Cold to Hot: Rational Design of a Minimalist Multifunctional

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Biological and Medical Applications of Materials and Interfaces

Cold to Hot: Rational Design of Minimalist Multifunctional PhotoImmunotherapy Nanoplatform toward Boosting Immunotherapy Capability Di Zhang, Jing Zhang, Qian Li, Aixin Song, Zhonghao Li, and Yuxia Luan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b09568 • Publication Date (Web): 20 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019

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ACS Applied Materials & Interfaces

Cold to Hot: Rational Design of Minimalist Multifunctional Photo-Immunotherapy

Nanoplatform

toward

Boosting

Immunotherapy Capability Di Zhang,† Jing Zhang,† Qian Li,† Aixin Song,‡ Zhonghao Li,‡ Yuxia Luan,† †

School of Pharmaceutical Science, Key Laboratory of Chemical Biology (Ministry of Education)

Shandong University, 44 West Wenhua Road, Jinan, Shandong Province 250012, China, E-mail: [email protected]. ‡

Key Laboratry of Colloid & Interface Chemistry, Shandong University, Ministry of Education,

250100, China.

Keywords: immunotherapy; IDO inhibitor; assembly; IR820; photothermal therapy

Abstract The concept of integrating immunogenic cell death (ICD) with tailoring immunosuppressive tumor microenvironment (TME) is promising for immunotherapy. Photothermal therapy (PTT) could efficiently induce ICD while indoleamine 2,3-dioxygenase (IDO) inhibitor could convert “cold” TME. Therefore, combination of PTT and IDO inhibitor is an attractive approach for immunotherapy. Unfortunately, combination of PTT and IDO inhibitor for tumor therapy is rarely reported. Herein, organic photothermal agent IR820 and IDO inhibitor 1-methyl-tryptophan (1MT) were, for the first time, designed to be an all-role-in-one molecule nanoplatform via molecular engineering strategy. The designed IR820-1MT molecule could self-assemble into nanoparticles with remarkably high dual-therapeutic agent loading (88.8 wt%). Importantly, poor water solubility 1

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of 1MT as well as inadequate target and short lifetime of IR820 were all well solved within as-prepared IR820-1MT nanoparticles. The laser-triggered IR820-1MT nanoparticles remarkably enhanced accumulation of cytotoxic T cells (CTLs), helper T cells (Ths), memory T cells and simultaneously suppressed proportion of regulatory T cells (Tregs), resulting in excellent immunotherapy against tumor metastasis and recurrence. Our molecular engineering strategy provides a promising alternative option for design of robust immunotherapy weapon against tumor metastasis and recurrence.

1. Introduction Cancer immunotherapy, attacking tumors by stimulating the immune system, has attracted increasing attention recently.1-3 In contrast to conventional therapies, immunotherapy could inhibit tumor metastasis and recurrence due to its systemic anti-tumor immune response as well as memory T cells.4 Indoleamine 2,3-dioxygenase (IDO), an intracellular enzyme overexpressed in tumor cells, could catalyze the degradation of tryptophan (Trp) into kynurenine (Kyn). Depletion of Trp could lead to anergy or apoptosis of T cells, and Trp starvation results in the differentiation from naïve CD4+ T cells to regulatory T cells (Tregs). So IDO is one of the culprits for the “cold” immunosuppressive tumor microenvironment (TME).5,6 The IDO inhibitor 1-methyl-tryptophan (1MT), is able to increase Trp and decrease Kyn for converting the “cold” immunosuppressive TME.7,8 However, its clinical application is hindered by deficient potency, poor water solubility, inadequate target and various side effects on normal tissues.9 Thus, improving the efficacy and reducing the restrictions of the IDO inhibitor have become an important theme for cancer immunotherapy. Photothermal therapy (PTT), photodynamic therapy (PDT) and some chemotherapies can not only kill primary tumors directly, but also induce immunogenic cell death (ICD) through release of 2


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tumor-associated antigens (TAAs) and damaged-associated molecular patterns (DAMPs) to activate immune response for synergistical immunotherapy.10 In comparison with PDT and chemotherapy for triggering ICD, PTT holds more advantages such as no-multidrug resistance and negligible systemic side effects in contrast to chemotherapy as well as no-relying on the oxygen microenvironment of cancer tissues in contrast to PDT.11 Moreover, the increase in blood flow and permeability caused by increased temperature of PTT could contribute to deep penetration and more internalization.12 Based on the precise-controlled near-infrared (NIR) laser to tumors, TAAs could be explosive released in situ as powerful supplements to native inefficient antigen.13,14 Therefore, combination of PTT and IDO inhibitor is an attractive strategy for synergistical immunotherapy. Equally

important,

such

in

situ

“vaccine-like”

strategy

might

be

promising

for

patient-individualized antigens against the false idea of “one size fits all” vaccine. Unfortunately, up to date, only two works were reported on the combination of PTT and IDO inhibitor for tumor therapy, which were based on the loading of the IDO inhibitor with graphene oxide or micelle carriers via noncovalent interaction.15,16 Although the carrier-based works are pioneering, it is still a challenge to achieve a robust strategy for combination of PTT and IDO inhibitor against the restrictions of carrier-based loading such as unknown carrier-related toxicity, disappointing drug loading, premature leakage, batch-to-batch variation and complexity of multi-composition. Therefore, a carrier-free, all-role-in-one molecule nanoplatform is highly demanded when translated to clinical application. To our knowledge, such all-role-in-one molecule nanoplatform combing PTT and IDO inhibitor via molecular engineering strategy has not been reported up to date. Herein, for the first time, we combined PTT and IDO inhibitor in an all-role-in-one molecule nanoplatform via molecular engineering strategy. New indocyanine green (IR820) was integrated 3



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with 1MT via sensitive ester bond for the formation of IR820-1MT molecule, which could self-assemble into nanoparticles with dual-therapeutic agent loading of 88.8 wt% (Scheme 1A). The IR820-1MT

nanoparticles

were

an

one-composition

nanoplatform

with

multifunctional

immunotherapy via the NIR-triggered domino effects to convert the “cold” immunosuppressive TME to the “hot” immunogenic TME (Scheme 1B). The IR820-1MT nanoparticles exhibited remarkably high efficacy toward inhibiting primary tumor, tumor metastasis and recurrence. To demonstrate the versatility of IR820-1MT nanoplatform applied as itself or in combination with other immunotherapy strategy, post-injected programmed death-ligand 1 antibody (aPD-L1) was further combined with IR820-1MT nanoparticles, resulting in excellent immunotherapy against tumor metastasis and recurrence. Therefore, our molecular engineering strategy provides a promising alternative option for design of robust immunotherapy weapon against tumor metastasis and recurrence.

4


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Scheme 1. (A) Illustration of preparation of IR820-1MT nanoparticles. (B) The schematic illustration for mechanism of IR820-1MT nanoparticles to inhibit primary tumor, metastasis and recurrence based on enhanced immunotherapy via synergistic photothermal therapy.

