated Teffs from
the spleen of the rechallenged mice in vitro36. The extracted Teffs
from the HNP-injected mice exhibited negligible cytotoxicity to
both 4T1 tumor cells and 3T3 normal cells with cell viabilities of
above 70% (Fig. 4n). In contrast, the cell viability of 4T1 cells after
incubation with Teffs from HPNP-injected mice was only 40% at
an E/T (effector T cells to target cells) ratio of 50 (Fig. 4o), which
is much lower than that of 3T3 cells (89%), indicating the
activation of specic antitumor immunity. These results validated
that HPNP-mediated sono-metabolic checkpoint trimodal cancer
therapy exhibited stronger specic antitumor and anti-recurrence
effects compared to HNP-mediated therapy.
The biosafety of nano-immunocomplex-mediated sono-
metabolic checkpoint trimodal cancer therapy was further
evaluated by monitoring the body weight and histologically
analyzing the major organs of mice. The body weights of mice
with different treatments did not show obvious changes
(Supplementary Fig. 18). The H&E staining images of the major
organs including heart, liver, spleen, and kidney also did not show
obvious damages at 14 days post-injection (Supplementary
Fig. 19). This indicated the good biosafety and biocompatibility
of these NPs.
In vivo irAEs evaluation. The immune-related adverse events
(irAEs) were studied in 4T1 tumor-bearing mice. 4T1 tumor-
bearing mice were intravenously injected with saline, aPD-L1, or
HPNP (injection dose: 200 μg per mouse aPD-L1). Then the
major organs (including heart, liver, lung, and kidney) and sera
were collected and analyzed after 2 days post-injection (Supple-
mentary Fig. 20a). The major indexes for liver function including
alanine transaminase (ALT) and aspartate aminotransferase
(AST) in aPD-L1-inejcted mice exhibited obvious increase com-
pared to that of saline- or HPNP-injected mice, indicating sig-
nicant damages to the liver tissues (Supplementary Fig. 20b, c).
The inammatory cytokines (including tumor necrosis factor
α(TNF-α), interferon-γ(IFN-γ), and interleukine-6 (IL-6)) in
serum of aPD-L1-inejcted mice also exhibited obvious increase
compared to that of saline- or HPNP-injected mice (Supple-
mentary Fig. 20df). These results validated that aPD-L1 treat-
ment could signicantly induce the liver damage and elicit
systemic inammatory response, while our nano-
immunocomplex effectively reduced the incidence of irAEs.
To deeply investigate irAEs after aPD-L1 treatment in 4T1
tumor-bearing mice, we detected the inltrated immune cells
(T cells and macrophages) and the inammatory cytokines (TNF-
α, IFN-γ, and IL-6) in major organs (including heart, liver, lung,
and kidney) after 2 days post-injection. The populations of
CD45+leukocytes, CD45+CD3+T cells, CD3+CD69+activated
T cells, F4/80+macrophages, and F4/80+CD80+M1-Macs in
heart, liver, lung, or kidney from aPD-L1-injected mice
signicantly increased compared to the saline- or HPNP-
injected mice (Supplementary Figs. 2124). Meanwhile, the
inammatory cytokines (TNF-α, IFN-γ, and IL-6) in heart, liver,
lung, or kidney exhibited signicant increase after aPD-L1
treatment in 4T1 tumor-bearing mice relative to that of the
saline or HPNP treatments (Supplementary Fig. 25). These results
further veried that aPD-L1 treatment could lead to the
inltration of inammatory immune cells and elevated cytokine
levels in major organs of 4T1 tumor-bearing mice, which could
not be found in HPNP-injected mice. Thus, our nano-
immunocomplex exhibited excellent biosafety and effectively
reduced the incidence of irAEs compared to the native aPD-L1.
In vivo mechanistic studies of sono-metabolic checkpoint tri-
modal cancer therapy. To study the mechanism of nano-
immunocomplex-mediated sono-metabolic checkpoint trimodal
cancer therapy, Teffs were rst detected by collecting the primary
and distant tumor tissues, blood, and spleens by ow cytometry
analysis. The populations of CD3+tumor-inltrating T lym-
phocytes (TILs), Teffs (CD8+), and activated Teffs
(CD8+CD69+) in primary and distant tumors of HPNP-injected
mice were higher than that of proteins-, HNP-, or PNP-injected
mice after sono-irradiation (Fig. 5ac, Supplementary
Figs. 2630). The frequencies and activation of Teffs in blood and
spleen of HPNP-injected mice also increased relative to that of
proteins-, HNP-, or PNP-injected mice after sono-irradiation
(Fig. 5d, Supplementary Figs. 31, 32). These results indicated that
nano-immunocomplex-mediated therapy greatly enhanced anti-
tumor Teffs immunity. To further verify the important roles of
Teffs in nano-immunocomplex-mediated antitumor immunity,
the antitumor effects were evaluated in 4T1 tumor-bearing
immunodecient NOD-Scid IL2rg/(NSG) mice, which lack
functional lymphocytes (Fig. 5e). The growths of primary tumors
in HPNP-injected and sono-irradiated mice were partially
inhibited owing to the sonodynamic antitumor activity of HPNP,
while the distant tumors exhibited negligible inhibition effects
compared to that of the unirradiated mice (Fig. 5f, g, Supple-
mentary Fig. 33). These data validated that nano-
immunocomplex-mediated therapy was dependent on the acidic
TME/sono-activation of Teff-mediated antitumor immunity.
