Light-enhanced O2-evolving Nanoparticles Boost Photodynamic

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Biological and Medical Applications of Materials and Interfaces 2

Light-enhanced O-evolving Nanoparticles Boost Photodynamic Therapy to Elicit Anti-tumor Immunity Tingting Wang, Hao Zhang, Yaobao Han, Hanghang Liu, Feng Ren, Jianfeng Zeng, Qiao Sun, Zhen Li, and Mingyuan Gao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 17 Apr 2019 Downloaded from http://pubs.acs.org on April 17, 2019

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ACS Applied Materials & Interfaces

Light-enhanced O2-evolving Nanoparticles Boost Photodynamic Therapy to Elicit Antitumor Immunity Tingting Wang, Hao Zhang, Yaobao Han, Hanghang Liu, Feng Ren, Jianfeng Zeng, Qiao Sun, Zhen Li*, Mingyuan Gao Center for Molecular Imaging and Nuclear Medicine, State Key Laboratory of Radiation Medicine and Protection, School for Radiological and Interdisciplinary Sciences (RAD-X), Soochow University, Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions Suzhou 215123, China Email: [email protected]

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ABSTRACT: Breast cancer remains to show high mortality and poor prognosis in women despite of significant progress in recent diagnosis and treatment. Herein, we report the rational design of highly efficient ultrasmall nanotheranostic agent with excellent photodynamic therapy (PDT) performance to against breast cancer and its metastasis by eliciting anti-tumor immunity. The ultrasmall nanoagent (3.1 ± 0.4 nm) was fabricated from polyethylene glycol modified Cu2-xSe nanoparticles, βcyclodextrin, and chlorin e6 under ambient conditions. The resultant nanoplatform (CSCD-Ce6 NPs) can be passively accumulated into the tumor to exhibit dramatic antitumor efficacy through the excellent PDT effect under near-infrared irradiation. The excellent PDT performance of this nanoplatform is owing to its role of a Fenton-likeHaber-Weiss catalyst for the efficient degradation of H2O2 within tumor to release hydroxyl radicals (OH) and very toxic singlet oxygen (1O2) under the irradiation. The generated vast amounts of reactive oxygen species not only killed primary tumor cells, but also elicited immunogenic cell death (ICD) to release damage-associated molecular patterns (DAMPs) and induced pro-inflammatory M1- macrophages polarization. Thereby antitumor immune responses against metastasis of breast cancer were robustly evoked. Our work demonstrates that ultrasmall Cu2-xSe nanoparticles based nanoplatform offers a promising way to prevent cancer metastasis via immunogenic effects through its excellent PDT performance. KEYWORDS: anti-tumor immunity, immunogenic cell death, photodynamic therapy, hypoxia, O2-evolving nanoparticles

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TABLE OF CONTENTS GRAPHIC

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1. INTRODUCTION Breast cancer is the leading type of cancer in women and it is usually treated by surgery, followed by chemotherapy, radiotherapy, and combined therapy.1 These methods cannot completely treat the cancer with a large size, nodal spread, and metastasis, however, leading to poor prognosis and high mortality.2 Therefore, it is urgent to develop complimentary methods to effectively prevent the metastasis of breast cancer. Immunotherapy as a promising method has been rapidly developed in the recent decade, because of its ability to “train” immune system to find out and kill the residual tumor cells to prevent their metastasis. Immunotherapy alone often fails to kill primary tumors,3 however. In order to enhance the cancer therapeutic effect, ablation of primary tumors with photothermal therapy (PTT),4 hyperthermia (HT),5 radiotherapy (RT)6 and photodynamic therapy (PDT)7 is firstly introduced and then followed by immunotherapy, because these methods are able to elicit immunogenic cell death (ICD) for antitumor immunity to prevent tumor metastasis and recurrence. The ICD process is usually accompanied by the release of danger-associated molecular patterns (DAMPs), such as calreticulin (CRT), adenosine triphosphate (ATP), and high-mobility group box 1 (HMGB1).8 Compared with conventional therapies, photodynamic therapy (PDT) has emerged as a promising method for the treatment of cancer,9 because of its various merits such as repeatability with less toxicity, noninvasiveness, better selectivity, and minimal side effects.10, 11 In addition to kill primary tumor cells, PDT treatment can 4 / 41


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elicit ICD to promote dendritic cells (DCs) maturation, active T lymphocytes and natural killer (NK) cells.12-15 Reactive oxygen species (ROS) generated during DPT treatment also could enhance polarization of pro-inflammatory M1-macrophages to produce anti-tumor immunity.16, 17 It has been known that highly plastic macrophages could experience M1 activation or alternative M2 activation under different stimulations, of which M1 macrophages can kill tumor cells through release of tumor necrosis factor alpha (TNF-α) and lnterleukin-6 (IL-6) to enhance anti-tumor immunity.18 Since the performance of PDT directly influences the induced immune responses to against tumor metastasis,19 it is crucial to rationally design and fabricate platforms with excellent PDT performance. A big issue for the current PDT treatments is the concentration of oxygen (O2) in tumor due to the hypoxia feature.20,

21

Therefore,

alleviation of the hypoxia in tumor has become the most important strategy for improving the PDT performance to kill local tumor cells and against metastasis to prevent recurrence.22,

23

Various methods have been used to alleviate hypoxia to

improve PDT efficacy, such as breathing pure O2 in a pressurized chamber24 and using O2-evolving agents to in situ produce O2 within tumor. The currently used O2-evolving agents include (1) highly reactive nanoparticles (e.g. CaO2), which can react with H2O within tumor to produce O2;25 (2) Fenton- and Fenton-like nanocatalysts (e.g. MnO2), which can degrade H2O2 within tumor to release O2;26-28 (3) metal-organic frameworks29 and catalases, which could also degrade H2O2 to release O2.30 Among

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these O2-evolving agents, Fenton and Fenton-like nanocatalysts have attracted considerable interest owing to the abundant H2O2 (100 μM – 1 mM) within tumor. Fe-, Mn-, and Cu-based nanoagents are the common Fenton-reaction nanocatalysts, of which Cu-based nanoagents are very attractive because Cu+ ions can degrade H2O2 in a broader pH window31, 32. The currently available Cu-based nanoagents usually have a large particle size, which could significantly influence the release of O2 and the efficacy of therapy, due to their low accumulation at tumor and slow reaction speed with H2O2. In this context, preparation of ultrasmall Cu-based Fenton-reaction agents with long blood circulation time for degradation of H2O2 is highly desirable. This article aims to explore the excellent PDT performance of ultrasmall Cu2-xSe nanoparticles to elicit anti-tumor immunity. The nanoparticles were modified with βcyclodextrin (CD NPs) and photosensitizer Ce6 (named as CS-CD-Ce6 NPs). The ultrasmall size and abundant Cu+ ions in CS-CD-Ce6 NPs ensure their excellence in degradation of H2O2 within tumor and generation of large amount of ROS under nearinfrared (NIR) irradiation. Large amount of ROS can not only elicit immunogenic cell death (ICD) to release of danger-associated molecular patterns (DAMPs), but also enhance pro-inflammatory M1-macrophages polarization to produce anti-tumor immunity. The results demonstrate that our ultrasmall CS-CD-Ce6 NPs can not only effectively kill primary tumor cells through their excellent PDT performance, but also can effectively elicit anti-tumor immunity to against metastasis and recurrence of treated tumor.

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2. EXPERIMENTAL PROCEDURES 2.1 Materials The reagents were used directly without any purification. Copper(II) chloride dehydrate (CuCl2·2H2O), selenium powder (Se, ≥99.5%), sodium borohydride (NaBH4, 99%), methanesulfonic acid (MSA, 99%), chlorin e6 (Ce6), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, Mono-(6-Mercapto-6-deoxy)-βCyclodextrin was purchased from Shandong Binzhou Zhiyuan Biotechnology Co.,Ltd. Dimercapto poly (ethylene glycol) (HS-PEG-SH, MW = 5000) was brought from Adamas. 2.2 Synthesis of ultrasmall Cu2-xSe -CD-Ce6 NPs 0.5 mmol (39.48 mg) Se was reduced rapidly by 1.5 mmol (56.75 mg) NaBH4 in 50 mL H2O at room temperature under magnetic stirring and the protection of N2. Then, a mixture of 1 mmol (170 mg) CuCl2·2H2O and 6.66 mmol (1 g) MSA in 5 mL H2O was added into the precursor solution of selenium to immediately form a black solution. After stirring for 1.5 h under the protection of N2, the solution was purified by ultrafiltration resulting in MSA-capped Cu2-xSe (referred to as CS) NPs. 1 g CD was added into the CS solution and stirred for 12 h at 4 °C. To improve their biocompatibility, 0.2 g HS-PEG-SH was introduced into the above solution and stirred at 0 °C for 12 h. The mixed solution was purified by ultrafiltration, the resultant PEGylated nanoparticles (referred as CS-CD NPs) were stored for further use. 10 mL of CS-CD NPs solution (400 μg/mL) and Ce6 (100 μg/mL in DMSO) were mixed and stirred for 5 h at room temperature, and then free Ce6 was removed by 7 / 41