2. Materials and methods 2.1 Materials IR820, 2,6-lutidine, tetrahydrofuran (THF) and N,N-dimethylformamide (DMF) were purchased from Beijing J & K Technology Co., Ltd. 1MT, 6-aminocaproic acid (EACA), triethylamine (TEA), 1,3-propanediol, di-tert-butyl dicarbonate (Boc), tert-butyldimethylsilyl trifluoromethanesulfonate

(TBSOTf),

4-dime-thylaminopyridine

(DMAP),

dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) were all purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. MTT was bought from Beijing Solarbio Technology Co., Ltd. Annexin V-FITC/PI Apoptosis Detection Kit and D-luciferin were bought from Shanghai Yeasen Corporation. Trp and Kyn were bought from Sigma Co., Ltd. Phytohemagglutinin (PHA-M) was bought from Dalian Meilun Biotechnology Co., Ltd. Hoechst, Alexa Fluor 488-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) and calreticulin rabbit monoclonal antibody were bought from Beyotime Biotechnology Co., Ltd. HSP70 antibody, β-Actin antibody, Goat Anti-Mouse IgG (H+L) HRP and Goat Anti-Rabbit IgG (H+L) HRP were bought from Affinity Biosciences. Mouse IL-4 ELISA kit, mouse IL-5 ELISA kit, recombinant murine granulocyte-macrophage colony stimulating factor (GM-CSF) and recombinant murine IL-4 were obtained from MultiSciences (Lianke) Biotech Co., Ltd. Carboxyfluorescein succinimidyl ester (CFSE) Cell Division Tracker Kit, LEGENDplex™ Mouse Th1 Panel (5-plex), Cell Division Tracker Kit, True-Nuclear™ Transcription Factor Buffer Set, IL-2, recombinant mouse interferon gamma (IFN-γ), APC anti-mouse CD3, PE anti-mouse CD8a, FITC anti-mouse CD4, PerCP/Cy5.5 anti-mouse/human CD44, APC anti-mouse CD122, anti-CD11c-FITC, anti-CD80-APC and 5



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anti-CD86-PE and Alexa Fluor® 647 anti-mouse FOXP3 were all bought from BioLegend, Inc. 2.2 Cell lines. The mouse melanoma cell line B16F10 and mouse breast cancer cell line 4T1 were obtained from Institute of Immunopharmacology & Immunotherapy of Shandong University. B16F10-luc was obtained from the Department of Pharmacology of Shandong University. B16F10 cells and 4T1 cells were cultured with the final medium (1640 medium with 1% 100 × penicillin-streptomycin solutions and 10% fetal bovine serum). B16F10-luc cells were cultured with the final medium (MEM medium with 1% penicillin-streptomycin solution, 10% fetal bovine serum, 1% 100 × Non-essential amino acids, 1% 200 mM 100 × L-Glutamine, 1% 100 mM Na Pytuvate and 1% 100 × MEM vitamin solution). Both of them were cultured at 37 °C in 5.0% CO2. 2.3 Animals. 6 to 8 weeks female C57BL/6 mice were bought from Beijing Huafukang Biotechnology Co., Ltd. All animal experiments were carried out according to the Health Guide for the Care and Use of Laboratory the Animals of National Institutes and Shandong University Animal Experiment Ethics Review. 2.4 Preparation of IR820-1MT Nanoparticles. To prepare IR820-1MT nanoparticles, 3 mg IR820-1MT was dissolved in 0.2 mL DMSO and then 2 mL distilled water was dropped into the solution slowly. After that, the mixture was moved to a 1 KD dialysis bag and dialyzed against distilled water for 24 h to eliminate DMSO. 2.5 Characterization of IR820-1MT Nanoparticles. Morphology of IR820-1MT Nanoparticles was characterized via transmission electron microscopy (JEM-200CX). Size, size distribution and zeta potential were measured via Zetasizer Nano ZS90 (Malvern Instrument). Stability study of IR820-1MT nanoparticles themselves and in the presence of serum were also carried out. The critical aggregation concentration (CAC) was evaluated via digital conductivity meter (DDS-11A). 2.6 In vitro release of 1MT. Standard curves of 1MT in PBS (pH 7.4, 6.5 and 5.0) were established 6


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via fluorescence spectrophotometer (F-7000, Hitachi High Technology, ex = 287nm, em = 360 nm). 1 mL IR820-1MT nanoparticle solution (25 μg 1MT) in 1 KD dialysis bag was put into PBS (20 mL, pH 7.4, 6.5 or 5.0). 2.0 μM papain, a proteolytic enzyme like lysosomal cathepsin B, was added into pH 5.0 PBS to test enzyme-sensitivity of IR820-1MT nanoparticles. Release behaviors were studied at 37 °C with shaking (100 rpm). Release mediums (1 mL) were removed and substituted with fresh medium at prearranged times. The amount of 1MT released was diluted and measured via fluorescence spectrophotometer. Free 1MT was also carried out for comparison. Furthermore, morphology of IR820-1MT nanoparticles after 24 h incubation with pH 5.0 PBS plus 2.0 μM papain was characterized via transmission electron microscopy (TEM). 2.7 In vitro photothermal performance. 1 mL IR820-1MT nanoparticle solution with series concentrations was irradiated with 660 nm lasers (1.0 W·cm-2, MW-GX-660/2000 mW). The temperature was recorded via digital thermometer at the predetermined times. Free IR820 (irradiation with 808 nm laser, 1.0 W·cm-2, MW-GX-808/10 W) was also carried out for comparison. The 660 nm laser was chosen for irradiation of IR820-1MT nanoparticles because their maximum absorption peak was around 663 nm, and the 808 nm laser was chosen for irradiation of free IR820 because their maximum absorption peak was around 820 nm. The photothermal performance of IR820 under 660 nm irradiation was operated for comparison. Furthermore, photographs were taken via the infrared imaging device (Testo 869). To investigate the photothermal stability of IR820 and IR820-1MT nanoparticles, both of them were irradiated under four laser on/off cycles, and the temperature variation curves were recorded. 2.8 In vitro cellular uptake. B16F10 cells were cultured in 6 - well plates in 2 mL (3 × 105 cells/well) overnight and then treated with 29 μM IR820 or IR820-1MT nanoparticles for 1, 2, 4 or 6 h. Fluorescence inverted microscope (ECLIPSE-Ti, Nikon) was used to perform qualitative 7



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research and flow cytometry (BD FACSAria III) was used to conduct quantitative research. Moreover, the lipo-soluble, low-molecular weight flourescein isothiocyanate (FITC) was used to replace 1MT in the experiment of the cellular uptake of free 1MT due to the lack of fluoresce of 1MT. 2.9 In vitro cytotoxicity. B16F10 cells were cultured in 96 - well plates in 200 μL (6 × 103 cells/well) overnight and then incubated with 1.8 to 35 μM 1MT, IR820 or IR820-1MT nanoparticles for 4 h. Afterwards, medium was removed and substituted with fresh medium. For phototoxicity, the cells were irradiated by 1 min NIR laser (1.0 W·cm-2, IR820 at 808 nm while IR820-1MT nanoparticles at 660 nm) and then incubated for 24 h. The cells without incubating with drugs were also carried out for comparison. For dark toxicity, the cells were then incubated for 24 h. 5% 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 10 μL) was added into the medium and then incubated for 4 h. Afterwards, medium was removed and substituted with 150 µL DMSO. Each sample was characterized by the microplate reader (RS-232C) at 570 nm. The cell inhibition ratio was determined by the following formula.