To gain insight into the high antitumor Teffs immunity of
nano-immunocomplex-mediated therapy, the corresponding
immunological processes including ICD induction, DC matura-
tion, and cytokine release of NPs-injected 4T1 tumor-bearing
mice were investigated and compared. The green uorescence
signals from FITC-labeled anti-HMGB1 antibodies were detected
in primary tumors of HNP- or HPNP-injected and sono-
irradiated mice at 7 days post-injection, which exhibited a similar
2.7-fold increase relative to that of the unirradiated mice (Fig. 5h,
Supplementary Fig. 34a). DC maturation was also studied by
detecting the population of matured DCs (CD80+CD86+)in
tumor-draining lymph nodes (TDLNs) by ow cytometry
analysis. The frequency of matured DCs in HNP- or HPNP-
injected and sono-irradiated mice increased by 2.0- or 2.2-fold
compared to that of the unirradiated mice (Fig. 5i, Supplementary
Fig. 35). The inammatory cytokines of the serum were further
detected via ELISA at 5, 7, or 14 days post-injection. Both the
serum concentration of IL-6 and TNF-αin HPNP-injected and
sono-irradiated mice reached maximum values at 7 days post-
injection, which increased by 11.6- and 23.6-fold relative to the
unirradiated mice, respectively (Supplementary Fig. 34b, c). These
results conrmed that HNP- or HPNP-mediated therapy
enhanced tumor immunogenicity to a similar level via inducing
ICD, promoting DC maturation and antigen presentation, and
stimulating inammatory cytokines release owing to their
analogical sonodynamic activity.
Afterward, the immunosuppressive TME was studied to
explore the difference between HNP- and HPNP-mediated
therapy. The immune suppressor cells including Tregs, M2 Macs,
and MDSCs play key roles in developing immunosuppressive
TME against antitumor Teffs immunity37, which were analyzed
in this study. The populations of Tregs (CD4+Foxp3+)in
primary and distant tumors of HPNP-injected and sono-
irradiated mice decreased by 80.6% and 72.5% compared to that
of the saline-injected mice (Fig. 5j, k, Supplementary Figs. 36, 37).
Notably, the ratios of Teff to Treg in primary and distant tumors
of HPNP-injected and sono-irradiated mice were ~10.6 and ~4.9,
which exhibited 75.5- and 37.7-fold increments compared to that
of the unirradiated mice, respectively (Fig. 5l, m). Moreover, the
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populations of the immunosuppressive M2 Macs and MDSCs in
primary tumors of HPNP-injected and sono-irradiated mice were
lower than the mice in other groups, which showed 9.3- and 7.9-
fold decreases relative to that of the saline-injected mice,
respectively (Fig. 5nq, Supplementary Figs. 3841). The
amounts of M2 Macs and MDSCs exhibited similar declining
trends in distant tumors of HPNP-injected and sono-irradiated
mice. The slight decreases of these immune suppressor cells
(including Tregs, M2 Macs, and MDSCs) and increases of
immune effector cells (Teffs) were also found in primary and
distant tumors of proteins-injected mice. This is because aPD-L1-
mediated ICB and ADA-mediated IMT can partially improve the
activity of Teffs and reprogram immunosuppressive TME. The
relative populations of immune effector cells (including matured
DCs, Teffs in tumor, and Teffs in blood) and immune suppressor
cells (including Tregs, M2 Macs, and MDSCs) further validated
that nano-immunocomplex-mediated therapy could leverage the
immune balance to the antitumor orientation (Fig. 5r). As a
12345678
0
5
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15
M2-Macs in primary tumors (%)
***
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0
5
10
MDSCs in primary tumors (%)
****
12345678
0.0
0.5
1.0
1.5
Tregs in primary tumors (%)
****
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0.0
0.5
1.0
1.5
2.0
CD3
+
CD8
+
T cells in blood (%)
****
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0
1
2
3
CD3
+
CD8
+
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in primary tumors (%)
****
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0
10
20
30
40
50
Granzyme B MFI (a.u.)
Primary tumors
Distant tumors ****
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0
10
20
30
40
50
HMGB1 MFI (a.u.)