ultrafiltration with Milli-Q water, resultant nanoparticles are referred to as CS-CD-Ce6 NPs. 2.3 Characterization The morphology of the nanoparticles was characterized by transmission electron microscopy (TEM, FEI Tecnai F20) at an acceleration voltage of 200 kV. The ultraviolet-visible-near-infrared (UV-vis-NIR) absorbance was recorded with a PerkinElmer Lambda 750 UV-vis-NIR spectrophotometer. The valence states of elements in nanoparticles were characterized by X-ray photoelectron spectroscopy (XPS, Thermo Scientific Sigma Probe) using Al Kα X-ray radiation and fixed analyzer transmission mode. 2.4 Fenton-like reaction by CS-CD-Ce6 NPs The degradation of H2O2 by CS-CD-Ce6 NPs was characterized by an indicator, which was composed by 24% Ti(SO4)2 (1.33 mL) and 8.33 mL H2SO4 in 50 mL H2O. 392 μM of H2O2 was mixed with 25 μg/mL CS-CD-Ce6 NPs in 1 mL H2O, and then irradiated with an 808 nm laser for 5 min. The residual H2O2 was characterized by measuring the absorbance of the mixture at 405 nm. The generation of O2 was measured with a dissolve oxygen meter (JPBJ-608). Dichlorofluorescein diacetate (DCFH-DA) was introduced to detect the total ROS produced. 2.5 In vitro experiments 2.5.1 Hypoxia condition at the cell level 4T1 cells were seeded on glass-bottom dishes at a density of 8 × 104 − 1 × 105 cells per well for 24 h to allow them to attach to the surfaces of wells. Different media 8 / 41


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containing reagent (at a concentration of 25 μg/mL) and a probe for hypoxia (50 nM) were added into 4T1 cells, respectively, and they were cultured at 37 °C under 5% CO2 for 4 h. The excess reagent was removed by washing three times with PBS. Then, the cells were irradiated with an 808 nm laser (0.75 W/cm2) for 5 min and then stained with Hoechst 33342 for 15 min, characterized by confocal laser scanning microscopy (CLSM, λex = 596 nm, λem = 670 nm). 2.5.2 Monitoring of different types of ROS at the cell level A singlet oxygen sensor green reagent (SOSG, S36002, Invitrogen) was used to detect 1O2. 4T1 cells were seeded on glass-bottom dishes at a density of 8 × 103 − 1 × 104 cells per well for 24 h. After incubation with Ce6 (2.5 μg/mL, 1 mL), CS-CD NPs (25 μg/mL, 1 mL), and CS-CD-Ce6 NPs (25 μg/mL, 1 mL) for 4 h. They were then cultured with SOSG (5 μM, 1 mL) for 30 min prior to the laser irradiation (808 nm laser, 0.75 W/cm2) for 5 min. Then stained with Hoechst 33342 for 10 min and characterized by confocal laser scanning microscopy. 2.5.3 Detection of crucial ICD biomarkers Immunofluorescence was introduced to assess the calreticulin (CRT) exposure onto the plasma membrane surface. 4T1 cells were seeded on glass-bottom dishes at a density of 8 × 104 − 1 × 105 cells per well for 24 h to allow them attaching to the surface of wells. Different media containing reagent (at a concentration of 25 μg/mL) were added. After 4 h incubation, cells were irradiated with an 808 nm laser (0.75 W/cm2) for 5 min, and the exposure of CRT was detected by using calreticulin antibody (FITC) (Fitzgerald). 9 / 41



The extracellularly released adenosine triphosphate (ATP) and high-mobility group box 1 (HMGB1) were examined with an ATP detection Kit (A22066) (Molecular Probes) and a HMGB1 detection Kit (Chondrex), respectively. 4T1 cells were cultured similarly by the above method, and then irradiated with an 808 nm laser (0.75 W/cm2) for 5 min. The released ATP and HMGB1 in the cell supernatant was detected with the above Kits by using the provided protocols, respectively. 2.5.4 Flow cytometry analysis of M1- and M2-Macrophages RAW264.7 cells were first seeded in 6-well plates at a density of 8 × 104 − 1 × 105 cells per well and cultured for 24 h in RPMI 1640 containing 10% FBS. Then the cells were washed twice with PBS and incubated with different reagents (at a concentration of 25 μg/mL) at 37 °C under 5% CO2 for 4 h. The cells were irradiated with an 808 nm laser (0.75 W/cm2) for 5 min and washed with PBS. After incubation for 24 h, the cells were collected, washed twice with PBS, and then re-dispersed in 100 μL of anti-CD86FITC (eBioscience) and anti-CD-206-APC (eBioscience) and incubated for 20 min on ice. The cells were washed twice with PBS, and then analysed with flow cytometry analysis (FCAS). 2.6 In vivo experiments 2.6.1 Mice tumor model A suspension of 4T1 cells (50 μL, 5×106 cells) was subcutaneously injected into the flank region of the right back of 5-week-old male BALB/c mice, which were used with protocols approved by the Laboratory Animal Center of Soochow University. After 10 days of inoculation with tumor cells, mice bearing tumors were used for treatment. 10 / 41


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2.6.2 In vivo therapy of tumour BALB/c mice bearing subcutaneous tumors with an average volume of 125 mm3 were divided into five groups. All groups of mice were injected with the same volume (200 μL) of solutions of PBS and CS-CD-Ce6 NPs. The solutions of CS-CD-Ce6 NPs contained 100 μg Cu. The irradiation was performed by using an 808 nm laser with a power density of 1.0 W/cm2 for 10 min (Hi-Tech Optoelectronics Co., Ltd. Beijing, China). The body weights and tumor sizes were measured every 2 days. 2.6.3 In vivo PA imaging of tumor For in-vivo PA imaging, nude mice bearing subcutaneous tumors were anesthetized with 1.5% isoflurane delivered via a nose cone, and injected with 200 μL of CS-CDCe6 NP solution (500 μg/mL Cu) via the tail vein. The PA signals of HbO2 at tumor sites were separated from PA images by the MSOT software. The PA images were collected before and after 4 h post-irradiation with 808 nm laser. The irradiation time was 10 min. 2.6.4 Histological analysis Tumors were dissected from mice on day 2 after treatment, and fixed in neutral buffered formalin (10%). Then, the tumors were sliced into pieces with a thickness of 4 μm for hematoxylin and eosin (H&E) and HIF-1α staining. The slices were observed with a Leica microscope (DM750). Tumor-bearing mice were injected with the solutions of PBS or CS-CD-Ce6 NPs (500 μg/mL of Cu, 200 μL) via their tail veins. After 12 h injection, the mice were intratumorally injected with DCFH-DA (10 μM, 200 μL), followed by irradiation with 11 / 41



808 nm laser for 10 min (0.75 W/cm2). Then the tumors from various groups of mice were harvested, and examined with CLSM to observe the fluorescence of DCF oxidized by ROS within tumor cells (ex/em= 488/525 nm).

3. RESULSTS AND DISCUSSION 3.1 Characterization of CS-CD-Ce6 NPs The ultrasmall Cu2-xSe NPs were synthesized in an aqueous solution at ambient conditions.33 As shown in Figure 1a, they were then modified with polyethylene glycol (PEG) and β-cyclodextrin (CD) (with the product referred to CS-CD NPs) to enable the loading of hydrophobic photosensitizer Ce6 into the cavity of CD through hydrophobic interactions.34 Both PEG and CD modification not only decreased the aggregation of Ce6 but also improved the biocompatibility of the nanoparticles.35 The nanoparticles further modified with Ce6 are denoted as CS-CD-Ce6 NPs and their morphology is characterized with transmission electron microscope (TEM). Figure 1a and Figure S1a in the Supporting Information show the particle size distribution of the resulting particles, according to which an average size of 3.1 ± 0.4 nm was obtained for the uniform spherical particles. As shown in Figure 1b, the zeta potential of CS NPs was measured to be about -9.0 mV, and it slightly increased to -7.6 mV after modification with thiol functionalized CD, then drastically decreased to -14.5 mV after uploading of Ce6, proving the successful modification of CS NPs with CD and Ce6. The ultravioletvisible-near infrared (UV-vis-NIR) absorbance in Figure 1c further demonstrates the