Cell inhibition ratio (%)  (1 

A A

sample

) 100%

blank

Where Apositive, Asample and Ablank referred to the absorbance of the positive control group, blank control group and sample, separately. 2.10 In vitro IDO inhibitory effect. Standard curve of Kyn in medium was established via microplate reader at 480 nm. B16F10 cells were cultured in 48 - well plates in 800 μL (2 × 104 cells/well) overnight and then treated with 10 to 90 μM 1MT or IR820-1MT nanoparticles. The medium contained 50 ng·mL-1 IFN-γ to stimulate the IDO expression and 100 μM L-Trp to provide more reactant. After 48 h incubation, 140 μL supernatants were moved into an EP tube with the addition of 15 μL 30% trichloroacetic acid for 30 min incubation at 50 °C to precipitate protein. 8


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After


10

min

centrifugation

(3000

g),

100

μL

supernatants

and

100

μL

p-dimethylaminobenzaldehyde/acetic acid were mixed and moved into a new 96 - well plate for measurement at 480 nm in a microplate reader. Cells without incubating with drugs were also carried out for comparison. 2.11 In vitro immunogenic cell death induction. B16F10 cells were cultured in 6 - well plates in 2 mL (3 × 105 cells/well) overnight and then incubated with PBS, 1MT, IR820 or IR820-1MT nanoparticles (29 μM) for 4 h. Afterwards, medium was removed and substituted with fresh medium. The two group cells were irradiated by 1 min NIR laser (1.0 W·cm-2, IR820 at 808 nm while IR820-1MT nanoparticles at 660 nm) and washed by cold PBS. Then it was added with 100 μL lysis buffers. After grinding for 30 min, the mixture was centrifugated to obtain proteins. Western blot analyses were carried out following protocols for the routine with HSP 70 and β-actin antibodies. In the study of calreticulin (CRT) exposure, the cell incubation procedure was same as the western blot except for irradiation for 30 s and then incubation for 24 h. Next, cells were washed by PBS and stained nuclei by Hoechst for 15 min and then fixed with immune staining fix solution for 15 min. After that, the cells were incubated with primary antibody for 1 h, and then incubated with Alexa 488-conjugated secondary antibody for 1 h and finally incubated with PI for 15 minutes. Fluorescence inverted microscope (ECLIPSE-Ti, Nikon) was used to do qualitative research and flow cytometry was used to doing quantitative research. PI-negative cells were gated. 2.12 In vitro DCs maturation. Bone-marrow-derived dendritic cells (BMDCs) were obtained from the marrow cavities of C57BL/6 mice and seeded in 6-well plat in 1640 medium (2 mL) with GM-CSF (20 ng·mL-1) and IL-4 (10 ng·mL-1). Maturation of BMDCs was studied using the transwell system. BMDCs (5 × 105 cells/well) were co-cultured with B16F10 cells (6 × 104 cells/well) after treatments of 29 μM PBS, 1MT, IR820 (with/without NIR laser) or IR820-1MT 9



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nanoparticles (with/without NIR laser). 24 h later, BMDCs were collected to stain with anti-CD11c-FITC, anti-CD80-APC and anti-CD86-PE antibodies and then analyzed by flow cytometry. The supernatant of the culture medium was collected to analyze the secretion of IL-6 and TNF-α. 2.13 In vitro T-cells proliferation. To examine the immune response in vitro, mixed leukocyte reactions (MLRs) were carried out. B16F10 cells were cultured in 6 - well plates in 1 mL (5 × 104 cells/well) overnight and then incubated with PBS, 1MT, IR820 or IR820-1MT nanoparticles (29 μM) for 4 h. Afterwards, medium was removed and substituted with fresh medium. The two group cells were irradiated by 1 min NIR laser (1.0 W·cm-2, IR820 at 808 nm while IR820-1MT nanoparticles at 660 nm). Peripheral blood mononuclear cells (PBMCs) contain lymphocytes, DCs, monocytes and other small numbers of cells, were used to simulate immune microenvironment in vivo. Density gradient centrifugation with Ficoll-urografin was used to obtain PBMCs. In detail, a certain amount of Ficoll-urografin, lymphocyte separation solution, was added to the glass tube. Afterwards, the mouse peripheral blood diluted with PBS in equal volume was added into Ficoll-urografin. There was a clear boundary with blood in the upper layer and Ficoll-urografin in the lower layer. After 25 min centrifugation (500 g), the intermediate white membrane layer (PBMCs) was collected and washed twice with PBS. The obtained PBMCs were cultured with the final medium (1640 medium with 1% 100 × penicillin-streptomycin solutions and 10% fetal bovine serum). Afterwards, 1 mL PBMCs from mice at a density of 2 × 105 cells/well, stained by CFSE, were added into wells. 100 μM L-try and 50 ng·mL-1 IFN-γ were added into the medium to stimulate the IDO expression. In addition, 10 μg·mL-1 PHA-M and 1 ng·mL-1 IL-2 were added into the medium to activate T cells. After co-culture for 4 days, the medium was centrifuged to collect PBMCs, which were subsequently stained with APC-anti CD3 antibody. Flow cytometry was used 10


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to test T cell proliferation. CFSE and CD3 dual-positive T cells were gated. 2.14 Apoptosis analysis with/without PBMCs. B16F10 cells were cultured in 6 - well plates in 1 mL (5 × 104 cells/well) overnight and then incubated with PBS, 1MT, IR820 or IR820-1MT nanoparticles (29 μM) for 4 h. Afterwards, medium was removed and substituted with fresh medium. The two group cells were irradiated by 1 min NIR laser (1.0 W·cm-2, IR820 at 808 nm while IR820-1MT nanoparticles at 660 nm). Afterwards, 1 mL PBMCs (2 × 105 cells/well) in the same medium as the T cells proliferation experiment were co-cultured with B16F10 cells. 3 days later, B16F10 cells were collected and re-suspended in 200 µL PBS and then stained by PI (10 µL) and Annexin V-FITC (5 µL) for 15 min. Flow cytometry was used to analyze apoptosis. Cells without PBMCs co-culturing were also carried out for comparison. 2.15 In vivo imaging and biodistribution. To establish primary tumor-bearing mice model, 6 to 8 weeks female C57BL/6 mice were subcutaneous injected with 1 × 106 B16F10 cells in the right anterior armpit. Tumor volumes were calculated based on the formula V = L × W2 /2 (L refers to the longest tumor diameter and W refers to the shortest tumor diameter). When the tumor volumes were about 100 mm3, B16F10 primary tumor-bearing mice were divided into two groups (n = 5) at random. The mice were injected with IR820 and IR820-1MT nanoparticles at an equal IR820 dose of 6 mg·kg-1. The mice were imaged by in vivo the imaging system (IVIS Kinetic) at predetermined times. 24 h later, the mice were sacrificed. Tumor and organs were imaged. 2.16 In vivo photothermal performance. Above-mentioned B16F10 primary tumor-bearing mice were irradiated with 1.0 W·cm-2 after 4 h of intravenous injection with normal saline (NS), IR820 (808 nm) or IR820-1MT nanoparticles (660 nm) for 5 min. The mice were imaged via an infrared thermal imaging camera at the predetermined time during irradiation. 2.17 In vivo efficacy on anti-primary tumor. When the tumor volume were about 100 mm3, 11