**** ****
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0
5
10
Teff/Treg ratio in distant tumors
****
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0
5
10
15
Teff/Treg ratio in primary tumor
s
****
02468101214
0
10
20
30
Relative primary tumor volume
Time (d)
Saline
HPNP
HPNP + US
***
02468101214
0
10
20
30
40
50
Relative distant tumor volume
Time (d)
Saline
HPNP
HPNP + US
ns
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0
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20
30
40
CD80
+
CD86
+
DCs (%)
****
****
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0
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2
3
4
5
Ade content (nmol/g)
****
abcd
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j
klm
nor
eCancer therapy in NSG mice
CD4
CD8
T cells in primary tumors
Saline HNP + US HPNP + US
CD4
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Saline HNP + US HPNP + US
Tregs in primary tumors
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Saline HNP + US HPNP + US
MDSCs in primary tumors
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Saline HNP + US HPNP + US
M2 Macs in primary tumors
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0
2
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6
CD3
+
T cells
in primary tumors (%)
****
qp s
1. Saline 2. Proteins 3. HNP 4. PNP 5. HPNP 6. HNP + US 7. PNP + US 8. HPNP + US
t
Immune
suppressor
cells
Immune
effector
cells
0.25 0.34 0.31 0.39 0.45 0.62 0.54 1
0.0041 0.47 0.044 0.032 0.069 0.13 0.12 1
0.13 0.25 0.2 0.19 0.17 0.31 0.18 1
1 0.39 0.99 0.96 0.96 0.85 0.97 0.19
1 0.58 1 0.95 0.95 0.98 0.95 0.097
1 0.22 0.82 0.69 0.73 0.64 0.71 0.11
12345678
Matured DCs
Teffs in tumor
Teffs in blood
Tregs
M2 Macs
MDSCs
0.0
0.20
0.41
0.61
0.81
1.0
Primary tumor implantation
Distant tumor implantation
Time (d )
i.v. injection of nanoparticles
US treatment
-7 -2 0 1 2 3 4 5 6 7
Fig. 5 In vivo mechanistic study of nano-immunocomplex-mediated activatable sono-metabolic checkpoint trimodal cancer therapy. a Flow cytometry
assay of tumor-inltrating T lymphocytes (CD8+and CD4+) and quantication of CD3+Tcells(b) and CD3+CD8+Teffs (c) in primary tumors (n=3).
8 versus other groups in band c:p<0.0001. dFlow cytometry quantication of CD3+CD8+Teffs in blood (n=3). 8 versus other groups: p< 0.0001.
eSchematic illustration of the schedule for implantation and treatment of 4T1 tumor-bearing immunodecient NSG mice. Growth curves of primary tumors
(f) and distant tumors (g) after different treatments (n=5). HPNP +US versus saline in f:p=0.0002; HPNP +US versus saline in g: not signicant (ns).
hQuantication of HMGB1 expression in primary tumors (n=5). 8 versus 5: p< 0.0001; 6 versus 3: p< 0.0001. iFlow cytometry quantication of matured
DCs (CD80+CD86+)inTDLNs(n=3). 8 versus 5: p< 0.0001; 6 versus 3: p< 0.0001. Flow cytometry assay (j)andquantication (k)ofCD4
+Foxp3+Tregs
in primary tumors (n=3). 8 versus 1: p< 0.0001. Quantication of Teff/Treg ratio in primary (l)anddistant(m)tumors(n=3). 8 versus other groups in
land m:p< 0.0001. Flow cytometry assay and quantication of F4/80+CD206+M2 Macs (nand o) and CD11b+Gr-1+MDSCs (pand q) in primary tumors
(n=3). 8 versus 1 in o:p=0.0004; 8 versus 1 in q:p<0.0001.rThe relative populations of immune effector cells and immune suppressor cells after different
treatments (n=3). sQuantication of granzyme B expression in primary tumors (n=5). 8 versus 5 in primary and distant tumors: p<0.0001. tThe Ade
content in primary tumors after different treatments (n=3). 8 versus other groups: p< 0.0001. Injection dose: 200 μL, [HP] =1 mmol/L, or [ADA] =40 U/
mL; sono-irradiation: 1.0 MHz, 1.2 W/cm2, 50% duty cycle for 6min. Statistical signicance in bd,h,i,km,o,q,s,andtwas calculated via one-way ANOVA
with a Tukey post-hoc test. Statistical signicance in fand gwas calculated via a two-tailed Studentst-test. ***p< 0.001, and ****p< 0.0001. The mean values
and SD are presented. Source data are provided as a Source Data le.
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result, the expression of granzyme B could be obviously detected
in primary and distant tumors of HPNP-injected and sono-
irradiated mice, and the MFIs were 3.4- and 3.0-fold higher than
that of the unirradiated mice, respectively (Fig. 5s, Supplementary
Fig. 42). Then the aPD-L1 release in tumor tissues was studied via
immunouorescence staining to verify the aPD-L1-mediated ICB.
