photodynamic therapy

Journal Pre-proofs CoWO4-x-based nanoplatform for multimode imaging and enhanced photothermal/photodynamic therapy Haixia Liu, Qingzhu Yang, Wei Guo, Huiming Lin, Feng Zhang, Jingxiang Zhao, Tianyue Ma, Le Zhao, Na Xu, Ruiwen Wang, Jingyao Yu, Fengyu Qu PII: DOI: Reference:

S1385-8947(19)33394-7 https://doi.org/10.1016/j.cej.2019.123979 CEJ 123979

To appear in:

Chemical Engineering Journal

Received Date: Revised Date: Accepted Date:

1 August 2019 25 December 2019 27 December 2019

Please cite this article as: H. Liu, Q. Yang, W. Guo, H. Lin, F. Zhang, J. Zhao, T. Ma, L. Zhao, N. Xu, R. Wang, J. Yu, F. Qu, CoWO4-x-based nanoplatform for multimode imaging and enhanced photothermal/photodynamic therapy, Chemical Engineering Journal (2019), doi: https://doi.org/10.1016/j.cej.2019.123979

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© 2019 Published by Elsevier B.V.

CoWO4-x-based

nanoplatform

for

multimode

imaging and enhanced photothermal/photodynamic therapy Haixia Liu, ‡a Qingzhu Yang, ‡b Wei Guo,*a Huiming Lin, a Feng Zhang, aJingxiang Zhao,a Tianyue Ma, a Le Zhao, a Na Xu, c Ruiwen Wang,*b Jingyao Yu, a Fengyu Qu*a

a

Key Laboratory of Photochemical Biomaterials and Energy Storage Materials, Heilongjiang

Province and College of Chemistry and Chemical Engineering, Harbin Normal University, Harbin, 150025, China b School c

of Life Science and Technology, Harbin Institute of Technology, Harbin, China.

Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, Nanjing, China

‡ Equal

contribution from Haixia Liu and Qingzhu Yang

Corresponding author: [email protected] (W. Guo) [email protected] (F. Y. Qu) Keywords: Immunoresistance, Photothermal therapy, Photodynamic therapy, HSP60, NRF2

1

Abstract

Although phototherapy has received widespread attention in recent years due to its high efficiency, low invasiveness, and minor side effects, there are still few studies on the relationship between phototherapy and immunotherapy. Therefore, non-stoichiometric CoWO4-x nanoparticles (NPs) were strategically designed and prepared, as these can simultaneously generate hyperthermia and reactive oxygen species (ROS) under near-infrared laser irradiation, illustrating that they can be used to realize photothermal therapy (PTT) and photodynamic therapy (PDT) on tumors. CoWO4-x NPs not only have strong near-infrared absorption, outstanding biocompatibility, and excellent photothermal stability but they also can be used as photoacoustic (PA) and Computer Tomography (CT) contrast agents for working with tumors. In particular, the relationships between phototherapy, immunogenic cell death (ICD) and immunoresistance are here discussed in depth, and enhanced phototherapy was achieved for the first time by injecting etoposide and ML385, which are two typical inhibitors for HSP60 and NRF2, respectively. Furthermore, the biocompatibility of materials in vivo was demonstrated through a variety of cell experiments, changes in mouse body weight, and histological analyses. Our work highlights the great potentials of non-stoichiometric CoWO4-x NPs as a kind of multifunctional therapeutic agent for implementing enhanced phototherapy in breast cancer by reducing the immunoresistance of HSP60 and NRF2 toward PTT and PDT.

2

1. Introduction Breast cancer is one of the most common malignant tumors in women, which has the characteristics of high morbidity, enormous harmfulness, and ease of metastasis

[1].

The current

standard-of-care treatment for breast cancer mainly relies on a combination of surgery and chemotherapy [2]. In theory, surgical treatment can altogether remove tumor tissue and cure cancer, but it is not effective in cases with diffuse or multiple tumors

[3].

For chemotherapy, most

chemotherapeutic drugs often lack specific targeting abilities for tumor cells and have strong side effects, creating adverse reactions in healthy cells and other organs while eliminating cancer cells [4].

At present, there are other emerging alternative anti-cancer therapies for the treatment of breast

cancer, such as phototherapy, magnetic hyperthermia, biocatalytic therapy, immunotherapy, and ultrasonic therapy [5-9]. Among these therapeutic techniques, phototherapy (including photothermal therapy (PTT) and photodynamic therapy (PDT)) has received extensive attention as a powerful technique for tumor therapy due to its low invasiveness, deep tissue penetration, and high efficiency

[10-15].

PDT is an oxygen-dependent treatment method, where the therapeutic effect

gradually diminishes with increasing consumption of tissue oxygen

[16].

In PTT, the process

involves a temperature rise in target cells, and the therapeutic effect increases progressively with time and is not affected by oxygen concentration [17]. Therefore, a combination of PDT and PTT should theoretically have synergistic efficacy [18]. To accomplish this antitumor synergistic impact, most previous studies developed nanoplatforms composed of a photosensitizer (PS) and photothermal agent (PTA) for PDT/PTT combination treatment. These studies had some shortcomings, including complex reaction processes, mutual interference, and mismatch of absorption between PTA and PS, for example

[19].