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successful uploading of Ce6. To calculate the loaded amount of Ce6 on the surface of CS-CD NPs, the dependence of UV-vis absorption of Ce6 on its concentration was measured and shown in Figure S1(b-c). The calculated mass ratio between Cu2-xSe nanoparticles and Ce6 is 7.7:1. This result illustrates that Cu2-xSe nanoparticles modified with PEG and CD can be efficiently loaded with hydrophobic Ce6 without sacrifice of their colloidal stability. Figure S1d presents the crystal structure of CS-CD and CS-CD-Ce6 NPs as determined by powder X-ray diffraction (XRD). Their diffraction peaks are well matched the characteristic peaks of cubic berzelianite (Cu2-xSe, JCPDS Card No. 06-0680), which illustrates that modification and uploading of Ce6 did not change the crystal structure of CS-CD NPs. The contents of PEG on the surfaces of nanoparticle was determined by thermogravimetric analysis (TGA), as shown in Figure S2. There is about 37 wt% weight loss in the range of 330−412 °C. To investigate their stability in simulated blood, we dispersed CS-CD-Ce6 NPs (25 μg/mL) in 10% fetal calf serum (FBS) and phosphate buffered saline (PBS) solutions, and then monitored the variation of their hydrodynamic size. As shown in Figure S3 (ab), there is no drastic difference in the time-dependent hydrodynamic size of CS-CDCe6 NPs in FBS and PBS media. The hydrodynamic size of CS-CD-Ce6 NPs in 10% FBS is smaller than that in PBS. In addition, CS-CD-Ce6 NPs were degraded in PBS solution after 2 days, which illustrates our nanoparticles could be degraded in mouse after intravenous injection. Furthermore, the release of Ce6 from CS-CD-Ce6 NPs in 13 / 41



PBS solution was also investigated and shown in Figure S4. Obviously, there is about 50% Ce6 released from the CS-CD-Ce6 NPs. The result demonstrates that modification of Cu2-xSe nanoparticles with PEG and CD not only benefits to the loading of hydrophobic Ce6, but also benefits to the release of Ce6 for PDT. Therefore, the organic coating is extremely important for the balance between the stability of nanoparticles and the loading and release efficiency of drugs. 3.2 Fenton-like-Haber-Weiss catalytic property of CS-CD-Ce6 NPs To evaluate the Fenton-like-Haber-Weiss catalyst property of CS-CD-Ce6 NPs, degradation of H2O2 was carried out. As shown in Figure 1d, about 89% of H2O2 was degraded within 150 min by CS-CD-Ce6 NPs with laser irradiation. However, the CSCD-Ce6 NPs without laser irradiation can degrade about 55% H2O2, 34% lower than that degraded with laser irradiation. It means that laser irradiation can speed up the degradation of H2O2. But the self-degradation of H2O2 is relatively slow and laser irradiation alone has limited effect on the degradation of H2O2 (no more than 30%). The results reveal that CS-CD-Ce6 NPs have excellent catalytic performance in degrading H2O2, which can be significantly enhanced by NIR irradiation. To more quantitatively describe the Fenton-like-Haber-Weiss catalytic effect, the valence state of Cu in the nanoparticles was determined by X-ray photoelectron spectroscopy (XPS) before and after reaction with H2O2 [Figure S5(a-b)]. Through quantitative analysis of the high-resolution XPS spectra, it was found that the proportion of Cu+ in the CS-CD-Ce6 NPs decreased from 74.9% to 22%, and the

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proportion of Cu2+ increased from 25.1% to 78% after they were mixed with 392 μM H2O2 with irradiated. In addition, the characteristic peak at 933.1 eV and the satellite peak at 942.3 eV of Cu2+ further demonstrate the oxidation of Cu+ into Cu2+. The above results demonstrate the degradation of H2O2 catalyzed by CS-CD-Ce6 NPs under NIR irradiation. The degradation products were determined. As shown in Figure 1e, about 0.83 ppm O2 was produced by CS-CD NPs under irradiation, which was measured with a dissolve oxygen meter, and more than that (0.5 ppm) produced without laser irradiation. Furthermore, no more than 0.05 ppm O2 was produced in the absence of CS-CD NPs, whether H2O2 was irradiated with or without laser. These results illustrate that NIR irradiation can significantly enhance the catalytic degradation of H2O2 to generate more O2 in the presence of CS-CD NPs. This proved that CS-CDCe6 can produce large amount of O2 with H2O2 (392 μM) under irradiation (808 nm, 0.75 W/cm2, 5 min) through Fenton-like-Haber-Weiss catalytic reactions. In addition to O2, OH radicals were also produced, which were detected by electron spin resonance spectroscopy (ESR) after addition of a specific spin-trapping agent (i.e., 5,5-dimethyl-1-pyrroline-N-oxide, DMPO) into the reaction mixture. The ESR spectrum of the DMPO-OH adduct (Figure 1f) clearly shows quadruplet peaks with an intensity ratio of 1:2:2:1, which indicates the production of OH radicals by CS-CDCe6 NPs under NIR irradiation.36 In contrast, no quadruplet peaks in the ESR spectrum of the adduct were recorded if the irradiation was performed in the absence of CS-CDCe6 NPs. The result further supports the proposal that CS-CD-Ce6 NPs can improve the degradation of H2O2 to produce ROS. Moreover, the generated O2 could be 15 / 41



efficiently transformed into 1O2 with assistance of photosensitizer Ce6 under NIR irradiation as shown in Figure 1g.37 Typical ESR spectrum of adduct of 2,2,6,6tetramethylpiperidine (TEMP) and 1O2 exhibits the characteristic triplet peaks, while there is no obvious signal in the absence of CS-CD-Ce6 NPs with irradiation. The result demonstrates CS-CD-Ce6 NPs could produce 1O2 very well under irradiation. The above results suggest that CS-CD-Ce6 NPs can efficiently produce O2 to relieve the hypoxia environment and larger amount of ROS to kill tumor cells. It is attributed to Fenton-like-Haber-Weiss catalytic effect of Cu+ ions from ultrasmall CS-CD-Ce6 NPs.38 The overall catalytic degradation of H2O2 to generate OH- and O2 can be described by Eqs. (1-3).39 The generated O2 can be excited to 1O2 with assistance of Ce6 to improve the total amount of ROS (Figure 1h). 𝐶𝑢 + + 𝐻2𝑂2→𝐶𝑢2 + + H𝑂 ― + 𝑂𝐻 



𝑂𝐻 + 𝐻2𝑂2→𝐻2𝑂 + 𝑂2― + 𝐻 +



𝑂2― + 𝐻 + + 𝐻2𝑂2→𝑂2↑ + 𝑂𝐻 + 𝐻2𝑂

(1) (2) (3)

The total ROS produced was quantified by fluorescence intensity of adducts of dichlorofluorescein diacetate (DCFH-DA) shown in Figure 1i.40 It shows that very strong fluorescence at 529 nm was observed in CS-CD-Ce6 group with irradiation, but no characteristic fluorescence of the adduct [i.e., dichlorofluorescein (DCF)] observed in the solutions of H2O2 with or without irradiation in the absence of CS-CD-Ce6 NPs. This is due to that degradation of H2O2 by CS-CD-Ce6 NPs is very fast under irradiation through the Fenton-like-Haber-Weiss catalytic reactions to produce O2, which can be easily transformed into 1O2 by Ce6 under the laser irradiation, and then react with 16 / 41


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DCFH-DA to form fluorescent DCF. It is well known that Ce6 can be efficiently excited with a 660 nm laser,41 however, it is not known about their excitation and sensitization effect under 808 nm irradiation. Therefore, the degradation of H2O2 by CS-CD-Ce6 NPs under excitation by 660 nm and 808 nm lasers was firstly compared. As shown in Figure 2a, in the presence of CSCD and CS-CD-Ce6 NPs, only 56.8% of the H2O2 was degraded under irradiation by a 660 nm laser with a power density of 0.75 W/cm2 for 5 min, which is obviously lower than for irradiation by an 808 nm laser under the same conditions. The results demonstrate that 660 nm NIR light is not as efficient as 808 nm NIR light for the degradation of H2O2. This is attributed to the strong absorbance of CS-CD-Ce6 at 808 nm, which is almost twice higher than that at 660 nm. Terephthalic acid (TA) is introduced to detect the OH radials generated during degradation of H2O2 (392 μM) by CS-CD-Ce6 NPs under NIR irradiation. TA can be oxidation by OH into 2-hydroxyterephthalic acid (TAOH).42 Thus, TAOH fluorescence intensity at 426 nm is dependent on the concentration of OH radicals. As shown in Figure 2b, the amounts of OH radicals produced under 808 nm irradiation are twice more than those obtained from 660 nm irradiation. This is consistent with the conclusion drawn from Figure 2a. 1,3-Diphenylisobenzofuran (DPBF) was used to quantitatively evaluate 1O2 and O2produced by CS-CDNPs and CS-CD-Ce6 NPs after 660 nm or 808 nm laser irradiation.43 As shown in Figure 2c, the lowest UV-Vis absorbance of DPBF after reaction with CS-CD-Ce6 NPs under 808 nm irradiation indicates that the largest 17 / 41