The immunouorescence staining images of the tumor tissues in
HPNP-injected and sono-irradiated mice exhibited obvious green
uorescence signals, indicating the effective release of aPD-L1
upon sono-irradiation (Supplementary Fig. 43). To further
conrm the immunosuppressive TME reprogramming by IMT,
the Ade content and immunosuppressive cyclic adenosine 3,5-
monophosphate (cAMP) level in tumor tissues were investigated
via HPLC analysis and ELISA, respectively38. After injection of
HPNP and subsequent sono-irradiation, the Ade contents in
primary tumors decreased by 94.5%, 91.3%, and 89.6% relative to
that of the saline-injected, unirradiated, and proteins-injected
mice, respectively (Fig. 5t). Meanwhile, the cAMP levels in
primary tumors of HPNP-injected and sono-irradiated mice
exhibited obvious decreases relative to that of the saline-injected
(69.0%), unirradiated (62.5%), and proteins-injected (37.9%) mice
(Supplementary Fig. 44). These results indicated that nano-
immunocomplex could induce more effective Ade and cAMP
depletion than the other groups via acidic TME/sono-activation
of IMT, leading to better immunosuppressive TME reprogram-
ming and stronger antitumor immunity. Although the intratu-
moral Ino contents should increase with the continuous catalytic
conversion of Ade by ADA, the exact mechanism of Ino action on
the immune system is still under investigation. Prior data
supports an immunosuppressive role for Ino, which can activate
A2AR signaling to inhibit inammation and antitumor
immunity39. However, inosine supplementation was recently
reported to enhance the antitumor efcacy of checkpoint
blockade therapy in specic mouse tumors that unable to
catabolize Ino to support cell growth40; Mager et al. also
demonstrated context-dependent actions of inosine on T cell
immunity, which boosted or inhibited Th1 differentiation of
naive T cells in the presence or absence of exogenous interferon-
γ, respectively41. Thus, whether the inosine action is relevant to
antitumor immunity deserves scrutiny.
To further study the mechanism of nano-immunocomplex-
mediated sono-metabolic checkpoint trimodal cancer therapy on
CT26 tumor-bearing mice, CD8+Teffs were rst detected by
collecting the primary and distant tumor tissues, blood, and
spleens by ow cytometry analysis. The populations of activated
CD69+T cells in primary and distant tumors, blood, and spleens
of HPNP-injected mice were obviously higher than that of HNP-
injected mice after sono-irradiation (Supplementary Fig. 45).
These results conrmed that nano-immunocomplex-mediated
therapy greatly enhanced antitumor Teffs immunity, which were
consistent with that on 4T1 tumor models. Then the correspond-
ing immunological processes including ICD induction and DC
maturation were investigated and compared. The green uores-
cence signals from FITC-labeled anti-HMGB1 antibodies were
detected in primary tumors of HNP- or HPNP-injected and
sono-irradiated mice at 7 days post-injection, which exhibited
2.1- and 2.5-fold increases relative to that of the unirradiated
mice, respectively (Supplementary Fig. 46a, b). DC maturation
was further studied by detecting the population of matured DCs
(CD80+CD86+) in TDLNs by ow cytometry analysis. The
frequency of matured DCs in HNP- or HPNP-injected and sono-
irradiated mice increased by 1.2- or 2.0-fold compared to that of
the unirradiated mice (Supplementary Fig. 46c).
Afterward, the immunosuppressive TME was studied to
investigate the mechanism of nano-immunocomplex-mediated
cancer therapy. The populations of Tregs (CD4+Foxp3+)in
primary and distant tumors of HPNP-injected and sono-
irradiated mice decreased by 68.1% and 63.1% compared to that
of the saline-injected mice, respectively (Supplementary Fig. 47a).
However, HNP-treated group only exhibited moderate decreases
of Tregs in primary (33.5%) and distant (38.9%) tumors relative
to that of the saline-injected mice. Moreover, the populations of
the immunosuppressive M2 Macs and MDSCs in primary tumors
of HPNP-injected and sono-irradiated mice were lower than the
mice in other groups, which decreased by 26.3% and 24.9%
relative to that of the saline-injected mice, respectively (Supple-
mentary Fig. 47b, c). Meanwhile, the amounts of M2 Macs and
MDSCs exhibited similar declining trends in distant tumors of
HPNP-injected and sono-irradiated mice. The expression of
granzyme B exhibited an obvious increase in primary and distant
tumors of HPNP-injected and sono-irradiated mice (Supplemen-
tary Fig. 47d). The MFIs were 3.2- and 5.4-fold higher than that
of the saline-injected mice, respectively. These results indicated
that nano-immunocomplex could induce more effective anti-
tumor immune responses than the other groups via enhancing
tumor immunogenicity and reprogramming immunosuppressive
TME in CT26 tumor model, which were consistent with that of
4T1 tumor model.