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B16F10 primary tumor-bearing mice were divided into six groups (n = 9) at random and intravenous injected with 1) NS, 2) 1MT (1.5 mg·kg-1), 3) αPD-L1 (3 mg·kg-1), 4) IR820 (6 mg·kg-1), 5) IR820-1MT nanoparticles (8.5 mg·kg-1) and 6) IR820-1MT nanoparticles (8.5 mg·kg-1) & αPD-L1 (3 mg·kg-1), respectively. According to clinical use strategies, the 1, 2, and 3th groups were treated without lasers, and the 4, 5, and 6th groups were treated with lasers. In detail, 4th group mice were irradiated (808 nm) and the 5th, 6th group mice were irradiated (660 nm) for 5 min with 1.0 W·cm-2. Furthermore, the 6th group mice were intravenous injected with αPD-L1 after irradiation. The mice were treated every 3 days and measured every 2 days for 15 days and sacrificed on the 16th day. The excised tumors were weighted, imaged and immunofluorescence stained. The tumors and organs were hematoxylin and eosin (H&E) stained and executed histology analysis. The tumor-draining lymph nodes (TDLNs), tumors, spleens and blood were used to analyze the immune response. 2.18 In vivo IDO inhibitory effect. To analyze the Trp and Kyn in tumor tissues, the above-mentioned excised tumors were cut and ground to obtain the homogenate. Afterwards, the homogenate were moved into an EP tube with the addition of 15 μL 30% trichloroacetic acid for 30 min incubation at 50 °C to precipitate protein. The following operation was same as in vitro IDO inhibitory effect. Trp and Kyn were measured by a microplate reader. 2.19 In vivo anti-tumor immune response. The above-mentioned excised TDLNs were ground and then centrifuged to obtain a cell suspension. DCs were stained with anti-CD11c-FITC, anti-CD80-APC and anti-CD86-PE antibodies and then analyzed by flow cytometry. The above-mentioned excised tumors were used to analyze intratumoral infiltration of T lymphocytes. Briefly, lymphocytes in tumors were obtained following the protocols for the routine included cutting, dissociation, grinding, percoll, red blood cell lysis and centrifugation. The obtained 12


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lymphocytes in tumors were stained following the protocols with APC anti-mouse CD3 and FITC anti-mouse CD4 to analyze helper T cells (Ths, CD3+ CD4+), APC anti-mouse CD3 and PE anti-mouse CD8a to analyze cytotoxic T cells (CTLs, CD3+ CD8+), FITC anti-mouse CD4 and Alexa Fluor® 647 anti-mouse FOXP3 to analyze regulatory T cells (Tregs, CD4+ FOXP3+). The above-mentioned excised spleens were used to analyze the system of T lymphocytes. The obtained lymphocytes in spleens were also stained following the protocols to analyze Ths, CTLs and Tregs. Moreover, lymphocytes in spleens were stained with FITC anti-mouse CD4, PE anti-mouse CD8a, PerCP/Cy5.5 anti-mouse/human CD44 and APC anti-mouse CD122 to analyze memory T cells (CD4+ CD44+ CD122+ and CD8+ CD44+ CD122+). Furthermore, the above-mentioned blood was used to analyze system of cytokines including tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin-2 (IL-2), interleukin-6 (IL-6) and interleukin-10 (IL-10) by a kit (LEGENDplex™ Mouse Th1 Panel). All were analyzed via flow cytometry. For control, Th2 cytokines interleukin-4 (IL-4) and interleukin-5 (IL-5) were also detected by ELISA kit. 2.20 In vivo efficacy on anti-abscopal tumor. To establish the abscopal tumor-bearing mice model, 6 to 8 weeks female C57BL/6 mice were subcutaneous injected with 6 × 105 B16F10 cells in the right anterior armpit as the 1st tumor and then 6 × 105 B16F10 cells in the left anterior armpit for 4 days later as the 2nd tumor. When the 1st tumor volumes were about 70 mm3, mice were divided into six groups (n = 5) at random. The treatment plan was same with the primary tumor-bearing mice model except changing intravenous injection into intratumoral injection to control variables better. The mice were treated every 3 days and measured every 2 days for 15 days and sacrificed on the 16th day. The excised tumors were weighted, imaged and analyzed for immune response. 2.21 In vivo antigen-specific immune responses. To further prove antigen-specific immune responses, the different two types of tumors bearing mice model were established. Specifically, 6 to 13



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8 weeks female C57BL/6 mice were subcutaneous injected with 6 × 105 B16F10 cells in the right anterior armpit as the 1st tumor and then 6 × 105 4T1 cells in the left anterior armpit for 4 days later as the 2nd tumor. The treatment plan was same with the abscopal tumor-bearing mice model. The mice was divided into two groups and treated with NS and IR820-1MT nanoparticles (8.5 mg·kg-1) & αPD-L1 (3 mg·kg-1), respectively. The tumor volume of the 2nd tumor was recorded. For comparison, C57BL/6 mice with the 1st tumor of 6 × 105 B16F10 cells and the 2nd tumor of 6 × 105 B16F10 cells were also carried out. 2.22 In vivo efficacy on anti-tumor lung metastasis. To establish the lung metastasis tumor-bearing mice model, the primary tumor-bearing mice model was used and intravenous injected with 6 × 105 B16F10-Luc cells after treatment for 15 days. The mice were bioluminescence imaged after intraperitoneal injection of D-luciferin solution at predetermined times. The lungs were excised, imaged and H&E stained on the 10th day. 2.23 In vivo efficacy on anti-tumor post-surgical recurrence. To establish the post-surgical tumor recurrence mice model, when the tumor volume reached about 100 mm3, the primary tumor-bearing mice model were divided into six groups (n = 5) at random. After that, 90% tumor was removed by surgery and wounds were sutured. The mice were treated every 3 days, measured every 2 days and imaged for 14 days. The treatment procedure was similar with primary tumor-bearing mice model. Survival curves were drawn after treatment. 2.24 Statistical analysis. Statistical analysis was assessed by GraphPad Prism (7.0). T-test was used to count the statistical significance of two populations, and log-rank test was used to compare the survival curves. Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001. 2.25 Synthesis of Boc-1MT; Synthesis of Boc-1MT-OH; Synthesis of IR820-COOH; Synthesis of IR820-1MT-Boc; Synthesis of IR820-1MT; Hemolysis test of IR820-1MT Nanoparticles; In vitro 14


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singlet oxygen generation detection (Supporting Information). 3. Results and Discussion IR820-1MT was obtained through an esterification reaction (Figure S1, Supporting Information). Briefly, 1MT was first amine-protected by Boc-group (Figure S2-S4, Supporting Information) and then reacted with a 1, 3-propanediol linker via the formation of an ester bond (Figure S5 and S6, Supporting Information). IR820 was modified with a carboxyl group by reacting with an EACA linker (Figure S7-S9, Supporting Information). Next, IR820-1MT-Boc was prepared via an ester bond between IR820-COOH and Boc-1MT-OH (Figure S10 and S11, Supporting Information). Finally, IR820-1MT was obtained after deprotection of Boc-group (Figure S12 and S13, Supporting Information). 1H NMR and ESI-MS results all demonstrated that IR820-1MT was synthesized successfully. As the hydrophilic IR820 was conjugated to hydrophobic 1MT, the amphiphilic IR820-1MT molecule could self-assemble into nanoparticles without any precipitation in aqueous solution via dialysis method. The loading amount of IR820 was 78.52%, and 1MT was 18.15% in the IR820-1MT nanoparticles. The TEM image showed the assembly was a spherical nanoparticle (Figure 1A). Furthermore, dynamic light scattering (DLS) results showed the size of IR820-1MT nanoparticles was 105.7 nm with PDI of 0.204 (Figure 1B). Their zeta potential was -33.1 mV (Figure S14, Supporting Information). Size around 100 nm and a negative charge on the surface endowed IR820-1MT nanoparticles a highly accumulation into the tumors because of the enhanced permeation and retention (EPR) effect and a good stability in blood circulation, beneficial for intravenous administration.17-19 The obtained nanoparticles did not show phase separation or precipitation after storing at 4 °C for 3 months and no significantly change of size after storing at 4 °C for 15 days, demonstrating their excellent stability (Figure S15-S17, Supporting Information). 15