amount of 1O2 and O2- was produced, due to the fact that Ce6 successfully excited O2 to 1O2 under 808 nm irradiation. As shown in Figure 2d, CS-CD-Ce6 NPs could produce the highest amount of ROS under irradiation by 808 nm laser, evidenced by the strongest fluorescence of DCF. This means that CS-CD-Ce6 NPs could efficiently produce ROS under irradiation by an 808 nm laser more than that irradiated under 660 nm laser. 3.3 In vitro cell experiments with CS-CD and CS-CD-Ce6 NPs All the above results highlight the great potential of CS-CD-Ce6 NPs in PDT, and that 808 nm NIR light is the better choice for our later experiments. To investigate their PDT performance on cellular level, their cytotoxicity towards 4T1 cells was assessed by the standard methyl thiazolyl tetrazolium (MTT) assay (Figure S6a).44 The cell viability was decreased against the concentrations of CS-CD NPs and CS-CD-Ce6 NPs. Both types of nanoparticles exhibit no obvious toxicity towards 4T1 cells when their concentration is lower than 50 μg/mL. In addition, the cytotoxicity of CS-CD-Ce6 NPs is slightly higher than that of CS-CD NPs, due to the fact that the photosensitizer Ce6 has some toxicity. As mentioned previously, CS-CD NPs could degrade H2O2 through a Fenton-likeHaber-Weiss catalytic reactions to produce O2, which can be used to alleviate the hypoxia of tumor cells. Therefore, 25 μg/mL CS-CD or CS-CD-Ce6 NPs were selected for experiments on the basis of the cytotoxicity results. A hypoxia/oxidative stress detection kit was introduced to indicate the intracellular hypoxia.45 CLSM were used to characterize the fluorescence intensity of the hypoxia probe in cells treated with 18 / 41


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different agents. The stronger fluorescence indicates the higher hypoxia. As shown in Figure 3a, strong fluorescence was observed in untreated cells, indicating their severe hypoxia. The fluorescence is becoming stronger when treated with Ce6. In sharp contrast, 4T1 cells treated with CS-CD NPs and NIR irradiation displayed very weak fluorescence, indicating the improvement of their hypoxia environment due to the generation of O2 from degradation of H2O2 within tumor cells, catalyzed by CS-CD NPs under laser irradiation. For the cells treated with CS-CD-Ce6 NPs, their fluorescence intensity is much stronger than that of cells treated with CS-CD NPs under the same irradiation, because of the consumption of O2 produced by Ce6. Without laser irradiation, however, as shown in Figure S7a there is no big difference in the fluorescence intensities of cells untreated or treated with CS-CD or CS-CD-Ce6 NPs. This is because CS-CD NPs can slowly produce O2 through the Fenton-like-HaberWeiss catalyst reaction without NIR irradiation, and Ce6 cannot excite O2 to 1O2 without NIR irradiation consistent with the group with Ce6 only and without irradiation. In order to detect 1O2 produced in 4T1 cells, singlet oxygen sensor green reagent (SOSG) was introduced (Figure 3b, Figure S7 b).46 4T1 cells were respectively incubated with Ce6, CS-CD NPs and CS-CD-Ce6 NPs for 4 h. After irradiation with 808 nm laser for 5 min, the cells treated with CS-CD-Ce6 NPs showed a very stronger green fluorescence of SOSG than those treated with CS-CD NPs and free Ce6, suggesting the generation of larger amount of 1O2. The results demonstrate that CSCD-Ce6 NPs not only improved the cellular uptake of Ce6 but also produced more O2 for transformation into 1O2. Obviously, very low or no 1O2 was produce without 808 19 / 41



nm irradiation in all cases as shown in Figure S7b, which is consistent with that NIR irradiation can enhance the catalytic performance of CS-CD NPs and CS-CD-Ce6 NPs for degradation of H2O2. To further assess the intracellular accumulation of total ROS, DCFH-DA as a fluorogenic probe was cultured with cells. The DCFH-DA can be immediately hydrolyzed to DCFH after it enters into cells, and is further oxidized into DCF in the presence of ROS. DCF exhibits bright green fluorescence under excitation, which can be detected by CLSM. 4T1 cells were treated with free Ce6, CS-CD NPs, or CS-CDCe6 NPs, with or without irradiation. Compared with the CLSM images in Figure 3c and Figure S7c, stronger Ce6 fluorescence emerged in 4T1 cells incubated with CSCD-Ce6 NPs than those incubated with free Ce6, indicating that ultrasmall CS-CD NPs could efficiently improve the cellular uptake of Ce6 through endocytosis. The fluorescence intensity of DCF in cells cultured with the medium only or with free Ce6 is very low and irrespective of laser irradiation, which means that only a very low level of ROS was produced in these cells. Furthermore, the DCF fluorescence in the cells cultured with CS-CD NPs and irradiated with the laser is much stronger than that of cells cultured with the medium. This is attributed to the photo-degradation of H2O2 catalyzed by CS-CD NPs under laser irradiation. The green fluorescence in cells cultured with CS-CD-Ce6 NPs is also much stronger than that cultured with CS-CD NPs under irradiation, which is due to the increase in total ROS arising from the additional 1O2 formed from O2 in the presence of Ce6 under NIR irradiation, and well in agreement with that shown in Figure 3a, b. These results further prove that CS-CD20 / 41


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Ce6 NPs as an O2-evolving agent could fight against cancer by producing large amounts of ROS through alleviation of the hypoxic environment. The above results demonstrate the great potential of CS-CD-Ce6 NPs for PDT. To distinguish the effects of PTT and PDT, uric acid (UA) as an antioxidant was used to protect cells from damage by ROS.47 The toxicity of UA on 4T1 cells was assessed by the standard MTT approach shown in Figure S6b confirm that UA had no obvious effect on the cell viability. Based on this result, different concentrations of CS-CD and CSCD-Ce6 NPs were cultured with 4T1 cells in the absence or presence of 0.25 mM UA with irradiated shown in Figure 3d. The viability of cells with UA gradually increased due to the fact that UA can efficiently consume ROS and protect cells from damage by ROS. Quantifying the contributions of PDT and PTT shows that the influence of CSCD and CS-CD-Ce6 NPs on cell viability was completely attributable to the PDT effect (Figure S6c-d). It also demonstrates that CS-CD-Ce6 NPs obviously have a much better PDT effect than CS-CD NPs under the same conditions, due to the generation of reactive 1O2 from O2 produced by CS-CD NPs through the Fenton-like-Haber-Weiss catalyst reaction. To further demonstrate the PDT effect of CS-CD-Ce6 NPs in cells, the cells were cultured with Ce6 (2.5 μg/mL), CS-CD NPs (25 μg/mL), and CS-CD-Ce6 NPs (25 μg/mL) for 4 h, respectively. Cell apoptosis was quantified by FCAS in Figure 3e. Without laser irradiation, the apoptosis rates of cells cultured with phosphate buffered saline (PBS), Ce6, CS-CD NPs, and CS-CD-Ce6 NPs were 6.61%, 14.93%, 58.36%, and 64.05%, respectively. With laser irradiation, the corresponding apoptosis rates were 21 / 41



increased to 15.75%, 28.31%, 66.17%, and 88.22%, which supports the significance of laser irradiation. Furthermore, live/dead staining result also presents that CS-CD-Ce6 NPs with laser irradiation could kill almost 4T1 cells (Figure S7d), which is consistent with the result shown in Figure 3e. This means that CS-CD-Ce6 NPs could produce large amount of ROS to against breast cancer. 3.4 Tumor immunotherapy caused by ROS produced by CS-CD-Ce6 NPs Previous studies have already reported that chemotherapy, radiotherapy and PDT can elicit immunogenic cell death (ICD) by inducing the dying tumor cells to release immunogenic signals of damage-associated molecular patterns (DAMPs),48 which can activate the antigen-presenting cells (APCs) to stimulate the tumor-specific effector T cells.49 DAMPs such as CRT, ATP and HMGB1 are shown in Figure 4a. Furthermore, in most cancers, macrophages exhibit as M2 phenotype with immunosuppressive property, which can interact with other immune cells and suppress innate and adaptive antitumor immune response. It has been proven that the ROS increase could induce M1 polarization to produce antitumor immunity, as illustrated in Figure 4b. To investigate whether CS-CD-Ce6 NPs can really mediate PDT triggering ICD, the detection of CRT exposure, ATP secretion and HMGB1 release was carried out after 4T1 cells were treated with CS-CD-Ce6 NPs and then irradiated with or without 808 nm laser. As shown in Figure 4c, 4T1 cells treated without CS-CD-Ce6 NPs showed very few or even no cell-surface CRT exposure, indicated by the extremely weak green fluorescence. However, very high cell-surface CRT exposure (or very strong green fluorescence) can be observed in the cells treated with CS-CD-Ce6 NPs and laser 22 / 41