To deeply investigate the impact of nano-immunocomplex-
mediated therapy on TME, the transcriptome landscape of the
tumor tissues at 14 days post-injection was proled. A total of
26703 genes were identied. Nano-immunocomplex (HPNP-
injected and sono-irradiated groups) treatment promoted a
dramatic shift of the transcription program in TME relative to
the saline-injected or unirradiated HPNP-injected groups. Both
the principal component analysis (PCA) and Venn diagram
indicated the signicant discrepancy of the transcriptome land-
scape between the nano-immunocomplex and saline treatment
groups (Fig. 6a, b). Compared to the saline treatment, 938
differentially expressed genes were found under a threshold with
absolute fold changes >2 and pvalues < 0.05 after nano-
immunocomplex treatment. Specically, 866 upregulated genes
and 72 downregulated genes were identied in TME in nano-
immunocomplex treatment relative to saline treatment (Fig. 6c).
Thereafter, these differentially expressed genes associated with
immune functions were sorted out (Fig. 6d). Nano-
immunocomplex treatment induced the upregulated expression
of genes associated with the adaptive immune system (for
example, Klhl41 and Asb18 for antigen presentation; Cd300lg and
Sell for immunoregulatory interactions; Cdc34 and Prkcq for T
cell receptor signaling), cytokine signaling (including Camk2a
and Camk2b for interferon signaling; Il16 and Il11ra1 for
interleukin signaling; Eda2r and TNFR2 for non-canonical NF-
kB (nuclear factor kappa-light-chain-enhancer of activated B
cells) pathway), and innate immune system (for instance, Wasf3
for Fcγreceptor (FCGR) dependent phagocytosis; Cd55 and Cfd
for complement cascade; Cd36 and Nkiras1 for Toll-like receptor
cascades). Moreover, immunosuppressive genes (such as Pdcd1lg2
for negative regulation of the adaptive immune response) were
downregulated after nano-immunocomplex treatment. After-
ward, Kyoto Encyclopedia of Genes and Genomes (KEGG)
pathway enrichment analysis of these differentially expressed
genes validated that several immune-associated signaling path-
ways (for example, mitogen-activated protein kinase (MAPK)
signaling pathway for regulating Th1- and Th2-type immune
responses; Ras-associated protein 1 (Rap1) signaling pathway for
regulating T cell functions; Fc gamma R-mediated phagocytosis
signaling pathway for regulating innate immune system) were
obviously affected after nano-immunocomplex treatment
(Fig. 6e). To further predict the functional interactions of these
differentially expressed genes in immunological processes,
GeneMANIA, a multiple association network integration
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algorithm, was performed42 (Fig. 6f). The complex gene networks
indicated the co-expression of these differentially expressed genes,
followed by physical interactions. GeneMANIA analysis of the
gene sets associated with cytokine receptor binding (45 detected
genes of 287 genes), chemokine receptor binding (36 of 71),
humoral immune response (28 of 199), adaptive immune
response (18 of 294), granulocyte migration (40 of 153),
lymphocyte migration (31 of 99), and mononuclear cell migration
(26 of 86) further conrmed that multiple immunological
processes were activated and interacted with each other to
promote antitumor immunity. As a result, these transcriptome
data provided evidence that nano-immunocomplex-mediated
therapy had the ability to promote a cascade of transcriptional
events in multiple immunological processes to reshape immuno-
suppressive TME and activate antitumor immunity.
Discussion
Precision combinational immunotherapy is a highly desired
cancer therapeutic modality to increase patient response rate and
minimize irAEs, but it remains lacking. Our nano-
immunocomplex represents an example of catalytical nanome-
dicine whose sonodynamic, checkpoint blockade, and immuno-
metabolic reprogramming activities are dual-locked and can be
remotely activated by sono-irradiation in TME. As traditional
sono-irradiation strategy utilizes micro-/nanobubble loaded
nanoparticles to induce instant drug burst at tumor site via US-
mediated cavitation22, it still encounters nonspecic releases at
normal tissue and thus off-target toxicity. In contrast, our design
addresses this issue by covalently immobilizing the therapeutic
agents into one entity via acid-cleavable and 1O
2
-activatable lin-
kers followed by precise sonodynamic scission. This covalently
immobilized nanostructure was found to silence the activities of
both aPD-L1 and ADA in physiological conditions, which how-
ever can be awakened in acidic TME under sono-irradiation:
increased PD-L1 blockade efciency from 45 to 99% and Ade
degradation efciency from 17 to 79% (Fig. 2eh). Notably, all the
major components (HP, aPD-L1, and ADA) of the nano-
immunocomplex are FDA-approved, natural-derived, or self-
originated, which ensures the high biosafety and in turn high
clinical translation potential.