Various photothermal agents, including

inorganic and organic ones, plasma metals, carbon-based nanomaterials, inorganic semiconductor

3

nanomaterials, organic polymers, or organic dyes, have been used in PTT. As a non-stoichiometric compound, CoWO4-x NPs have the following advantages: (i) Wide optical absorption band. Compared with most other photoactive materials, CoWO4-x NPs have a full spectral absorption in the range of 200-2500 nm. (ii) They possess both photothermal and photodynamic characteristics. Not all photothermal materials have both photothermal and photodynamic properties, and only a few of them have both characteristics. Recently, some novel materials, such as CsxWO3, TiO2-x and Cu2(OH)PO4, have been identified as potentially good candidates as both PS and PTA agents, which can be used in PDT/PTT combination treatment because they can produce both reactive oxygen species (ROS) and hyperthermia at the same time under single wavelength laser irradiation [20-23].

(iii) Multimodal imaging abilities. CoWO4-x NPs have excellent near-infrared absorption

properties and contain tungsten elements with high atomic number (Z = 74), making them ideal photoacoustic (PA) and computed tomography (CT) contrast agents. The phototherapy of tumors can stimulate the immune response of an organism, so characterization of agents for PTT and PDT is indispensable

[24].

As a cell death mode,

immunogenic cell death (ICD) is characterized by the release of high mobility group protein B1 (HMGB1), which can serve as the “eat me” signal of the innate immune system, as well as the exposure of Calreticulin and secretion of adenosine triphosphate (ATP) [25]. According to reports in the literature, both PTT and PDT can induce ICD, but it was difficult to completely ablate solid tumors by relying on such a cell death mode alone, and may also lead to the metastasis of cancer cells [26]. We speculate that the immunoresistance of an organism to phototherapy may be one of the most important aspects for the efficacy of PDT and PDT. The expression changes of heat shock protein (HSP) and NF-E2 related factors (NRF2) are critical immune responses of the tumor microenvironment to PTT and PDT, respectively, and both HSP and NRF2 belong to the cell’s

4

“protective switch” [27, 28]. PTT and PDT can lead to an increase in the expression of HSP and NRF2 around tumor cells, respectively. HSP is abundant in cells and widely distributed in various organisms [29]. When an organism is stressed by a threat to its internal or external environment, the organism regulates the expression of HSP, thereby achieving self-protection. Based on the relative molecular weight of HSPs and their degree of homology, it can be divided into HSP60, HSP70, and HSP90, and so on [30]. NRF2 is an important transcription factor for regulating the oxidative stress response of cells, and it is also a central regulator for maintaining intracellular redox homeostasis [31]. By inducing and regulating the constitutive and inducible expression of a series of antioxidant proteins, NRF2 can reduce the cell damage caused by ROS and electrophiles, keeping the cells in a stable state and maintaining the dynamic redox balance of an organism [32]. However, most studies on the expression of HSP and NRF2 have been carried out at the cellular level, while studies of the changes in HSP and NRF2 expression at the tumor level have been relatively rare during phototherapy. Therefore, it is of considerable significance to monitor changes in the expression levels of HSP and NRF2 at the solid tumor level at various stages of phototherapy. Additionally, depending on the trend of the expression levels of HSP and NRF2, appropriate molecular drugs can be selected to inhibit their expression, thereby achieving phototherapy and immune synergistic treatment. In this work, we developed a novel multifunctional theranostic agent based on Poly (ethylene oxide-b-methacrylic acid) (PEO-b-MAA) functionalized non-stoichiometric CoWO4-x (CoWO4x@PEO-b-MAA),

which showed strong NIR absorbance, for multimodal imaging and highly

effective in vivo enhanced phototherapy of breast tumors for the first time. These CoWO4-x NPs can not only function role as excellent CT and PA imaging contrast agents, but they also realize NIR laser-induced PTT and PDT. To improve the stability and biocompatibility of CoWO4-x

5

nanoparticles, PEO-b-MAA was modified on its surface through ultrasonic self-assembly. As shown in Schematic 1, phototherapy can induce ICD, which leads to an increase in the expression of HMGB1 and Calreticulin, but in general, it was difficult to obtain the desired therapeutic effect. Meanwhile, phototherapy can also stimulate the immunoresistance of an organism to phototherapy, as PTT and PDT induced an increase in the expression of HSP60 and NRF2, respectively. As specific inhibitors of PTT and PDT respectively, etoposide and ML385 were used to weaken the protective effects of the two “protective switches,” thus ultimately achieving phototherapy and synergistic immune therapy for breast tumors. Consequently, we hypothesize that the results presented here will stimulate advances in the use of CoWO4-x@PEO-b-MAA NPs for highly practical applications in fighting breast cancer tumors using PDT and PTT.