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irradiation, which indicates the production of enough ROS to induce ICD. Next, luciferin-based ATP assay was introduced to detect ATP release from 4T1 cells after treated with CS-CD-Ce6 NPs and irradiated with or without laser. As shown in Figure 4d, ATP secretion from 4T1 cells was significantly enhanced after treated with CS-CDCe6 NPs and laser irradiation, which is about two times of that released in cells treated only with CS-CD-Ce6 NPs, much higher than that in cells without any treatment. HMGB1 release can be analyzed by enzyme-linked immunosorbent assay (ELISA) shown in Figure 4e. Similar to CRT exposure and ATP secretion, the cells treated with CS-CD-Ce6 NPs and laser irradiation released the highest HMGB1 (837 pg/mL), in comparison with cells without treatment or treated under other conditions. All these results prove that the CS-CD-Ce6 NPs internalized by tumor cells can produce much more ROS under NIR irradiation to trigger ICD and promote DC maturation.50 To determine whether CS-CD-Ce6 NPs can really induce M1 polarization by large amount of ROS produced, we used CD86 as a marker of M1 macrophages and CD206 as a marker of M2 macrophages to perform FCAS (Figure 4f).51 Clearly, there are almost no M1 macrophages in the groups of control, control + NIR, CS-CD-Ce6 group, but more than 90% of M1 macrophages are presented in the CS-CD-Ce6 + NIR group. This is attributed to that CS-CD-Ce6 NPs in the cells produced much more ROS under laser irradiation to induce M1 polarization, which can improve the immune system in tumor to produce antitumor immunity. Furthermore, IL-12 (produced by M1 macrophages) and IL-10 (produced by M2 macrophages) were detected by ELASA in vitro (Figure 4 g and Figure 4 h). Obviously, IL-12 concentration is the highest in the 23 / 41



group of cells cultured with CS-CD-Ce6 NPs and then irradiated with 808 nm laser, which illustrates that large amount of ROS can really induce M1 polarization. However, IL-10 concentration is almost the same, which demonstrates the similar amount of M2 macrophages in different groups. The results are consistent with those get from FCAS in Figure 4f. TNF-а and IL-6 also detected and shown in Figure S8, which illustrate that large amount of ROS produced by CS-CD-Ce6 NPs with 808 nm NIR irradiation can induce M1 polarization leading the TNF-а and IL-6 releasing amount increased to enhance anti-tumor immunity. 3.5 Tumor-targeted light-enhanced O2-evolving CS-CD-Ce6 NPs Prior to in vivo experiment, we first measured the blood circulation time of CS-CDCe6 NPs after they were intravenously injected into 4T1 tumor-bearing BALB/c nude mice (200 μL, 500 μg/mL). Blood samples were collected at different time after tail vain injection of our nanoparticles, and the copper concentration in the blood was detected by ICP-MS. The result in Figure S9 show that CS-CD-Ce6 NPs exhibited a relatively long blood circulation with a half-life of 6.95 h due to their ultrasmall size and surface modification, which can make them escape from phagocytosis by macrophages. Then PA imaging was performed to determine the optimal time for the treatment. CS-CD-Ce6 NPs (200 μL, 500 μg/mL) was intravenously injected into 4T1 tumor-bearing BALB/c nude mice with a tumor volume of 125 mm3. Figure 5a and Figure 5b display the PA images of tumor and their signals collected at different time.52 Obviously, PA signal in the area of tumor before the injection of CS-CD-Ce6 NPs was very weak, which was increased gradually with the circulation of nanoparticles in the 24 / 41


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body after injection. The maximum PA signal, achieved at 12 h post-injection, was about 2.2 times of pre-contrast image. The time-dependent PA signal illustrates the dynamic accumulation of CS-CD-Ce6 NPs in tumor, and the optimal treatment time is 12 h post injection of nanoparticles. PA imaging was also used to evaluate the vascular saturated O2 within 4T1 solid tumors. As shown in Figure 5c and Figure 5d, the intratumor injection of CS-CD-Ce6 NPs could increase the blood oxygen saturation significantly in the tumor up to 1.5 times than that without irradiation, and 6 times of that without injection of CS-CD-Ce6 NPs. Significantly, it proves that CS-CD-Ce6 NPs can relieve the hypoxia by produce O2 under irradiation (808 nm of 1 W/cm2 for 10 min). Furthermore, hypoxia-inducible factor-1 (HIF-1α) as an important central mediator of adaptive responses to tumors in a hypoxic environment was detected. As shown in Figure 5e, CS-CD NPs can alleviate the tumors’ hypoxic environment by generation of O2 from degradation of H2O2 within tumor through the Fenton-like-Haber-Weiss catalytic reactions. The generated O2 could be excited to 1O2 mediated by Ce6 under irradiation. The in vivo PDT effect was also demonstrated by intratumor injection of fluorescent probe (DCFH-DA) at 12 h post intravenous-injection of CS-CD-Ce6 NPs.53 Strong green fluorescence (Figure 5f) demonstrates the generation of large amount of ROS produced in the tumor from the mice injected with CS-CD-Ce6 NPs and irradiated with 808 nm laser, in comparison with other groups. Obviously, with the increase of generated ROS, the fluorescence of CD86-FITC (a marker of M1 macrophages) become stronger, which illustrates the apparent polarization of M1 within tumor. The 25 / 41



result is consistent with that obtained from cells in vitro. Both results demonstrate that ROS can induce M1 polarization. Based on the above result, 4T1 tumor-bearing BALB/c nude mice with tumor volumes of 125 mm3 were divided into four groups and received different treatments, i.e., 1) injected with PBS solution (200 μL) only (PBS group); 2) injected with PBS solution (200 μL) followed by laser irradiation (PBS + NIR group); 3) injected with CS-CD-Ce6 NPs only (500 μg/mL, 200 μL) (CS-CD-Ce6 NPs group); 4) injected with CS-CD-Ce6 NPs followed by laser irradiation (500 μg/mL, 200 μL) (CS-CD-Ce6 NPs + NIR group). After 12 h tail vein injection, irradiation was performed with an 808 nm laser (1.0 W/cm2, 10 min). The temperatures of the tumors from different groups of mice were recorded by an infrared thermal camera. Typical images are shown in Figure S10a, and Figure S10b show that the temperature increments in the PBS group and CSCD-Ce6 NPs group are smaller than 10 °C. Unlike conventional PTT, where high temperature above 50 °C is required in order to completely kill cancer cells,54, 55 the temperature of the tumor area in our study was kept below 50 °C to minimize its damage to healthy tissue. The volumes of their tumors (Figure 6a), the weights of mice (Figure 6b), and their photographs (Figure S11) were measured and collected every 2 days. The lack of any obvious weight loss in the four groups of mice indicates the low toxicity of our nanoparticles. The tumor volumes from PBS group and CS-CD-Ce6 group show that the tumors grew very quickly without laser irradiation. With laser irradiation, the tumors in the PBS group became smaller, which demonstrates the role of laser in suppressing tumor growth. In the CS-CD-Ce6 + NIR group, the tumors gradually 26 / 41


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disappeared within 16 days, which is attributed to the perfect PDT property. Consequently, the average lifespans and survival rates of mice that received laser irradiation were longer than those without laser irradiation, as shown in Figure 6c. The mice from CS-CD-Ce6 + NIR group successfully survived and lived well, while the mice from the other groups eventually died. To demonstrate differences in the tumors from the mice receiving different treatments, the tumors were harvested after treated for 2 days and stained with hematoxylin and eosin (H&E).56 The staining results in Figure 6d indicate that the tumor tissues were damaged more seriously in the CS-CD-Ce6 + NIR group compared with the other groups. To further prove that the ROS generated by CS-CD-Ce6 NPs can not only destroy the local tumor cells but also can suppress the tumor metastasis, lung photographs and H&E staining of lung are displayed in Figure 6e and Figure 6f. Clearly, there are no nodules of lung metastasis are observed in mice from the CS-CD-Ce6 + NIR group than others (Figure 6e). The H&E staining also show no metastasis in the lung of mice from the CS-CD-Ce6 + NIR group. The results prove that CS-CD-Ce6 NPs can not only kill local tumor cells, but also can suppress the tumor metastasis. This is not only due to the release of immunogenic signals and activation of APCs to elicit the anti-tumor immunity, but also due to M1 polarization induced by ROS generated by CS-CD-Ce6 NPs under NIR irradiation by an 808 nm laser. The polarization of tumor associated macrophages (TAMs) into M1 phenotype induced by ROS also can be confirmed by the cytokines detected in serum (Figure S12). Obviously, ROS generated by CS-CD-Ce6 NPs changed the activity of TAMs to produce IL-12, 27 / 41



especially after irradiation with an 808 nm laser. The result is consistent with the polarization of M1 macrophages. Meanwhile, the decrease of IL-10, which was produced by M2, further proved that CS-CD-Ce6 NPs can generate a large amount of ROS under NIR irradiation to induce M1 polarization to enhance antitumor immunity. Both in vitro and in-vivo results support our hypothesis that ROS generated from CSCD-Ce6 NPs can induce M1 polarization. Furthermore, Figure 6g and Figure 6h show the concentrations of TNF-α, IL-6 in serum acquired at 48 h from the mice in the CS-CD-Ce6 group treated with and without irradiation. Both TNF-α and IL-6 from the mice injected with CS-CD-Ce6 NPs and irradiated with 808 nm laser are much higher than those obtained in other groups. The results demonstrate that CS-CD-Ce6 NPs could really induce a higher level of tumorspecific immunity through their excellent PDT performance. Additionally, the toxicity of CS-CD-Ce6 NPs was assessed by hematoxylin-eosin staining of tissues of major organs (i.e. heart, liver, spleen, lung, kidney) of mice, which were sacrificed at different time of postinjection. There was no obvious damage to the major organs after injection of CS-CD-Ce6 NPs, in comparison with healthy mice Figure S13, which demonstrates the good biocompatibility of CS-CD-Ce6 NPs.57