Enzymatic therapy has been utilized to afford more durable
response than small-molecule drugs for a broad range of
diseases4345. Our nano-immunocomplex represents a remote-
controlled enzymatic nanomedicine to reprogram the immuno-
suppressive TME precisely and persistently for cancer immu-
notherapy. Although several phase I/phase II clinical trials
combining Ade blockade therapy with chemotherapy or ICB have
been launched (e.g., NCT03549000, NCT03884556,
NCT02754141, NCT02740985, etc.)46,47, preclinical results
demonstrated their limited therapeutic efcacy in mouse
models48. The major reason is the existence of multiple Ade
184 204
276
1249
317 238
13431
HPNP Saline
HPNP + US
Camk2a
Camk2b
Il11ra1
Nkiras1
Il16
Eda2r
Grb10
Nkiras1
Cd36
Cfd
Gzmm
Cd55
Mapk12
Wasf3
Cdc34
Prkcq
Clec4d
Prss2
Lcn2
Art1
Cxcl3
Cxcl2
Sell
Retn
Pgm2
Fpr1
Dok3
Aldoa
Agl
Ppbp
Gstp2
Stbd1
Pigr
Svip
Naprt
Dpp7
Nos3
-1.620
-0.7640
0.09200
0.9480
1.804
2.660
Cdc34
Prkcq
Pdcd1lg2
Rasgrp3
Asb18
Asb5
Klhl41
Kbtbd13
Fbxl22
Asb16
Fbxo27
Prkn
Asb10
Asb11
Klhl13
Fbxl16
Cd36
Asb2
Fbxo40
Fbxo32
Tuba4a
Tuba8
Sell
Cd300lg
Rasgrp2
Btnl9
Adaptive immune system
Innate immune system
Cytokine
signaling
cGMP-PKG signaling pathway
Rap1 signaling pathway
PPAR signaling pathway
Proteoglycans in cancer
Regulation of lipolysis in adipocytes
Adipocytokine signaling pathway
MAPK signaling pathway
Calcium signaling pathway
Leukocyte transendothelial migration
Oxidative phosphorylation
AMPK signaling pathway
Fc gamma R-mediated phagocytosis
1.0 1.5 2.0
77
93
43
89
29
37
121
76
50
57
54
40
Enrichment ratio
50
100
150
Count
0
0.01
0.02
0.03
0.04
0.05
FDR
-5 0 5
0
1
2
3
4
Asb18
Wasf3
Asb5
Fbxl16
Cd300lg
Cfd
Sell
Fbxo27
Rasgrp3
Gzmm
Tuba8
Il16
Cxcl3
Mapk12
Nos3
Il11ra1
Pdcd1lg2
Prss2
Klhl41
Eda2r
Fbxo40
Camk2a
Cd55
Asb16
Cd36
-log
10
FDR
log
2
(Fold Change)
abc
d
e
f
Granulocyte migration
Cytokine receptor binding
Chemokine receptor binding
Humoral immune response
Adaptive immune response
Lymphocyte migration
Mononuclear cell migration
Functions
Physical interactions
Co-expression
Networks
Fig. 6 Transcriptome analysis of nano-immunocomplex-mediated sono-metabolic checkpoint trimodal cancer therapy. a Principal component analysis
(PCA) score plot of the expressed genes in TME of saline-injected, HPNP-injected, or HPNP-injected and sono-irradiated mice (n=3). bVenn diagram of
the identied differentially expressed genes. cVolcano plot showing 938 differentially expressed genes (26703 total genes) in TME from HPNP-injected
and sono-irradiated mice compared to TME from saline-injected mice. 866 upregulated genes and 72 downregulated genes were differentially expressedin
TME in HPNP-injected and sono-irradiated mice. dExpression of selected genes related to the adaptive immune system, cytokine signaling, and innate
immune system in TME from HPNP-injected and sono-irradiated mice compared to TME from saline-injected mice. eKEGG enrichment analysis of the
identied differentially expressed genes for studying the pathways of immune responses. fGeneMANIA analysis for predicting gene interactions between
differential genes in the immunological processes. Source data are provided as a Source Data le.
NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-022-31044-6 ARTICLE
NATURE COMMUNICATIONS | (2022) 13:3468 | https://doi.org/10.1038/s41467-022-31044-6 | www.nature.com/naturecommunications 11
Content courtesy of Springer Nature, terms of use apply. Rights reserved
production sources (including the CD39/CD73 axis, alkaline
phosphatase, and prostatic acid peptidase) and diverse purinergic
receptors (A2AR and A2BR)25. By virtue of the durable conver-
sion of Ade to Ino via the catalysis of ADA, our nano-
immunocomplex can potently and completely deplete Ade to
eliminate a spectrum of immunosuppressive adenosinergic sig-
naling pathways. It is validated by the higher Ade depletion
efciency (ca. 94%) of the nano-immunocomplex relative to that
of free proteins (ca. 47%) and unirradiated group (ca. 36%) in
4T1 tumor mouse model (Fig. 5t). Notably, this Ade depletion
efciency (ca. 99%) in 4T1 cells with additional Ade is also higher
than the reported studies using various anti-CD39/CD73 anti-
bodies (ca. 60%) in vitro49. Such a durable and broad elimination
of adenosinergic signaling pathways also leads to 10-times lower
dosage (ADA, 2 mg/kg) for the nano-immunocomplex relative to
that of the reported studies on anti-CD39/CD73 antibodies
(1020 mg/kg). In comparison with bimodal therapies with lim-
ited immunomodulation, the trimodal therapy based on adeno-
sinergic signaling elimination in combination with SDT and ICB
modulates the whole immunological processes (including
ICD induction, TAAs release, DC maturation, immunosuppres-
sive TME reprogramming, and immune effector cells activation).