Schematic 1 Schematic illustration of enhanced phototherapy of breast cancer by reducing immunoresistance of HSP60 and NRF2 toward PTT and PDT. 2. Results and discussion

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2.1. Synthesis and characterization of CoWO4-x@PEO-b-PMAA NPs Figure 1a shows the synthesis path for creating CoWO4-x@PEO-b-PMAA NPs and their multifunctional biological applications. The transmission electron microscope (TEM) image in Figure 1b displays that the CoWO4 NPs, with a particle size ~ 50 nm, were prepared using a previously reported hydrothermal method. HR-TEM study showed that they exhibited bright lattice fringes, and the lattice parameter was observed to be 0.288 nm, which was assigned to the (1 1 1) plane of the monoclinic crystal of the CoWO4 (Figure S1). The non-stoichiometric CoWO4-x structure was achieved through the reduction of CoWO4 at 550 °C for 2 h under a hydrogen/argon atmosphere. After this reduction process, the morphology and crystal structure of CoWO4-x NPs were well preserved based on TEM (Figure 1c) and X-ray diffraction (XRD) analyses (Figure 1e). X-ray photoelectron spectroscopy (XPS) analysis (Figure 1f and g) was further employed to examine the chemical states of tungsten in the CoWO4 and CoWO4-x NPs. For the CoWO4 NPs, there were two peaks located at 37.3 eV (W4f5/2) and 35.2 eV (W4f7/2), which could be assigned to the spinorbit coupling of W6+ ions. In contrast, for the CoWO4-x NPs, except for the W6+ ions at the same location, there also existed peaks with lower binding energy at 34.4 eV and 36.5 eV, which could be attributed to the W 4f5/2 and W 4f7/2 core levels from W+5, implying the successful preparation of non-stoichiometric CoWO4-x NPs [33]. To improve the dispersity and stability of CoWO4-x NPs in physiological conditions, PEOPMAA was utilized to modify their surface using an ultrasonic self-assembly method to prevent particle aggregation, and TEM of these CoWO4-x@PEO-b-PMAA NPs are shown in Figure 1d [34].

To certify the successful modification of PEO-PMAA on the surface of CoWO4-x NPs, Fourier

transform infrared (FT-IR) spectroscopy was utilized to explore the chemical compositions of CoWO4-x@PEO-b-PMAA. As displayed in Figure S2, the absorption bands at 3465 and 2923 cm-1

7

could be assigned to the O-H stretching and C-H stretching vibration modes, respectively, while the bands located at 1710 cm-1 belonged to the C=O stretching vibration mode, and all of the above three bands resulted from the surface PEO-b-PMAA ligand [35,36].

Figure 1. (a) Schematic illustration of the synthetic method and multi-functional biological applications. (b) TEM image of CoWO4. (c) TEM image of CoWO4-x. (d) TEM image of CoWO4x@PEO-b-PMAA.

(e) XRD patterns of CoWO4 and CoWO4-x. (f) W 4f XPS spectra of CoWO4.

(g) W 4f XPS spectra of CoWO4-x. Additionally, the weight loss variance between CoWO4-x and CoWO4-x@PEO-b-PMAA originated from the PEO-b-PMAA modification, which indicated that the PEO-b-PMAA layer

8

accounted for 2.3% of the weight of the CoWO4-x@PEO-b-PMAA NPs (Figure S3). As can be seen in Figure S4, dynamic light scattering (DLS) indicated that the hydrodynamic size of CoWO4 NPs increased from ~ 122 nm to ~164 nm after PEO-PMAA modification. Moreover, the zetapotential value increased from -23.9 ± 7.36 eV for CoWO4 NPs to -16.3 ± 5.98 eV. The above data further indicated successful PEO-b-PMAA modification. 2.2. Optical and photothermal/photodynamic properties of CoWO4-x@PEO-b-PMAA NPs Excellent

optical

absorption

in

the

near-infrared

region

and

associated

photothermal/photodynamic properties are essential requirements for the implementation of phototherapy by CoWO4-x@PEO-b-PMAA NPs. As can be seen in Figure 2a, CoWO4 powders exhibited a high absorption in the UV-vis spectra, corresponding to a band gap of approximately 2.8 eV. In contrast to CoWO4 powders, an additional absorption band beyond 578 nm extending to the infrared region was observed for the CoWO4-x powders. According to the literature, this phenomenon can be caused for two reasons: (1) oxygen vacancies produced by hydrogen reduction and (2) W5+ on the surface of the CoWO4-x powders [37,38]. These results indicated that CoWO4-x had better application prospects as a NIR phototherapy agent than CoWO4. We further evaluated the absorbance of the CoWO4-x@PEO-b-PMAA dispersions at various concentrations. As illustrated in Figure 2b, CoWO4-x@PEO-b-PMAA dispersions showed a concentration-dependent extinction increase from the UV to the NIR region, suggesting their photothermal heating potential for the phototherapy. The photothermal conversion capability of CoWO4 NPs and CoWO4x@PEO-b-PMAA

NPs were evaluated in aqueous dispersions.