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The CAC for IR820-1MT was evaluated to be 5.53 × 10-5 mol·L-1 by conductivity measurement (Figure 1C). Hemolysis ratios determined for IR820-1MT nanoparticles were below 3% (Figure 1D), showing their high biosafety and biocompatibility. The TEM image of IR820-1MT nanoparticle had no obvious difference after 7 days’ incubation in pH 7.4 PBS (Figure S18, Supporting Information). Moreover, the size of IR820-1MT nanoparticles illustrated no significantly change under PBS (pH 7.4) containing 10% fetal bovine serum (FBS) after storing at 4 °C for 15 days, demonstrating the stability in the blood circulation (Figure S19, Supporting Information). In contrast, the TEM image of IR820-1MT nanoparticle had a significant change in size after 12 h incubation in pH 5.0 PBS with 2.0 μM papain (Figure 1E), which could be attributed to degradation of the ester bond of IR820-1MT in acid and esterase-containing environment. The particles in Figure 1E might be the in-stable co-assembled structure from the two part of broken IR820-1MT based on their π-π interaction.20 Furthermore, release behavior of 1MT was studied (Figure 1F). According to standard curves (Figure S20, Supporting Information), more than 95% 1MT had been released in pH 7.4 PBS at 4 h from free 1MT, showing unfavorably burst release in blood circulation. But fortunately, only 5.90% 1MT was released from IR820-1MT nanoparticles in PBS (pH 7.4) for 60 h, proving their high stability in the blood circulation. 9.67% 1MT was released from IR820-1MT nanoparticles in PBS (pH 6.5) for 60 h. In comparison, a higher 1MT (27.86%) release from IR820-1MT nanoparticles was observed in PBS (pH 5.0) for 60 h, demonstrating their good pH sensitive release behavior. Additionally, a significantly higher 1MT (61.76%) release from IR820-1MT nanoparticles was observed in PBS (pH 5.0) with 2.0 μM papain at 60 h, verifying the good enzyme-sensitive release behavior. The tumor lysosome has a slightly acidic environment and a variety of enzymes, so the PBS (pH 5.0) plus papain could well simulate the lysosome.21 The release behavior of IR820 from 16


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IR820-1MT nanoparticles was the same as that of 1MT due to molecular structure of IR820-1MT. Therefore, our IR820-1MT nanoparticles with pH- and enzyme-sensitive capability could have responsive release in cancer cells, ensuring the low side effects and high anti-tumor activities resulting from their high stability in blood circulation and sensitive release in the tumor environment. The UV-vis-NIR absorbance spectra (Figure S21, Supporting Information) demonstrated that IR820-1MT had the maximum absorption peak at 663 nm while IR820 at 820 nm. The blue shift of IR820-1MT nanoparticles may be due to the extension of π system and the extended conjugation length.22,23 Furthermore, for photothermal performance, IR820-1MT nanoparticle solutions were irradiated with 660 nm lasers while IR820 with 808 nm based on the maximum absorption peak. The photothermal performance in vitro was tested and the results were shown in Figure 1G to 1I and Figure S22 to S24 (Supporting Information). IR820-1MT nanoparticles exhibited similar photothermal ability with IR820. For instance, the temperature of IR820-1MT nanoparticles increased by 20.4 °C and IR820 increased by 22.2 °C at the same concentration after irradiation. Therefore, IR820-1MT nanoparticles could be used as efficient nanoplatform toward photothermal therapy. Moreover, the photothermal performance of IR820 under 660 nm irradiation also has been assessed. As shown in Figure S23 , the temperature of IR820 solutions (118 μM) increased by 10.9 °C after 5 min irradiation, which is weaker than under 808 nm (22.2 °C). Therefore, wavelength selection was suitable based on the best photothermal performance. After four on/off cycles of consecutive irradiation, there was no significant changes in photothermal performance, showing the good photothermal stability of IR820 and IR820-1MT nanoparticles (Figure 1H and Figure S24, Supporting information).24 The capability of generation of singlet oxygen was also investigated, and the results were shown in Figure S25 and S26. The capability of IR820 and IR820-1MT 17



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nanoparticles to generate singlet oxygen was negligible in comparison with the conventional photodynamic agent Chlorin e6 (Ce6). Therefore, the anti-tumor effect could be the photothermal effect rather than the photodynamic effect.25,26

Figure 1. (A) TEM image of IR820-1MT nanoparticles. (B) Size distribution and Tyndall phenomenon of IR820-1MT nanoparticles. (C) The electrical conductivity of IR820-1MT solution with various concentrations. (D) Hemolysis ratios of IR820-1MT nanoparticles (n = 3). Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001. (E) TEM image of IR820-1MT nanoparticles after 12 h incubation in PBS (pH 5.0) plus 2.0 μM papain. (F) In vitro 1MT release of various conditions. (G) Temperature changes in IR820-1MT nanoparticle solutions after irradiation (1.0 W·cm-2, 660 nm). (H) Temperature variation curves of IR820-1MT nanoparticle solutions under four cycles of irradiation. (I) Infrared thermal photographs of IR820-1MT nanoparticle and IR820 solutions after irradiation. 18


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Cellular uptake was investigated by flow microscope and fluorescence cytometry (Figure 2A and 2B). In comparison with free IR820, IR820-1MT nanoparticles showed a significantly enhanced internalization ability, resulting from the efficient endocytosis of nanoparticles in contrast to passive diffusion of free IR820. Moreover, cellular uptake of IR820 and IR820-1MT nanoparticles both exhibit time-dependence profile. FITC was used to replace 1MT for the study of the cellular uptake behavior of 1MT. IR820-1MT nanoparticles also showed much more enhanced internalization ability than free 1MT (Figure S27, Supporting Information). The in vitro cytotoxicity was investigated via MTT assay. IR820-1MT presented higher inhibition ratio than IR820 (Figure 2C) due to the higher internalization ability of nanoparticles. Moreover, IR820-1MT nanoparticles showed similar low dark cytotoxicity as that of IR820 (Figure S28, Supporting Information). Here, 1MT alone almost showed no inhibition to B16F10 cells because they should work together with the immune system via suppressing IDO pathway. Therefore, we measured the IDO inhibitor effect in B16F10 cells (Figure 2D) on the level of Kyn (Figure S29, Supporting Information), the downstream product of IDO pathway. After treatment, the lower Kyn concentration represented the better IDO inhibitor effect.27 The IDO inhibitor effect was increased with the increase of 1MT concentration within the scope of the study. Furthermore, IR820-1MT nanoparticles showed a similar IDO inhibitor effect with free 1MT due to the enhanced internalization ability but slower 1MT release behavior of IR820-1MT nanoparticles. These results fully confirmed that the PTT-IDO inhibitor strategy was feasible and the combination therapy efficacy would be reflected by the following MLRs and in vivo experiments.