4. CONCLUSIONS In summary, versatile ultrasmall O2-evolving CS-CD-Ce6 NPs were successfully developed to boost the performance of photodynamic therapy to kill breast cancer cells and to elicit antitumor immunity for reducing their metastasis. These ultrasmall 28 / 41


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nanoparticles can overcome tumor hypoxia and generate large amount of ROS due to their strong near-infrared (NIR) absorbance and Fenton-like-Haber-Weiss catalytic property. The generated ROS not only can kill primary breast cancer cells, but also induce ICD to release DAMPs from tumor cells to elicit anti-tumor immunity. In addition, M1-polarization was also induced by ROS produced by CS-CD-Ce6 NPs under NIR irradiation. The activation of anti-tumor immunity successfully inhibited the recurrence and metastasis of cancer. Our research provides new insights into the design of highly efficient nanotheranostic agents with excellent PDT performance, especially copper chalcogenide based Cu2-xE (E = S, Se, Te, 0 ≦ x ≦ 1) nanomaterials, to against cancer and its metastasis.

ASSOCIATED CONTENT Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOUR INFORMATION Corresponding Author * To whom correspondence should be addressed: Email: [email protected] ACKNOWLEDGEMENT Z. Li acknowledges support from the National Key Research and Development Program of China (2018YFA0208800), National Natural Science Foundation of China 29 / 41



(81471657, 81527901), the 1000 Plan for Young Talents, Jiangsu Specially Appointed Professorship, and the Program of Jiangsu Innovative and Entrepreneurial Talents. The authors also are grateful for support from the Jiangsu Provincial Key Laboratory of Radiation Medicine and Protection, the Priority Academic Development Program of Jiangsu Higher Education Institutions (PAPD). The authors would like to thank Dr. Tania Silver for critical reading of the manuscript.

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REFERENCES 1. Müller, A.; Homey, B.; Soto, H.; Ge, N.; Catron, D.; Buchanan, M. E.; Mcclanahan, T.; Murphy, E.; Yuan, W.; Wagner, S. N. Involvement of Chemokine Receptors in Breast Cancer Metastasis. Nature 2001, 410, 50−56. 2. Yamamoto, K. N.; Nakamura, A.; Haeno, H. The Evolution of Tumor Metastasis During Clonal Expansion with Alterations in Metastasis Driver Genes. Sci. Rep. 2015, 5, 15886. 3. Ng, C. W.; Li, J.; Pu, K. Recent Progresses in Phototherapy-Synergized Cancer Immunotherapy. Adv. Funct. Mater. 2018, 28, 1804688. 4. Bear, A. S.; Kennedy, L. C.; Young, J. K.; Perna, S. K.; Joao Paulo, M. A.; Lin, A. Y.; Eckels, P. C.; Drezek, R. A.; Foster, A. E. Elimination of Metastatic Melanoma Using Gold Nanoshell-Enabled Photothermal Therapy and Adoptive T Cell Transfer. Plos One 2013, 8, e69073. 5. Duan, X.; Chan, C.; Lin, W. Nanoparticle-Mediated Immunogenic Cell Death Enables and Potentiates Cancer Immunotherapy. Angew. Chem. Int. Edit. 2019, 58, 670−680. 6. Chen, Q.; Chen, J.; Yang, Z.; Xu, J.; Xu, L.; Liang, C.; Han, X.; Liu, Z. Nanoparticle-Enhanced Radiotherapy to Trigger Robust Cancer Immunotherapy. Adv. Mater. 2019, 1802228. 7. Yu, X.; Gao, D.; Gao, L. Inhibiting Metastasis and Preventing Tumor Relapse by Triggering Host Immunity with Tumor-Targeted Photodynamic Therapy Using Photosensitizer-Loaded Functional Nanographenes, ACS Nano 2017, 11, 10147−10158. 8. Panzarini, E.; Inguscio, V.; Dini, L. Immunogenic Cell Death: Can it be Exploited in Photodynamic Therapy for cancer? BioMed. Res. Int. 2013, 2013, 482160. 9. Lucky, S. S.; Soo, K. C.; Zhang, Y. Nanoparticles in Photodynamic Therapy. Chem. Rev. 2015, 115, 1990−2042. 10. Zhou, Z.; Song, J.; Nie, L.; Chen, X. Reactive Oxygen Species Generating Systems Meeting Challenges of Photodynamic Cancer Therapy. Chem. Soc. Rev. 2016, 45, 6597−6626. 11. Fan, W.; Huang, P.; Chen, X. Overcoming the Achilles' heel of Photodynamic Therapy. Chem. Soc. Rev. 2016, 45, 6488−6519. 12. Lu, K.; He, C.; Guo, N.; Chan, C.; Ni, K.; Weichselbaum, R. R.; Lin, W. ChlorinBased Nanoscale Metal-Organic Framework Systemically Rejects Colorectal Cancers via Synergistic Photodynamic Therapy and Checkpoint Blockade Immunotherapy. J. Am. Chem. Soc. 2016, 138, 12502−12510. 13. He, C.; Duan, X.; Guo, N.; Chan, C.; Poon, C.; Weichselbaum, R. R.; Lin, W. Coreshell Nanoscale Coordination Polymers Combine Chemotherapy and Photodynamic Therapy to Potentiate Checkpoint Blockade Cancer Immunotherapy. Nat. Commun. 2016, 7, 12499; 14. Agostinis, P.; Berg, K.; Cengel, K. A.; Foster, T. H.; Girotti, A. W.; Gollnick, S. O.; Hahn, S. M.; Hamblin, M. R.; Juzeniene, A.; Kessel, D.; Korbelik, M.; Moan, J.; Mroz, P.; Nowis, D.; Piette, J.; Wilson, B. C.; Golab, J. Photodynamic Therapy of Cancer: An Update. CA-Cancer J. Clin. 2011, 61, 250−281 15. Castano, A. P.; Mroz, P.; Hamblin, M. R. Photodynamic Therapy and Anti-Tumour 31 / 41