9

Figure 2. (a) UV-vis-NIR absorbance spectra of CoWO4 and CoWO4-x powders. (b) UV-Vis-NIR absorption spectra of CoWO4-x@PEO-b-PMAA dispersions at different concentrations. (c) Photothermal heating curves of CoWO4-x@PEO-b-PMAA dispersions under laser irradiation (808 nm, 1 W/cm2). (d) Comparison of the photothermal performance of CoWO4 and CoWO4-x@PEOb-PMAA. (e) Recycling heating profiles of CoWO4-x@PEO-b-PMAA NPs dispersions using an 808 nm laser (1.0 W/cm2). (f) Absorption changes of a DPBF probe under different irradiation time. As shown in Figure 2c, under the same laser irradiation (1 W/cm2,10 minutes), the temperature of the CoWO4-x@PEO-b-PMAA dispersions with different concentrations increased rapidly compared to pure water, indicating their excellent photothermal properties. As shown in Figure 2d, the temperature increase of CoWO4-x@PEO-b-PMAA dispersions with the same mass concentration (1 mg/mL) was higher than that of CoWO4 dispersions, which demonstrated that CoWO4-x@PEO-b-PMAA dispersions had superior photothermal performance. The photothermal

10

conversion efficiency (ɳ) of CoWO4-x@PEO-b-PMAA NPs was calculated to be 72.75% by a previously reported method (Figure S5)

[39].

To investigate the photothermal stability of the as-

prepared phototherapeutic agent, CoWO4-x@PEO-b-PMAA dispersions were irradiated by an 808 nm laser for 10 minutes, and then naturally cooled for 10 minutes in a cycle manner. Based on the recorded temperature values (Figure 2e), we found that five cycles showed little significant change in temperature, indicating that CoWO4-x@PEO-b-PMAA had excellent photothermal stability. To clarify whether CoWO4-x@PEO-b-PMAA NPs had sound photodynamic effects in aqueous solution, we applied a 1,3-diphenylisobenzofuran (DPBF)-based spectroscopic method to measure the formation ability of ROS in CoWO4-x@PEO-b-PMAA dispersions during laser irradiation. The DPBF probe reacts with ROS to be decomposed into 1,2-dibenzoylbenzene, thus leading to a decrease in optical absorption intensity at 410 nm [40]. As shown in Figure 2f, pure water resulted in minimal ROS production under laser irradiation. In contrast, the absorbance of DPBF dispersed in a CoWO4-x@PEO-b-PMAA solution (500 μg/mL) decreased significantly with prolonged irradiation time, indicating that CoWO4-x@PEO-b-PMAA induced abundant ROS. In addition, the absorbance of DPBF dispersed in a CoWO4 solution with the same mass concentration was lower than that of CoWO4-x@PEO-b-PMAA dispersions, indicating that CoWO4-x@PEO-b-PMAA dispersions had enhanced photodynamic property. As can be seen in Figure S6, CoWO4-x@PEOb-PMAA NPs possessed a very similar hydrodynamic diameter after dispersion in PBS for different time (~ 164 nm), which indicated they had a good colloidal stability in PBS. 2.3. In vitro cell cytotoxicity and phototherapeutic treatment Before in vitro PDT/PTT combination therapy in 4T1 cells, we first examined the cytotoxicity of the as-prepared CoWO4-x@PEO-b-PMAA NPs using a standard MTT assay. As shown in

11

Figure 3a, our results showed that with an increase in the concentration of CoWO4 and CoWO4x@PEO-b-PMAA

NPs, the survival rate of the cells gradually decreased, which demonstrated a

dose-dependent effect in vitro.

Figure 3. (a) Relative cell viability after treatment with various concentrations of CoWO4 or CoWO4-x@PEO-b-PMAA for 24 h. (b) Relative cell viability after different treatments, including the control group, PDT group, PTT group, and PTT + PDT group. (c) Fluorescence microscope images of Calcein-AM and PI co-stained 4T1 cells after receiving different treatments (Scale bars

12

= 500 µm). (d) Fluorescence microscope images of ROS generation in 4T1 cells after receiving different treatments (Scale bars = 100 µm). (e) Detection of cell membrane permeability in different groups by EB staining (Scale bars = 50 µm). (f) Detection of mitochondrial potential changes in different groups by JC-1 staining (Scale bars = 50 µm). The cell viabilities of 4T1 cells were 82.18% and 85.09%, respectively, when the concentrations of CoWO4 and CoWO4-x@PEO-b-PMAA were 1000 μg/mL, suggesting that these compounds had low cytotoxicity. In this work, CoWO4-x@PEO-b-PMAA NPs could simultaneously produce hyperthermia and ROS under a NIR laser irradiation and lead to the synergistic effect of PDT/PTT in 4T1 cells. The cellular uptakes of CoWO4-x@PEO-b-PMAA NPs were investigated by inductively coupled plasma optical emission spectrometry (ICP-OES) analysis. As shown in Figure S7, as the incubation time increased, the cellular uptake of CoWO4-x@PEO-b-PMAA NPs gradually increased, and there was no significant difference between uptake by 12 and 24 h, which proved that the material might have reached its maximum cellular uptake by this time. To distinguish the contribution of individual PDT or PTT to the overall therapeutic effect, we divided the experimental groups into a control group, a PTT only group, a PDT only group, and a PTT combined with PDT group. In the PTT only group, the photodynamic properties of the material were eliminated by adding the ROS quenching agent sodium azide [20]. For the PDT only group, the photothermal properties of the material were eliminated by placing the cells on an ice cube. As displayed in Figure 3b, for a single therapeutic effect in the PDT or PTT group, the inhibition rates of 4T1 cells were only 31.2% and 59.8%, respectively, under an 808 nm laser irradiation. In contrast, the inhibition rate of 4T1 cells in the PTT combined with PDT experimental group was 88.2%, which proved that CoWO4-x@PEO-b-PMAA NPs could simultaneously produce photothermal and photodynamic therapy for 4T1 cells under near-infrared laser irradiation. Next,