19



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Figure 2. (A) Fluorescence microscopy images and (B) Flow cytometric results of cell uptake of IR820-1MT nanoparticles and IR820. The scale bars were 50 μm. (C) Cell inhibition ratio of 1MT, IR820 + laser and IR820-1MT nanoparticles + laser at different concentrations. (D) IDO inhibitory effect on the level of Kyn of different treated B16F10 cells. (n = 3) Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001. PTT cannot only kill cancer cells directly, but promote anti-tumor immune responses via ICD. Briefly, necrosis or apoptosis tumor cells release TAAs as in situ “vaccine-like” component and DAMPs, resulting in “eat me” signals to the immune system. Usually, the “eat me” signals can be indicated by cell surface expression of CRT and expression of HSP70. CRT was usually expressed in the endoplasmic reticulum. However, it would expose on the cell surface after PTT. Then, the cell surface exposed CRT would combine with immune cells to activate immune responses.28 HSP70 is positively associated with activation of the immune response after PTT.29 Flow cytometry 20


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was used to study the exposure of CRT by testing the fluorescence intensity of Alexa 488, a molecule conjugated with antibody to sign CRT. A significant CRT exposure was observed in the group of IR820-1MT nanoparticles + laser while less or even none exposure exhibited in other groups (Figure S30 and S31, Supporting Information). Fluorescence microscopy images exhibited the same results (Figure 3A). HSP70 from B16F10 cells was tested via western blot. Compared with the dark groups, more HSP70 expressions were displayed in laser groups (Figure 3B). Additionally, the HSP70 expression of IR820-1MT nanoparticles was more than that of IR820, which was due to the stronger cancer cell killing effect. Therefore, the domino enhanced immunotherapy effects were turned on by NIR laser. In order to assess the effect of antigen presentation after ICD, transwell system was used to co-culture B16F10 cells and BMDCs, and the mature DCs (mDCs, CD80+ CD86+) were further measured by flow cytometry (Figure 3C). There was 44.5% and 51.5% mDCs in the group of IR820 and IR820-1MT nanoparticles after irradiation, which was more than that without irradiation. The content of secretion of IL-6 and TNF-α were also tested. As shown in Figure S32 in supporting information, the two laser groups exhibited the highest levels of cytokines secretion, in consistent with the results of cell phenotypic. Therefore, ICD would enhance the antigen presentation in the immune response. To further investigate the activation of immune system by the combination of an IDO inhibitor and PTT, T-cells proliferation was tested by MLRs.30,31 As shown in Figure 3D, the proliferation index (PI) of NS group was as low as 1.62% while that of the 1MT group was 16.2%, confirming effectively preventing T cells anergy of 1MT via suppressing IDO pathway. Comparing the dark and laser groups, PI of IR820 increased from 8.34% to 20.7% whereas IR820-1MT nanoparticles increased from 26.1% to 41.5%, verifying the activation effect of the immune system by PTT. The peak based on the weaken CFSE index, was drawn to show the new generation of T 21



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cells. Compared with others, the group treated with IR820-1MT nanoparticles + laser presented the highest amount of T cells generations, demonstrating the most efficient T cells proliferation. To simulate the whole anti-tumor process of combination therapy in vitro, typical apoptosis analysis was presented in Figure 3E and the statistical result (n=3) for apoptotic index was calculated (Figure S33, Supporting Information). In the experiment without PBMCs, both IR820 and IR820-1MT nanoparticles with laser irradiation had a higher apoptosis and necrosis than without irradiation, agreeing with the cell inhibition results. Furthermore, the apoptosis index (the sum of Q2 and Q3 to the number of all collected cells) was higher for the sample co-cultured with PBMCs than that with no PBMCs. For instance, the apoptosis index of IR820-1MT nanoparticles without PBMCs was 35.59% and it increased to 72.37% with PBMCs. Consequently, there was a synergistic cycle in anti-tumor response. On the one hand, IR820-1MT nanoparticles with lasers can not only kill tumors but also cause T cells proliferation. On the other hand, proliferated T cells can continue to kill tumors with the synergistic cycle, achieving a more effective anti-tumor result.

22


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Figure 3. (A) Fluorescence microscopy images of CRT exposure of different treated B16F10 cells. Nucleus: blue color, CRT: green color. The scale bars were 12.5 μm. (B) Western blots of HSP70 expression of different treated B16F10 cells. (n = 3) Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001. (C) DCs maturation after co-culture with different treated B16F10 cells. (D) T cell proliferation of PBMCs with different treated B16F10 cells. (E) Apoptosis and necrosis of different treated B16F10 cells with or without PBMCs. Q1 means necrotic cells; Q2 means late apoptotic cells; Q3 means early apoptotic cells; and Q4 means live cells. 23



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Fluorescence imaging and biodistribution of IR820-1MT nanoparticles were investigated via the primary tumor-bearing mice model in contrast with IR820 (Figure S34, Supporting Information). There was a highest fluorescent signal in 4 h in both two groups, so we irradiated tumors after 4 h of injection. Compared with free IR820, IR820-1MT nanoparticles always maintain a higher fluorescence intensity. This is due to passive targeting via excellent EPR effect of IR820-1MT nanoparticles in contrast to rapid elimination of free IR820.32 After 24 h, tumor and main organs were excised for fluorescence imaging (Figure 4A and Figure S35, Supporting Information). Due to lack of tumor targeting, IR820 had more distribution in normal tissues than IR820-1MT nanoparticles, which could cause additional side effects. Therefore, IR820-1MT nanoparticles owned a higher accumulation in tumors than free IR820, which was advantageous for anti-tumor in vivo. The in vivo PTT capability was further investigated via the primary tumor-bearing mice model (Figure 4B and 4C). After irradiation, temperature increased by 18 °C in tumor with IR820-1MT nanoparticles in contrast to only 7.4 °C with free IR820 due to a higher accumulation of nanoparticles in tumor. According to the temperature requirement for PTT, this temperature increase was able to kill tumors effectively. Temperature increased by only 3.9 °C in tumor of mice treated with only laser irradiation, which cannot kill tumors. So the group of only laser was not necessary for further in vivo study. Furthermore, the increased temperature contributed to blood flow inside tumors and cells member permeability, which were beneficial to deep penetration in tumor tissues and internalization of tumor cells.33 The efficacy of IR820-1MT nanoparticles on anti-primary tumor was then tested (Figure 4D). Programmed death-ligand 1 (PD-L1), one of the inhibitory checkpoint receptors, could limit the anti-tumor effect of cytotoxic lymphocytes. Therapy combined with aPD-L1 is promising in clinical treatment.34,35 To demonstrate the versatility of IR820-1MT nanoplatform applied as itself or in 24


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combination with other immunotherapy strategy, aPD-L1 was further combined with IR820-1MT nanoparticles. aPD-L1 was post-injected as soon as the laser irradiation was done.36 The free aPD-L1 was carried out for comparison. Here, the primary tumor-bearing mice were treated by NS, 1MT, IR820, αPD-L1, IR820-1MT or IR820-1MT nanoparticles & αPD-L1 every 3 days. The IR820-1MT without irradiation was not assessed due to the poor cell inhibition ratio (Figure S28, Supporting Information). The tumor volume, tumor weight and inhibition ratio were measured every 2 days for 15 days (Figure 4E-4G and Figure S36, Supporting Information). The NS group showed a rapid tumor growth rate. The 1MT group showed a weak anti-tumor effect (inhibition ratio 14%) because of poor targeting and single treatment. The αPD-L1 group performed a delay of tumor growth (inhibition ratio 37%). Additionally, the IR820 group only indicated a moderate inhibition of tumor (inhibition ratio 50%), which was consistent with its poor photothermal effect in vivo. However, the IR820-1MT nanoparticles group exhibited remarkably higher anti-tumor effect (inhibition ratio 70%). Moreover, IR820-1MT nanoparticles combined with αPD-L1 could further enhance the anti-tumor effect with the inhibition ratio as high as 87%. The mice treated with NS, 1MT, αPD-L1, IR820, IR820-1MT or IR820-1MT nanoparticles & αPD-L1 exhibited 0%, 0%, 0%, 11%, 44%, 78% survival rate on 40th day (Figure 4H). Based on the above results, it was clear that IR820-1MT nanoparticles amplified the therapy efficacy in comparison with 1MT and IR820. Furthermore, IR820-1MT nanoparticles could further enhance the therapeutic effect of αPD-L1, demonstrating its versatility used as itself or in combination with other immunotherapy strategy. Compared with the NS group, there were no obvious changes in body weight in other groups (Figure S37, Supporting Information). The hematoxylin and eosin (H&E) stained images presented no visible damage of main organs in every group (Figure S38, Supporting Information). These results further verify the excellent biocompatibility of IR820-1MT nanoparticles in vivo. 25