Immunity. Nat. Rev. Cancer 2006, 6, 535−545. 16. Tan, H. Y.; Wang, N.; Li, S.; Hong, M.; Wang, X.; Feng, Y. The Reactive Oxygen Species in Macrophage Polarization: Reflecting Its Dual Role in Progression and Treatment of Human Diseases. Oxid. Med. Cell. Longev. 2016, 2016, 2795090. 17. Zhou, Y.; Que, K. T.; Zhang, Z.; Yi, Z. J.; Zhao, P. X.; You, Y.; Gong, J. P.; Liu, Z. J. Iron Overloaded Polarizes Macrophage to Proinflammation Phenotype Through ROS/acetyl-p53 pathway. Cancer Med. 2018, 7, 4012−4022. 18. Shi, C.; Liu, T.; Guo, Z.; Zhuang, R.; Zhang, X.; Chen, X. Reprogramming TumorAssociated Macrophages by Nanoparticle-Based Reactive Oxygen Species Photogeneration. Nano Lett. 2018, 18, 7330−7342. 19. Castano, A. P.; Mroz, P.; Wu, M. X.; Hamblin, M. R. Photodynamic Therapy Plus Low-Dose Cyclophosphamide Generates Antitumor Immunity in A Mouse Model. P. Nat. Acad. Sci. USA. 2008, 105, 5495−5500. 20. Dang, J.; He, H.; Chen, D.; Yin, L. Manipulating Tumor Hypoxia Toward Enhanced Photodynamic Therapy (PDT). Biomater. Sci. 2017, 5, 1500−1511; 21. Dai, Y.; Xu, C.; Sun, X.; Chen, X. Nanoparticle Design Strategies for Enhanced Anticancer Therapy by Exploiting the Tumour Microenvironment. Chem. Soc. Rev. 2017, 46, 3830−3852. 22. Xue, P.; Hou, M.; Sun, L.; Li, Q.; Zhang, L.; Xu, Z.; Kang, Y. Calcium-Carbonate Packaging Magnetic Polydopamine Nanoparticles Loaded with Indocyanine Green for Near-Infrared Induced Photothermal/Photodynamic Therapy. Acta Biomater. 2018, 81, 242−255. 23. Zuo, H.; Tao, J.; Shi, H.; He, J.; Zhou, Z.; Zhang, C. Platelet-Mimicking Nanoparticles Co-Loaded with W18O49 and Metformin Alleviate Tumor Hypoxia for Enhanced Photodynamic Therapy and Photothermal Therapy. Acta Biomater 2018, 80, 296−307. 24. Qun, C.; Zheng, H.; Hua, C.; Howard, S.; Jill, B.; Hetzel, F. W. Improvement of Tumor Response by Manipulation of Tumor Oxygenation During Photodynamic Therapy. Photochem. Photobiol. 2010, 76, 197−203. 25. Liu, L. H.; Zhang, Y. H.; Qiu, W. X.; Zhang, L.; Gao, F.; Li, B.; Xu, L.; Fan, J. X.; Li, Z. H.; Zhang, X. Z. Dual-Stage Light Amplified Photodynamic Therapy against Hypoxic Tumor Based on an O2 Self-Sufficient Nanoplatform. Small 2017, 13, 1701621. 26. Fan, W.; Bu, W.; Shen, B.; He, Q.; Cui, Z.; Liu, Y.; Zheng, X.; Zhao, K.; Shi, J. Intelligent MnO2 Nanosheets Anchored with Upconversion Nanoprobes for Concurrent pH-/H2O2-Responsive UCL Imaging and Oxygen-Elevated Synergetic Therapy. Adv. Mater. 2015, 27, 4155−4161. 27. Wang, Z.; Zhang, Y.; Ju, E.; Liu, Z.; Cao, F.; Chen, Z.; Ren, J.; Qu, X. Biomimetic Nanoflowers by Self-assembly of Nanozymes to Induce Intracellular Oxidative Damage Against Hypoxic Tumors. Nat. Commun. 2018, 9 3334. 28. Jia, Q.; Ge, J.; Liu, W.; Zheng, X.; Chen, S.; Wen, Y.; Zhang, H.; Wang, P. A Magnetofluorescent Carbon Dot Assembly as an Acidic H2O2 -Driven Oxygenerator to Regulate Tumor Hypoxia for Simultaneous Bimodal Imaging and Enhanced Photodynamic Therapy. Adv. Mater. 2018, 30, 1706090. 32 / 41


Page 32 of 41

Page 33 of 41 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


29. Zhang, Y.; Wang, F.; Liu, C.; Wang, Z.; Kang, L.; Huang, Y.; Dong, K.; Ren, J.; Qu, X. Nanozyme Decorated Metal-Organic Frameworks for Enhanced Photodynamic Therapy. ACS Nano 2018, 12, 651−661. 30. Liu, T.; Zhang, N.; Wang, Z.; Wu, M.; Chen, Y.; Ma, M.; Chen, H.; Shi, J. Endogenous Catalytic Generation of O2 Bubbles for In Situ Ultrasound-Guided High Intensity Focused Ultrasound Ablation. ACS Nano 2017, 11, 9093−9102. 31. Nieto-Juarez, J. I.; Katarzyna, P. A.; Andrzej, S.; Tamar, K. Inactivation of MS2 Coliphage in Fenton and Fenton-like Systems: Role of Transition Metals, Hydrogen Peroxide and Sunlight. Environ. Sci. Technol. 2010, 44, 3351−3356; 32. Bokare, A. D.; Choi, W. Review of Iron-Free Fenton-like Systems for Activating H2O2 in Advanced Oxidation Processes. J. Hazard. Mater. 2014, 275, 121−135. 33. Zhang, S.; Sun, C.; Zeng, J.; Sun, Q.; Wang, G.; Wang, Y.; Wu, Y.; Dou, S.; Gao, M.; Li, Z. Ambient Aqueous Synthesis of Ultrasmall PEGylated Cu2-xSe Nanoparticles as a Multifunctional Theranostic Agent for Multimodal Imaging Guided Photothermal Therapy of Cancer. Adv. Mater. 2016, 28, 8927−8936. 34. Paul, S.; Heng, P. W.; Chan, L. W. pH-dependent Complexation of Hydroxypropyl-Beta-cyclodextrin With Chlorin e6: Effect on Solubility and Aggregation in Relation to Photodynamic efficacy. J. Pharm. Pharmacol. 2016, 68, 439−449. 35. Rajeswari, C.; Alka, A.; Javed, A.; Khar, R. K. Cyclodextrins in Drug Delivery: An Updated Review. Aaps Pharmscitech 2005, 6, E329−E357. 36. Zhang, L.; Wan, S. S.; Li, C. X.; Xu, L.; Cheng, H.; Zhang, X. Z. An Adenosine Triphosphate-Responsive Autocatalytic Fenton Nanoparticle for Tumor Ablation with Self-Supplied H2O2 and Acceleration of Fe(III)/Fe(II) Conversion. Nano Lett. 2018, 18, 7609−7618. 37. Yu, M.; Guo, F.; Wang, J.; Tan, F.; Li, N. A pH-Driven and Photoresponsive Nanocarrier: Remotely-Controlled by Near-Infrared Light for Stepwise Antitumor Treatment. Biomaterials 2016, 79, 25−35. 38. Liu, Y.; Zhen, W.; Jin, L.; Zhang, S.; Sun, G.; Zhang, T.; Xu, X.; Song, S.; Wang, Y.; Liu, J.; Zhang, H. All-in-One Theranostic Nanoagent with Enhanced Reactive Oxygen Species Generation and Modulating Tumor Microenvironment Ability for Effective Tumor Eradication. ACS Nano 2018, 12, 4886−4893. 39. Paschoalino, M.; Guedes, N. C.; Jardim, W.; Mielczarski, E.; Mielczarski, J. A.; Bowen, P.; Kiwi, J. Inactivation of E. coli Mediated by High Surface Area CuO Accelerated by Light Irradiation >360 nm. J. Photoch. Photobio. 2008, 199, 105−111. 40. Chen, J.; Luo, H.; Liu, Y.; Zhang, W.; Li, H.; Luo, T.; Zhang, K.; Zhao, Y.; Liu, J. Oxygen-Self-Produced Nanoplatform for Relieving Hypoxia and Breaking Resistance to Sonodynamic Treatment of Pancreatic Cancer. ACS Nano 2017, 11, 12849−12862. 41. Bharathiraja, S.; Manivasagan, P.; Moorthy, M. S.; Bui, N. Q.; Lee, K. D.; Oh, J. Chlorin e6 Conjugated Copper Sulfide Nanoparticles for Photodynamic Combined Photothermal Therapy. Photodiagn. Photodyn. 2017, 19, 128−134. 42. Chang, K.; Liu, Z.; Fang, X.; Chen, H.; Men, X.; Yuan, Y.; Sun, K.; Zhang, X.; Yuan, Z.; Wu, C. Enhanced Phototherapy by Nanoparticle-Enzyme via Generation and Photolysis of Hydrogen Peroxide. Nano Lett. 2017, 17, 4323−4329. 33 / 41