13

we evaluated the laser-activated therapeutic effect of CoWO4-x@PEO-b-PMAA NPs using a live/dead double-staining method. Briefly, Calcein AM, which had green emission, and propidium (PI), which had red emission were employed to stain living and dead 4T1 cells, respectively. As shown in Figure 3c, the cells of the control group, NIR laser group, and CoWO4-x@PEO-b-PMAA group showed strong green fluorescence, implying either a NIR laser or CoWO4-x@PEO-b-PMAA alone did not cause cell death. In contrast, in the CoWO4-x@PEO-b-PMAA combined with laser irradiation group, we observed a large number of cell deaths, and as the duration of irradiation increased, the number of cell deaths increased significantly. The PDT performance of CoWO4x@PEO-b-PMAA

in 4T1 cells was evaluated using a dichlorofluorescein diacetate (H2DCFDA)

probe, which was commonly used to detect intracellular ROS production. 4T1 cells without any treatment were used as a negative control and 4T1 cells treated with H2O2 (50 mM) were used as a positive control. As shown in Figure 3d, the negative control and CoWO4-x@PEO-b-PMAA treated cells had no observable green fluorescence, while the NIR irradiated group had only a weak fluorescent signal. In contrast, H2O2 and CoWO4-x@PEO-b-PMAA combined with laser irradiation groups emitted strong green fluorescence, suggesting that a large number of ROS were produced. When the ROS quencher sodium azide was added to the 4T1 cells in which CoWO4x@PEO-b-PMAA

nanoparticles were dispersed, the cells did not show green fluorescence under

NIR irradiation, which was due to the ROS produced during irradiation having been quenched. The above results clearly indicated that the CoWO4-x@PEO-b-PMAA NPs were a promising candidate for both realizing combined PTT /PDT treatments. We hypothesized that cell membrane permeability and mitochondrial membrane potential might change during photothermal therapy. To test our hypothesis, we used ethidium bromide (EB) and JC-1 probes to detect the cell membrane permeability and mitochondrial membrane potential of the CoWO4-x@PEO-b-PMAA

14

treated 4T1 cells under irradiation, respectively [41]. As illustrated in Figure 3e, the cells of the control group, NIR laser group, and CoWO4-x@PEO-b-PMAA group showed little red fluorescence. For the CoWO4-x@PEO-b-PMAA combined with the laser irradiation group, 4T1 cells showed strong red fluorescence, mainly due to the damage of the cell membrane, and then EB entered the cells through the damaged cell membrane and bound to nucleic acids. Based on previous literature reports, the JC-1 probe can be used to assess the function of mitochondria, as it can stain normal mitochondria and damaged mitochondria with red and green fluorescence, respectively. Among the four groups, a large number of damaged mitochondria were observed in the CoWO4-x@PEO-b-PMAA combined with the laser irradiation group (Figure 3f). The above two experimental results clearly demonstrated that photothermal therapy can kill cancer cells by changing the membrane permeability of cancer cells and damaging mitochondria. 2.4. PA and CT imaging Among the numerous extant multi-mode imaging techniques, the dual-imaging modalities of PA and CT imaging have critical applications in the field of accurate disease diagnosis

[42].

The

main reason is that CT imaging has the advantages in creating 3D visual images and deep tissue penetration, while PA imaging can provide increased details about the microstructure of a disease site [43]. Considering that CoWO4-x@PEO-b-PMAA NPs have excellent near-infrared absorption properties and contain tungsten elements, which has a high atomic number (Z = 74), they should theoretically be ideal PA and CT contrast agents. As expected, the PA signal value of CoWO4x@PEO-b-PMAA

was positively correlated with increased concentration, indicating that CoWO4-

x@PEO-b-PMAA

should be a promising PA imaging candidate (Figure 4a).