Thus, the trimodal therapy (nano-immunocomplex treatment)
promotes obvious increments of immune effector cells (1.9- and
1.1-folds for matured DCs and effector TILs respectively,
Fig. 5ac, i) and remarkable decreases of immune suppressor cells
(51.3, 83.3, and 50.0% for Tregs, M2 Macs and MDSCs respec-
tively, Fig. 5jq) compared to the bimodal therapy (synergistic
aPD-L1 and ADA treatment), leading to the effective inhibition of
tumor growth, metastasis, and recurrence (Fig. 4).
In summary, we report a catalytical nano-immunocomplex
equipped with cancer-specic and sono-activatable activities for
precision sono-metabolic checkpoint trimodal cancer therapy.
Apart from its easy nano-formulation and FDA-approved safe
composition, the nano-immunocomplex possesses long-desired
advantages including whole immunological modulation, durable
and amplied reprogramming of immunosuppressive TME, and
dual-lock induced tumor specicity, holding a high promise for
translation. Such an effective design can be generalized for precise
intratumoral modulation of other immune-associated metabolic
pathways (such as glycolysis, glutaminolysis, hypoxia, lipid
metabolism, etc.)50,51, permitting precision multimodal cancer
immunotherapy.
Methods
Chemicals. All the chemicals were purchased from Sigma-Aldrich unless otherwise
specic indicated. Hematoporphyrin was purchased from MedChemExpress. Anti-
mouse PD-L1 antibodies were purchased from Bio X Cell. TrypsinEDTA (0.05%),
penicillinstreptomycin (10,000 U/mL), fetal bovine serum (FBS), RPMI 1640
medium, ACK lysis, type I collagenase, and type IV collagenase were purchased
from Gibco. The mouse PD-1[Biotinylated]:PD-L1 inhibitor screening assay kit
was purchased from BPS Bioscience, Inc. Mouse granulocyte macrophage-colony-
stimulating factor (GM-CSF) was purchased from i-DNA Biotechnology Pte Ltd.
SOSG was purchased from Molecular Probes Inc. (Carlsbad, CA, USA). 3-(4,5-
Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetra-
zolium, inner salt (MTS) solution was purchased from Promega Corp. (Madison,
WI, USA). Granzyme B antibody (ab255598, dilution: 1:200) and secondary
antibody Alexa Fluor 488 conjugated goat anti-rabbit IgG H&L (ab150077, dilu-
tion: 1:500) were purchased from Abcam Inc. (Cambridge, CA, USA). ELISA kits
for cAMP detection, HMGB1 antibody (Catalog no. 3935 S, dilution: 1:100), and
cleaved caspase-3 antibody (Catalog no. 9661 L, dilution: 1:500) were purchased
from Cell Signaling Technology. ELISA kits for IL-6 and TNF-αdetection, Zombie
UVxable viability kit (Catalog no. 423108, dilution: 1:500), puried anti-mouse
CD16/32 (Catalog no. 156604, dilution: 1:200), AF700 anti-mouse CD45 antibody
(Catalog no. 147716, dilution: 1:200), FITC anti-mouse CD3 (Catalog no. 100204,
dilution: 1:50), APC anti-mouse CD8a (Catalog no. 100712, dilution: 1:80), PE
anti-mouse CD4 (Catalog no. 130310, dilution: 1:80), BV605 anti-mouse CD69
(Catalog no. 104530, dilution: 1:40), APC anti-mouse CD11c (Catalog no. 117310,
dilution: 1:80), FITC anti-mouse CD80 (Catalog no. 104706, dilution: 1:50), PE
anti-mouse CD86 (Catalog no. 105008, dilution: 1:20), PerCP anti-mouse CD4
(Catalog no. 100432, dilution: 1:80), AF647 anti-mouse Foxp3 (Catalog no. 126408,
dilution: 1:50), PE anti-mouse CD11b (Catalog no. 101208, dilution: 1:80), AF488
anti-mouse F4/80 (Catalog no. 123120, dilution: 1:50), AF647 anti-mouse CD206
(Catalog no. 141712, dilution: 1:100), BV605 anti-mouse Gr-1 (Catalog no. 108440,
dilution: 1:40), and were purchased from Biolegend.
Material characterization. Electrospray ionization-mass spectrometry (ESI-MS)
spectra were conducted with a ThermoFinnigan LCQ quadrupole ion trap mass
spectrometer (Thermo Fisher Corporation) equipped with a standard ESI source.