43. Li, Z.; Wu, M.; Bai, H.; Liu, X.; Tang, G. Light-Enhanced Hypoxia-Responsive Nanoparticles for Deep Tumor Penetration and Combined Chemo-Photodynamic Therapy. Chem. Commun. 2018, 54, 13127−13130. 44. Jiang, X.; Zhang, S.; Ren, F.; Chen, L.; Zeng, J.; Zhu, M.; Cheng, Z.; Gao, M.; Li, Z. Ultrasmall Magnetic CuFeSe2 Ternary Nanocrystals for Multimodal Imaging Guided Photothermal Therapy of Cancer. ACS Nano 2017, 11, 5633−5645. 45. Jiang, J.; Auchinvole, C.; Fisher, K.; Campbell, C. J. Quantitative Measurement of Redox Potential in Hypoxic Cells Using SERS Nanosensors. Nanoscale 2014, 6, 12104−12110. 46. Zhang, H.; Wang, T.; Liu, H.; Ren, F.; Qiu, W.; Sun, Q.; Yan, F.; Zheng, H.; Li, Z.; Gao, M. Second Near-Infrared Photodynamic Therapy and Chemotherapy of Orthotopic Malignant Glioblastoma with Ultra-small Cu2-xSe Nanoparticles. Nanoscale 2019, DOI:10.1039/C9NR01789E. 47. Wen, Y.; Zhang, W.; Gong, N.; Wang, Y. F.; Guo, H. B.; Guo, W.; Wang, P. C.; Liang, X. J. Carrier-Free, Self-Assembled Pure Drug Nanorods Composed of 10hydroxycamptothecin and Chlorin e6 for Combinatorial Chemo-Photodynamic Antitumor Therapy in vivo. Nanoscale 2017, 9, 14347−14356. 48. Chen, Z.; Liu, L.; Liang, R.; Luo, Z.; He, H.; Wu, Z.; Tian, H.; Zheng, M.; Ma, Y.; Cai, L. Bioinspired Hybrid Protein Oxygen Nanocarrier Amplified Photodynamic Therapy for Eliciting Anti-tumor Immunity and Abscopal Effect. ACS Nano 2018, 12, 8633−8645. 49. Huang, P.; Wang, X.; Liang, X.; Yang, J.; Zhang, C.; Kong, D.; Wang, W., Nano-, Micro-, and Macroscale Drug Delivery Systems for Cancer Immunotherapy. Acta Biomater. 2019, 85, 1−26. 50. Liang, R.; Liu, L.; He, H.; Chen, Z.; Han, Z.; Luo, Z.; Wu, Z.; Zheng, M.; Ma, Y.; Cai, L. Oxygen-Boosted Immunogenic Photodynamic Therapy with Gold Nanocages@Manganese Dioxide to Inhibit Tumor Growth and Metastases. Biomaterials 2018, 177, 149−160. 51. Deng, G.; Sun, Z.; Li, S.; Peng, X.; Li, W.; Zhou, L.; Ma, Y.; Gong, P.; Cai, L. Cell-Membrane Immunotherapy Based on Natural Killer Cell Membrane Coated Nanoparticles for the Effective Inhibition of Primary and Abscopal Tumor Growth. ACS Nano 2018, 12, 12096−12108. 52. Zhang, H.; Wang, T.; Qiu, W.; Han, Y.; Sun, Q.; Zeng, J.; Yan, F.; Zheng, H.; Li, Z.; Gao, M. Monitoring the Opening and Recovery of the Blood−Brain Barrier with Noninvasive Molecular Imaging by Biodegradable Ultrasmall Cu2−xSe Nanoparticles. Nano Lett. 2018, 18, 4985-4992. 53. Zhou, F.; Feng, B.; Wang, T.; Wang, D.; Cui, Z.; Wang, S.; Ding, C.; Zhang, Z.; Liu, J.; Yu, H.; Li, Y. Theranostic Prodrug Vesicles for Reactive Oxygen SpeciesTriggered Ultrafast Drug Release and Local-Regional Therapy of Metastatic TripleNegative Breast Cancer. Adv. Funct. Mater. 2017, 27, 1703674. 54. Hang, Q.; Zhang, S.; Zhang, H.; Han, Y.; Liu, H.; Ren, F.; Sun, Q.; Li, Z.; Gao, M. Boosting the Radiosensitizing and Photothermal Performance of Cu2-xSe Nanocrystals for Synergetic Radiophotothermal Therapy of Orthotopic Breast Cancer. ACS Nano 34 / 41


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2019, 13, 1342−1353. 55. Wang, M.; Deng, K.; Lu, W.; Deng, X.; Li, K.; Shi, Y.; Ding, B.; Cheng, Z.; Xing, B.; Han, G.; Hou, Z.; Lin, J. Rational Design of Multifunctional Fe@gamma-Fe2O3 @H-TiO2 Nanocomposites with Enhanced Magnetic and Photoconversion Effects for Wide Applications: From Photocatalysis to Imaging-Guided Photothermal Cancer Therapy. Adv. Mater. 2018, 30, 1706747. 56. Zhang, S.; Huang, Qian.; Zhang, L.; Zhang, L.; Zhang, H.; Han, Y.; Sun, Q.; Cheng, Z.; Qin, H.; Dou, S.; Li, Z. Vacancy Engineering of Cu2−xSe Nanoparticles with Tunable LSPR and Magnetism for Dual-Modal Imaging Guided Photothermal Therapy of Cancer. Nanoscale 2018, 10, 3130-3143. 57. Hao, X.; Li, C.; Zhang, Y.; Wang, H.; Chen, G.; Wang, M.; Wang, Q. Programmable Chemotherapy and Immunotherapyagainst Breast Cancer Guided by Multiplexed FluorescenceImaging in the Second Near-Infrared Window. Adv. Mater. 2018, 30, 1804437.

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Figure 1. Characterization of CS-CD-Ce6 NPs: a) TEM image, b) zeta potential, c) UV-vis-NIR absorbance. d) Degradation of H2O2 (392 μM) with CS-CD-Ce6 NPs (25 μg/mL) with or without irradiation (808 nm laser, 0.75 W/cm2, 5 min) (n=3). e) Generation of O2 from H2O2 and CS-CD NPs (25 μg/mL) with or without irradiation, (f-g) ESR spectra of adducts of (f) DMPO/OH, and (g) TEMP/1O2 to specifically detect OH and 1O2 generated from degradation of H2O2 with and without CS-CD-Ce6 NPs under irradiation, h) Schematic illustration of CS-CD-Ce6 NPs to produce ROS. i) Fluorescence spectra of DCFH-DA mixed with CS-CD-Ce6 NPs with or without irradiation.

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Figure 2. Under irradiation by 660 nm or 808 nm laser (0.75 W/cm2, 5 min), the degradation of H2O2 (392 μM) catalyzed by CS-CD-Ce6 NPs (25 μg/mL), and the detection of degradation products: a) Degradation of H2O2, b) fluorescence spectra of 2-hydroxy-terephalic acid (TAOH) for detecting OH, c) degradation of DPBF for detecting 1O2 and O2-, d) fluorescence spectra of DCF for detecting total ROS.

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Figure 3. CLSM images of 4T1 cells after incubation with Ce6 (25 μg/mL), CS-CD NPs (25 μg/mL) or CS-CD-Ce6 NPs (25 μg/mL), and irradiation with 808 nm laser (0.75 W/cm2, 5 min) (scale bar is 10 μm): a) hypoxia indicator, b) SOSG indicator of 1O , c) DCFH-DA indicator of total ROS. d) Viability of 4T1 cells cultured with 2 different concentration of CS-CD and CS-CD-Ce6 NPs in the presence or absence of UA with irradiation. e) Cell apoptosis of 4T1 cells cultured with Ce6 (2.5 μg/mL), CSCD NPs (25 μg/mL), or CS-CD-Ce6 NPs (25 μg/mL) with or without irradiation.

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Figure 4. In vitro anti-tumor immunity triggered by ROS during PDT. a) Schematic illustration of the dying tumor cells undergoing release of the DAMPs (CRT, ATP and HMGB1), triggered by large amount of ROS produced during degradation of H2O2 by CS-CD-Ce6 NPs under irradiation. b) Schematic illustration of PDT inducing proinflammatory M1- macrophages polarization. c) CLSM of CRT exposed on the surface of tumor cells. d) Detection of extracellular ATP secreted. e) Detection of extracellular HMGB1 released. f) FCAS of M1-macrophage polarization (CD86) and M1macrophage polarization (CD206). Cytokine levels in vitro, g) IL-12 produce by M1. h) IL-10 produced by M2. Those results suggested the polarization of TAM induced by ROS generated by CS-CD-Ce6.

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Figure 5. PA imaging guided in vivo therapy of tumors with different treatments: a) PA images of tumor (highlighted by red circles) from a 4T1 tumor-bearing mouse collected before and after tail vein injection of CS-CD-Ce6 NPs solution (500 μg/mL, 200 μL) at different time points. b) Relative PA signals on PA images shown in (a). c) Representative PA images of solid tumors characterized by HbO2 after injection of CSCD-Ce6 for 12 h before and after irradiation. d) Relative PA signals of HbO2 shown in (c). e) Images of hypoxia-inducible factor-1 (HIF-1α) (scale bar: 100 μm), and f) DCFH-DA and CD86 stained tumor sections from different groups of mice sacrificed after 2 days of treatments (scale bar: 50 μm).

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Figure 6. a) Relative tumor volume normalized to the initial volumes. b) Weight of mice. c) Survival rate of mice. d) Images of H&E stained tumor sections from different groups of mice sacrificed after 2 days of treatments (scale bar: 100 μm). e) Photographs of whole lungs from different groups of mice after treated for 23 days. The black circles denote the metastatic tumors. f) H&E of metastatic nodules in lung tissue. The read circles denote the metastatic tumor (scale bar: 200 μm). g, h) Pro-inflammatory cytokine (TNF-α and IL-6) levels in the sera of mice treated with CS-CD-Ce6 NPs and irradiated with or without 808 nm laser.

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Light-enhanced O2-evolving Nanoparticles Boost Photodynamic

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