15

Figure 4. (a) PA signal of CoWO4-x@PEO-b-PMAA solutions at different concentrations. (b) CT signal of CoWO4-x@PEO-b-PMAA solutions at different concentrations. (c) PA images of 4T1 tumor-bearing mice before and after intratumoral injection of CoWO4-x@PEO-b-PMAA (2 mg/mL, 50 μL) for different time. (d) CT images of 4T1 tumor-bearing mice before and after intratumoral injection with CoWO4-x@PEO-b-PMAA solutions (4 mg/mL, 50 μL). Tumor sites are marked with white circles. In order to test this in vivo, a CoWO4-x@PEO-b-PMAA dispersion (1 mg/mL, 50 μL) was injected into the tumors of 4T1 tumor-bearing mice by intratumoral injection and then imaged using a PA imaging system. As shown in Figure 4c, almost no PA signal was detected in the tumor site before the injection of the CoWO4-x@PEO-b-PMAA dispersion, and a relatively clear PA signal could be detected one hour after dosing with CoWO4-x@PEO-b-PMAA. The intensity of the PA signal at the tumor site of the mouse gradually increased from 1 h to 6 h, and slightly decreased by 24 h, indicating an excellent PA effect in vivo. Figure 4b displays the Hounsfield unit (HU) values of CoWO4-x@PEO-b-PMAA solutions at different concentration, which showed a good linear relationship with CoWO4-x@PEO-b-PMAA concentration increase. We then injected

16

a CoWO4-x@PEO-b-PMAA dispersion into the tumor of a mouse and detected the changes in CT signal at the tumor site of mice at different time. The results showed that the strongest CT signal in a tumor appeared after 6 h of material injection, and this trend was consistent with the results of PA imaging, which proved the potential of CoWO4-x@PEO-b-PMAA NPs for use in CT imaging. The changes of CT and PA signals may have been mainly related to the diffusion of the NPs after intratumoral administration. After intratumoral injection, the NPs reached their maximum diffusion level in the tumor around 6 h, after which they may have spread through the blood vessels inside the tumor to other locations. 2.5. In vivo enhanced phototherapeutic and immunological investigation Encouraged by the results of CoWO4-x@PEO-b-PMAA in vitro therapy, we further assessed its therapeutic effects on 4T1-tumor mice using in vivo experiments. After intratumoral injection of 100 μL of PBS or CoWO4-x@PEO-b-PMAA into a mouse, the tumor site was irradiated using an 808 nm laser with a power density of 1 W/cm2, and the surface temperature of the tumor was monitored with an IR photothermal camera. As displayed in Figure 5a and b, the tumor of CoWO4x@PEO-b-PMAA

injected mice exhibited a significant increase in temperature within 10 minutes

after exposure to an 808 nm laser, which increasing from 35 °C to 61.7 °C, while the tumor of PBS injected mice showed only a mild temperature change from 33.6 °C to 42.9 °C. When the tumor volume of mice reached 200-300 mm3, the tumor-bearing mice were first randomly divided into four groups: (i) control group, (ii) NIR laser group, (iii) CoWO4-x@PEO-b-PMAA group, and (iv) NIR laser + CoWO4-x@PEO-b-PMAA group (five mice per group). We evaluated the inhibitory effect of each group on tumors by calculating the relevant volume of the tumors in each group of mice at different treatment time.

17

Figure 5. (a) Infrared thermal images of 4T1 tumor-bearing mice and (b) corresponding temperature curves. (c) Relative tumor volumes of mice after different treatments. (d) Representative photographs of mice after the various treatments on the 14th day. (e) Western blotting analysis of tumors from the treatment procedure and (f) corresponding quantitative analysis of the Western blotting data. (g) Schematic diagram of immunoadjuvant therapy and obtained corresponding experimental results. (h) Quantitative measurement of tumor volume in mice with different treatments. (i) Body weight changes of mice during the treatment procedure. As shown in Figures 5c, 5d and S8, tumors in the control group, NIR laser group, and CoWO4x@PEO-b-PMAA

group were continuously increasing, indicating that NIR laser or CoWO4-

18

x@PEO-b-PMAA

treatment alone did not effectively inhibit tumor growth. For the NIR laser +

CoWO4-x@PEO-b-PMAA group, we found that the 4T1 tumors were significantly inhibited during the first week, but curiously, the tumors recurred after this time. It is well known that solid tumors are less able to withstand heat than healthy tissues, and when tumor cells are exposed to a temperature above 48 °C, they can undergo irreversible damage within 4-6 minutes. Moreover, if a material can produce ROS under NIR irradiation, the resulting ROS can penetrate tumor cells to achieve synergistic therapeutic goals with PTT. Additionally, both PTT and PDT can induce ICD, which further caused a large amount of HMGB1 and calreticulin (CALR) release around cancer cells. As shown in Figure 5e, we examined the expression of HMGB1 and CALR in tumors on 1, 3, 7, and 14 days of treatment. It can be seen that the expression levels of these two proteins increased over treatment time, suggesting that the innate immune system of the mouse had released an “eat me” signal in the tumors. The above three conditions were beneficial for the ablation of solid tumors, but why did tumors recur after one week of treatment? According to reports in the literature, this might be mainly related to the immunoresistance of HSP60 and NRF2 toward phototherapy [44]. PTT and PDT can respectively stimulate the tumor cell microenvironment to overexpress HSP60 and NRF2 to achieve self-protection against external aggression, which was very unfavorable for treatment and may be the leading cause of cancer recurrence. Therefore, we examined the expression of HSP60 and NRF2 at tumor sites at different treatment time. From Figure 5e, we found a positive correlation between their expression and treatment time. Moreover, compared with the group before treatment, the expression levels of HSP60 and NRF2 were 1.9 times and 4.4 times of their initial values, respectively (Figure 5f). To test our above hypothesis, we used etoposide and ML385 to reduce the immunoresistance of the organism to phototherapy, thus achieving enhanced phototherapy. As a specific inhibitor of HSP60 and NRF2, respectively,