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Figure 4. (A) Fluorescence images of tumor and organs after 24 h intravenous injections. (B) Infrared thermal images of mice and (C) Temperature-change curves of tumor being irradiation after intravenous injection. (D) Schematic illustration of anti-primary tumor experiment. (E) Change of average tumor volume, (F) Tumor weight and tumor inhibition ratio, (G) Images of excised tumors, (H) Survival curves of different treated mice. (n = 9) Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001. The mechanism of immune response in vivo was further investigated by flow cytometry and immunofluorescence. TDLNs are where tumor immunity occurs. DCs in TDLNs were collected to stain for flow cytometry analysis. As shown in Figure 5A, the expression of CD86 marker in mDCs was used for the quantification of the DCs maturation. Both IR820-1MT nanoparticles and combined with aPD-L1 groups promoted a much higher level of DCs maturation than other groups. The intratumoral infiltration of T lymphocytes was analyzed via flow cytometry (Figure 5B, 5C, 5D and Figure S39, Supporting Information). It was reported that cytotoxic T cells (CTLs, CD3+ CD8+) could kill tumors directly and helper T cells (Ths, CD3+ CD4+) was of vital importance in the 26


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process of immune regulation. Thus, both CTLs and Ths played an important role in anti-tumor immune responses.37 However, regulatory T cells (Tregs, CD4+ FOXP3+) could suppress the anti-tumor immune responses.38 From Figure 4I and 4J, there was a remarkable increase of CTLs and Ths after IR820-1MT nanoparticles treatment. For example, the proportions of CTLs for the IR820-1MT itself and IR820-1MT & αPD-L1 group were 1.97- and 2.33-fold higher than that of the NS group, respectively. The proportions of Ths for the IR820-1MT itself and IR820-1MT & αPD-L1 group were 1.66- and 1.93-fold higher than that of the NS group. In contrast to CTLs and Ths, the proportions of Tregs were largely reduced after treatment with IR820-1MT itself and IR820-1MT & αPD-L1 group. For example, the ratio of CTLs to Tregs was 5.10, 7.84, respectively for the IR820-1MT itself and IR820-1MT & αPD-L1 group in comparison with the NS group (1.21). The ratio of Ths to Tregs was 4.18, 6.34, respectively for the IR820-1MT itself and IR820-1MT & αPD-L1 group in comparison with the NS group (1.18) (Figure 5D). Based on these results, it was clear that IR820-1MT nanoplatform applied as itself or in combination with aPD-L1 could efficiently

induce

immune

response

to

boosting

immunotherapy.

Immunofluorescence

characterization was further used to study the typical IR820-1MT & αPD-L1 group (Figure 5E). Compared with the NS group, primary tumors presented more infiltration of CD4+ and CD8+ T cells after IR820-1MT & αPD-L1 treatment, agreeing with the flow cytometric results. In vivo IDO inhibitory effect was analyzed via the Trp to Kyn ratio in tumors. As shown in Figure 5F, the ratio of Trp to Kyn of the IR820-1MT itself and IR820-1MT & αPD-L1 group was 1.94- and 2.00-fold higher than that of the NS group, demonstrating the good IDO inhibitory effect. To verify whether the treatment based on IR820-1MT nanoparticles can activate the systemic anti-tumor immune response, lymphocytes in the spleens of the mice were analyzed (Figure S40 and S41, Supporting Information). It showed proportions of CTLs and Ths were remarkably 27



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increased and the proportions of Tregs was downregulated after IR820-1MT nanoparticles treatment in comparison with NS, 1MT, αPD-L1 and IR820 groups. Proportions of CTLs and Ths could be further enhanced when the IR820-1MT nanoparticles was combined with αPD-L1. Ratios of CTLs or Ths to Tregs were also increased when the IR820-1MT nanoparticles or IR820-1MT nanoparticles & αPD-L1 were compared with NS, 1MT, αPD-L1 and IR820 group (Figure S41). Blood from different treated mice was collected for analysis of cytokines. Th2 cytokines including IL-4 and IL-5, which was tested for comparison.39 As shown in Figure 5G and 5H, there was no significant difference of different treated mice. The immune-associated cytokines (Th1) also play an important role in anti-tumor immune response. Th1 mainly mediate cellular immune response and promote the killing effect of CTLs. Therefore, systemic concentrations of circulating Th1 including TNF-α, IFN-γ, IL-2, IL-6 and IL-10 were analyzed via LEGENDplex™ Mouse Th1 Panel (Figure 5I). The Quantitative data were shown in Table S1 in Supporting Information. The groups of IR820-1MT itself or in combination with αPD-L1 performed the higher level of TNF-α, IFN-γ, IL-2, IL-6 and IL-10 in contrast to other groups. As a result, the treatment rendered immune responses towards Ths1 rather than Ths2. Therefore, our IR820-1MT nanoparticles could be used as a versatile nanoplatform to activate the immune system.

28


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Figure 5. (A) DCs maturation in the tumor-draining lymph node of different treated mice. The proportions of (B) CTLs and (C) Ths in lymphocytes of the same weight of primary tumors in different groups. (D) Ratios of CTLs and Ths to Tregs in lymphocytes of the same weight of primary tumors in different groups. (E) The immunofluorescence stained images of tumors after different treatment. Nucleus: blue color, CD4: green color, CD8: read color. The scale bars were 25 μm. (F) Intratumoral Kyn to Trp ratio of different treated mice. Concentrations of (G) IL-4 levels and (H) IL5 levels in blood of different treated mice. (I) Concentrations of Th1 cytokines in blood of different treated mice. 1) NS, 2) 1MT, 3) αPD-L1, 4) IR820, 5) IR820-1MT nanoparticles and 6) IR820-1MT nanoparticles & αPD-L1. (n = 3) Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001. To study the therapeutic efficacy of IR820-1MT nanoparticles against anti-abscopal tumor, we constructed abscopal tumor-bearing mice model via subcutaneous injection (Figure 6A). B16F10 cells were subcutaneous injected into the right anterior armpit as the established 1st tumor of mice. Then, B16F10 cells were subcutaneous injected into the left anterior armpit for 4 days later as the 29