Proton nuclear magnetic resonance (1H NMR) spectra were conducted on a Bruker
BBFO 400 MHz system (Bruker Physik AG, Germany). TEM images were captured
using a JEM 1400 transmission electron microscope (JEOL, Tokyo, Japan). DLS
and Zeta potential measurements were performed on a Malvern Nano-ZS Particle
Sizer (Malvern Instruments, Southborough, UK). Absorption and uorescence
spectra were measured on a UV-2450 spectrophotometer (Shimadzu, Japan) and a
Fluorolog 3-TCSPC spectrouorometer (Horiba Jobin Yvon), respectively. HPLC
analyses and purication were performed on an Agilent 1260 system using
methanol (MeOH)/water (H
2
O) as the eluent. Confocal images were captured
using an LSM800 confocal laser scanning microscope (Carl Zeiss, Germany). Flow
cytometry assay was performed on Fortessa X20 (BD Biosciences) and analyzed by
FlowJo v10. The absorbance or chemiluminescence intensities of each well in a 96-
well plate were measured using a SpectraMax M5 microplate reader. In vivo animal
uorescence images were captured using an IVIS imaging system (IVIS-CT
machine, PerkinElmer). Tissues were cut into sections using a cryostat (Leica). The
tissue sections were examined on a Nikon ECLIPSE 80i microscope (Nikon
Instruments). NMR spectra were analyzed using Mestre Nova LITE v5.2.5-
4119 software (Mestre lab Research S.L.).
Synthesis of HP-NH
2
. HP was purchased from MedChemExpress company and
characterized by 1H NMR. 1H NMR (DMSO-d
6
, 400 MHz): δ10.4911.00 (m,
4H), 6.59 (d, J=3.6 Hz, 2H), 4.41 (s, 4H), 3.673.76 (m, 12H), 3.19 (t, J=6.6 Hz,
4H), 2.07 (s, 6H).
Then, a mixture of HP (335.81 mg, 0.5 mmol), o-benzotriazole-N,N,N ,N-
tetramethyluroniumhexauoro-phosphate (HBTU, 455.10 mg, 1.2 mmol),
1-hydroxybenzotriazole (HOBt, 162.16 mg, 1.2 mmol), diisopropylethylamine
(DIPEA, 198.3 μL, 1.2 mmol), and N-Fmoc-ethylenediamine hydrobromide
(435.90 mg, 1.2 mmol) was dissolved in DMF under N
2
atmosphere and stirred at
room temperature for 6 h. Purication of the residue by silica gel column
chromatography gave the compound HP-NHFmoc (purple solid, 461.9 mg, yield
82%). 1H NMR (DMSO-d
6
, 400 MHz): δ10.3510.85 (m, 4H), 7.77 (t, J=6 Hz,
4H), 7.54 (t, J=6.8 Hz, 4H), 7.297.34 (m, 4H), 7.207.24 (m, 4H), 6.54 (s, 2H),
6.54 (s, 2H), 4.364.37 (m, 4H), 4.074.15 (m, 8H), 3.623.72 (m, 12H), 3.06 (t,
J=6.2 Hz, 4H), 2.952.97 (m, 4H), 2.702.75 (m, 2H), 2.112.16 (m, 6H). ESI (m/
z): calcd for C
68
H
7
0N
8
O
8
, 1126.5 [M]; found, 1149.6 [M +Na]+.
The obtained HP-NHFmoc (60 mg, 0.053 mmol) was further stirred in 4 mL of
piperidine/DMF (20%, V/V) solution at room temperature for 2 h. Purication of
the residue by HPLC using methanol/water as eluents gave the compound HP-NH
2
(purple solid, 18.8 mg, yield 52%). 1H NMR (DMSO-d
6
, 400 MHz): δ10.2510.76
(m, 4H), 6.516.56 (m, 2H), 6.54 (s, 2H), 4.234.44 (m, 12H), 3.673.71 (m, 12H),
3.003.08 (m, 4H), 2.952.97 (m, 4H), 2.142.18 (m, 6H). ESI (m/z): calcd for
C
38
H
50
N
8
O
4
, 682.4 [M]; found, 342.1 [1/2(M +2H)]+.
Synthesis of thioketal-NHS. A mixture of mercaptoacetic acid (9.2 g, 100 mmol),
acetone (3.2 g, 55 mmol), and 20 μL triuoroacetic acid (TFA) was stirred at 0 °C
for 3 h. The reaction mixture was washed by diethyl ether for three times to give
the compound thioketal-COOH (white solid 21.1 g, yield 95%). 1H NMR (DMSO-
d
6
, 400 MHz): δ12.60 (s, 2H), 3.56 (s, 4H), 1.53 (s, 6H). ESI (m/z): calcd for
C
7
H
12
O
4
S
2
, 224.0 [M]; found, 225.1 [M +H]+.
Then, a solution of obtained thioketal-COOH (2.5 g, 11.1 mmol) in anhydrous
tetrahydrofuran (THF, 60 mL) was slowly added with NaBH
4
(2.5 g, 66 mmol) in
an ice bath. Subsequently, I
2
(10 g, 28.5 mmol) in anhydrous THF (50 mL) were
added dropwise in the mixture and was heated to reux for 24 h and cooled to
room temperature. Methanol (~40 ml) was