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ML385, with a concentration of 30 mg/kg, and etoposide with a level of 10 mg/kg, were intravenously injected into mice the day before treatment, and phototherapy was performed next day (Figure 5g). The inhibition effects were firstly evaluated by measuring the average tumor size after treatments with etoposide, ML385 or etoposide + ML385. As shown in Figure S9, tumors in the etoposide, ML385 or etoposide + ML385 treatment group continued to grow during the 14 days of treatment compared with control group, indicating that there was no significant inhibition of tumor growth through intravenous injection of etoposide, ML385 or etoposide + ML385. Next, we examined the expression levels of HMGB1, CALR, HSP 60 and NRF2 in tumors with etoposide, ML385 or etoposide + ML385 treatments. As shown in Figure S10, the etoposide only, ML385 only or etoposide + ML385 displayed nearly negligible influence on the expression of HMBG1 and CALR in tumors on 1, 7, and 14 days of treatment. Moreover, the expression of HSP60 or NRF2 in the tumor site significantly decreased after one day of injection of etoposide or ML385 through the tail vein. As time increases, the downward trend of expression of HSP or NRF2 was gradually reduced, which may be due to the progressive metabolism of etoposide or ML385 in mice. As expected, the expression of both HSP60 and NRF2 in the tumor site remarkablely decreased after one day of intravenous injection of etoposide + ML385. And the downward trend of expression of HSP or NRF2 was also gradually decreased from the first day to the 14th day. As shown in Figures 5g, h, and 6, compared with the phototreatment group without inhibitor injection, the treatment effect in the inhibitors injected group was significantly enhanced, and no tumor recurrence occurred after one week of treatment. Then, the expression changes of the four different proteins during the first two days. As shown in Figure S11, the expression levels of HMGB 1 and CALR were continually increased from the first day to the second day, which may

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be due to the immune response of the innate immune system. And the expression levels of HSP60 and NRF2 showed a significant decrease on the first day and reached almost the same level with the control group the next day, which proved that pre-injection of specific inhibitors weaken the protective effects of the two “protective switches” (HSP60 and NRF2) for tumors. Therefore, it can be concluded that the immunoresistance of the organism to phototherapy was an essential cause of tumor recurrence, and the reduction of immunoresistance can achieve enhanced tumor phototherapy outcomes.

Figure 6. H&E staining of organs collected from different groups of mice after 14 days of treatment and tumor slices collected from different groups of mice after 2 days of treatment. We also monitored the weight changes of mice in each experimental group during the 14 days of treatment, and our results demonstrated that the weight of mice in each experimental group increased slightly, which proved that the CoWO4-x@PEO-b-PMAA had excellent biosecurity (Figure 5i). As shown in Figure 6, the H&E staining of organs collected from different groups

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showed no visible damage, suggesting that the therapeutic method and materials in this work had little toxicity in mice. 3. Conclusions This work constructed and applied a novel non-stoichiometric CoWO4-x@PEO-b-PMAA with excellent NIR absorption for enhanced phototherapy outcome under an 808 nm laser irradiation. On the basis of its excellent NIR absorption and high atomic number (Z = 74) element content, these CoWO4-x@PEO-b-PMAA NPs can be employed for PA imaging and CT imaging. Moreover, Western blotting was utilized to explore the mechanisms of enhanced phototherapy. The results showed that although phototherapy led to enhanced expression of ICD-related proteins during a 14-day treatment, tumor recurrence still occurred, mainly due to overexpression of HSP60 and NRF2, which facilitated tumor immunoresistance toward to PTT and PDT. Enhanced phototherapy can be achieved if the expression of HSP60 and NRF2 was reduced using specific HSP60 and NRF2 inhibitors. Therefore, this work demonstrated a method for improving the efficacy of phototherapy, and we speculate that it will be promising if better specific inhibitors are developed in the future. 4. Experimental 4.1. Materials All of the reagents were used without further purification unless otherwise indicated. Sodium tungstate (Na2WO4·2H2O), cobalt chloride (CoCl2·6H2O), ethidium bromide (EB) and 1,3diphenylisobenzofuran were obtained from Aladdin. 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), calcein acetoxymethyl ester (Calcein-AM), propidium iodide (PI), 3-(4,5dimethylthialzol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and etoposide were purchased