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established 2nd tumor of mice. To avoid the therapeutic effects of 1MT on the abscopal directly and more clearly observe the effect of local treatment on the inhibition of abscopal tumors, we only treated the 1st tumor with intratumoral injection and controllable NIR irradiation. Figure 6B showed there was no obvious weight change of mice in each group. For the 2nd tumor, the treatment with 1MT or αPD-L1 delayed tumor growth slightly compared with the NS group (Figure 6C to 6F). Although IR820 and IR820-1MT nanoparticles had similar ability to PTT and ICD response via intratumoral injection, IR820-1MT nanoparticles showed much higher efficacy on anti-abscopal tumor, demonstrating the superiority of responsive release of 1MT in IR820-1MT nanoparticles to trigger strong immune responses. Moreover, there was a better tumor inhibition effect when IR820-1MT nanoparticles were combined with αPD-L1 due to prevent T cells from exhaustion and immune escape. Furthermore, infiltration of CTLs and Ths significantly increased in the 2nd tumor and the infiltration of Tregs was largely reduced after the treatment compared to that of the NS group (Figure 6G-6J and Figure S42, Supporting information), indicating the efficient abscopal anti-tumor immune response and effective T cells homing to tumors achieved by local treatment. Therefore, our IR820-1MT nanoparticles provide a versatile nanoplatform for fighting abscopal tumor via activated systemic anti-tumor immune response. To further prove antigen-specific immune responses, different two types of tumors bearing mice model were established (Figure S43, Supporting Information). After same treatments, the volume of 2nd tumor of B16F10-B16F10 mice was significantly less than that of B16F10-4T1 mice. It was because that the treatment of the 1st B16F10 tumor lead to the antigen-specific immune responses, which would kill the 2nd B16F10 tumor but not 4T1 2nd tumor, proving the induction of antigen-specific immune responses in vivo.

30


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Figure 6. (A) Schematic illustration of anti-abscopal tumor experiment. (B) Change in body weight, (C) Average volume of 1st tumor, (D) Average volume of 2nd tumor, (E) Images of excised tumors, (F) Weight and tumor inhibition ratio of 2nd tumor. (n = 5) Proportions of (G) CTLs and (H) Ths in 2nd tumor. Flow cytometric analyses of the proportions of (I) CTLs and (J) Ths in lymphocytes of the same weight of 2nd tumors after NS, 1MT, αPD-L1, IR820, IR820-1MT or IR820-1MT nanoparticles & αPD-L1 treatment. (n = 3) Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001. To study the therapy efficacy of our IR820-1MT nanoparticles against metastasis, the lung 31



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metastasis tumor-bearing mice model was established (Figure 7A). Besides activation of the systemic immune response, immunologic memory is also important for anti-tumor efficacy especially for against tumor metastasis.40-42 To study whether immunologic memory had been established, memory T cells (CD4+ CD44+ CD122+ and CD8+ CD44+ CD122+) were obtained from spleens in the primary tumor-bearing mice (Figure 7B, 7C and Figure S44, Supporting Information). Compared with the NS, 1MT, αPD-L1 and IR820 group, both proportions of CD4+ CD44+ CD122+ and CD8+ CD44+ CD122+ memory T cells were increased after treatment with IR820-1MT nanoparticles or IR820-1MT nanoparticles & αPD-L1. This demonstrated that our IR820-1MT nanoparticles were superior platforms to establish immunologic memory. In the lung metastasis tumor-bearing mice model, B16F10-Luc cells were intravenous injected to the above-mentioned primary tumor-bearing mice after 15 days treatment. Based on the study of the memory T cells, IR820-1MT & αPD-L1 showed the best immunologic memory. Therefore, mice treated with IR820-1MT & αPD-L1 were used to investigate the efficacy on anti-tumor lung metastasis. The NS group was also carried out as the control group. Bioluminescence images were collected to study tumor lung metastasis without further treatment (Figure 7D). When rechallenged with tumor cells, untreated mice (the NS group) presented an obvious lung metastasis while the mice treated with IR820-1MT nanoparticles & αPD-L1 presented lung metastasis on the 5th day. However, on the 10th day, the lung metastasis was eliminated in the IR820-1MT nanoparticles & αPD-L1 group in contrast to the NS group. As showed in bioluminescence images (Figure 7E), camera images (Figure 7F) and H&E stained images (Figure 7G) of the lungs, there were almost no lung metastasis in the IR820-1MT nanoparticles & αPD-L1 group in contrast to the large amount of lung metastasis in the NS group. Therefore, our IR820-1MT nanoparticles were very useful to treat metastatic tumors. 32


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Figure 7. (A) Schematic illustration of anti-tumor metastasis experiment. The proportions of effector memory T cells (B) CD4+ CD44+ CD122+ and (C) CD8+ CD44+ CD122+ memory T cells in spleens after different treatment. (D) Bioluminescence images of lung metastasis tumor-bearing mice at different times after treatment. (E) Bioluminescence images, (F) Camera images and (G) H&E stained images of the lung at the last day of treatment. The scale bars were 200 μm. Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001. In clinical therapy, surgical resection was the first step in the treatment of tumors regularly. However, surgery was prone to cause immune suppression and wound healing factors would stimulate tumors growth.43 Therefore, subsequent tumor recurrence was a big problem for tumor therapy. To study the therapeutic efficacy of IR820-1MT nanoparticles against anti-tumor recurrence, the post-surgical tumor recurrence mice model was established. The 10% remaining tumors after surgery were treated and investigated (Figure 8A-8C). Compared with others, mice treated with IR820-1MT itself and in combination with αPD-L1 group both presented stronger ability to prevent tumor recurrence with higher survival rate (Figure 8D). Therefore, our 33



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IR820-1MT plus PTT nanoparticles was an effective strategy to inhibit cancer recurrence.

Figure 8. (A) Schematic illustration of anti-tumor recurrence experiment. (B) Images of mice during the treatment. (C) Change in average tumor volume, (D) Survival curves of different treated mice. (n = 5) Statistical significance: *p < 0.05, **p < 0.01, and ***p < 0.001.

4. Conclusion In summary, organic photothermal agent IR820 and IDO inhibitor 1MT were, for the first time, designed to be an all-role-in-one molecule nanoplatform via molecular engineering strategy. The obtained IR820-1MT molecule could self-assemble into negative-surface-charged nanoparticles with high dual-therapeutic agent loading (88.8 wt%) for immunotherapy. Particularly, deficient potency and poor water solubility of 1MT as well as inadequate target and short lifetime of IR820 were all well solved within our IR820-1MT nanoparticles. The 1MT in IR820-1MT nanoparticles demonstrated responsive release in tumor cells resulting from the degradation of ester bonds by the 34


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low-pH and enzyme-rich microenvironment. By combination of PTT and IDO inhibitors in IR820-1MT nanoparticles, the PTT-triggered ICD induced immunogenicity whereas the controlled released of 1MT from IR820-1MT nanoparticles could reverse “cold” immunosuppressive TME to the “hot” immunogenic TME. Consequently, our IR820-1MT nanoparticles not only significantly enhanced accumulation of CTLs, Ths and memory T cells but also efficiently suppressed the proportion of Tregs, demonstrating an excellent immunotherapy against tumor metastasis and recurrence. Our molecular engineering strategy toward all-role-in-one molecule nanoplatform provides a promising alternative option for design of a robust immunotherapy weapon against tumor metastasis and recurrence.

Associated content Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Detailed synthesis procedure, hemolysis test procedure, in vitro singlet oxygen generation detection procedure, characterizations of 1H NMR, ESI-MS, flow cytometry analysis and additional figures as described in the text.

Author information Corresponding Authors ∗ E-mail: [email protected]. Tel: (86) 531-88382007. Fax: (86) 531-88382548. Notes The authors declare no competing financial interest.

Acknowledgment This work was supported by the National Natural Science Foundation of China (NSFC, No. 35



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21872083 and 21573134), Shandong Provincial Major Science & Technology Innovation Project (2018CXGC1411). The Fundamental Research Funds of Shandong University (2018JC019).

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Cold to Hot: Rational Design of a Minimalist Multifunctional

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