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from Sigma-Aldrich. Poly (ethylene oxide-b-methacrylic acid) (PEO-PMAA, Mw = 7500-b15,000) was obtained from Polymer Source Reagent Co. ML385 was obtained from Selleck. 4.2. Synthesis of CoWO4 nanoparticles The CoWO4 NPs were prepared via a hydrothermal method 45. First, 0.66 g of Na2WO4·5H2O was dissolved in 25 mL of deionized water under magnetic stirring for 0.5 h to obtain a colorless solution. Then, 0.178 g of CoCl2·6H2O was added to the deionized water (25 mL) under magnetic stirring for 0.5 h to achieve a pink solution. Next, the obtained cobalt chloride solution was added dropwise to the sodium tungstate solution under magnetic stirring for another 0.5 h. After that, the resulting solution was transferred into a 100 mL Teflon-lined autoclave, and the hydrothermal reaction was maintained at 160 °C in an electric oven for 24 h. The obtained blue products were collected by centrifuge, further alternately washed with water and ethanol three times, and finally stored at room temperature for future use. 4.3. Synthesis of CoWO4-x@PEO-b-PMAA nanoparticles Briefly, dehydrated CoWO4 powders were firstly calcined at 550 °C for 2 h under a hydrogen/argon atmosphere. Then, 18 mg of the as-prepared CoWO4-x nanoparticles and 60 mg of PEO-PMAA were added to 25 mL of deionized water under continuous ultrasonic agitation for 30 min. Finally, the resulting dispersed solution was stored at 4 °C for future use. 4.4. Characterization Transmission electron microscopy (TEM) was performed on a FEI Tecnai G2 F20 microscope under 200 kV acceleration voltage. The phase composition of the sample was recorded by X-ray diffraction analysis (XRD, Bruker AXS D8 Advance). The chemical valence of W ions was determined by X-ray photoelectron spectroscopy (XPS, Perkin Elmer PHI 5600). The optical properties were recorded on a U-4100 spectrophotometer (Hitachi, Japan). The temperature

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changes of tumors were obtained using an infrared camera (FLIR E6). MTT experiments were recorded using a microplate reader (Infinite M200, Tecan). 4.5. Cell culture 4T1 cells were cultured in RPMI (Roswell Park Memorial Institute)-1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco), 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C in a humidified atmosphere with 5% CO2. 4.6. In vitro cell cytotoxicity assay The viability of 4T1 cells in the presence of nanoparticles was evaluated using a standard MTT assay. 4T1 cells were seeded into 96-well plates at a density of 1 × 104 per well in 200 μL of RPMI medium and grown overnight. Then, cells were sequentially incubated with various concentrations of nano-materials for another 24 h. Subsequently, 20 µL of MTT solution (5 mg/mL) was added to each well and incubated with the 4T1 cells for 4 h. When the media with MTT was removed, 150 µL of DMSO was added to each well for dissolving the formazan crystals at room temperature for 30 min, and the absorbance was then measured at 490 nm using a multi-detection microplate reader. The phototherapeutic effect of different treatment was also measured using the MTT method. For the in vitro PTT group, 4T1 cells were incubated with CoWO4-x@PEO-b-PMAA (250 µg/mL) in a 96-well plate for 24 h. Then, 50 μL of sodium azide (10 μM) was added to the cells and cells were irradiated with an 808 nm laser at a power density of 1.0 W/cm2 for 2, 6 and 10 min. For in vitro PDT experiments, 4T1 cells cultured in 96-well plates were incubated with CoWO4-x@PEOb-PMAA (250 µg mL) for 24 h and then irradiated with an 808 nm laser at a power density of 1.0 W/cm2 on an icebox for 2, 6 and 10 min. For the in vitro PDT/PTT combined experiment, experimental conditions such as cell density, the concentration of CoWO4-x@PEO-b-PMAA,

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irradiation time and power density of laser remain unchanged, but neither sodium azide nor ice box was applied. 4.7. Detection of ROS The ROS detection in solution consisted of two groups, which included pure water with 808 nm NIR laser irradiation, CoWO4 solution (500 μg/mL) with 808 nm NIR laser irradiation and CoWO4-x@PEO-b-PMAA solution (500 μg/mL) with 808 nm NIR laser irradiation. Briefly, 20 μL of DPBF solution (1 mg/mL, solvent: N,N-dimethylformamide), was employed as a probe to detect ROS generation, was added to 3 mL of above three solutions. Then, they were irradiated with a NIR laser for different time. After centrifugation, the collected supernatant was assayed using a spectrophotometer. The DCFH-DA probe was used to evaluate the intracellular ROS generation in 4T1 cells. Briefly, 4T1 cells with a density of 5 × 103 per dish were seeded into a 35-mm culture dish and incubated at 37 °C overnight. Subsequently, the medium was discarded, and cells were incubated with fresh medium containing CoWO4-x@PEO-b-PMAA (250 μg mL) for 4 h. After being washed with the PBS, the cells were stained with DCFH-DA (50 μL, 10 mM) for another 1 h. Cells were then washed with PBS and irradiated for 10 min (808 nm, 1.0 W/cm2), and fluorescence images were obtained using a fluorescent microscope. Untreated cells were used as a negative control group, while the cells incubated with H2O2 (200 μL, 50 mM) at 37 °C for 1 h were employed as a positive control group. 4.8. In vitro living-dead staining 4T1 cells were seeded into a 35-mm culture dish and incubated at 37 °C until ~90% confluence. Then, 2.0 mL of fresh medium or medium containing the CoWO4-x@PEO-b-PMAA (250 μg/mL) was added to the culture dish to replace the previous culture medium. After incubation for another 6 h, the cells were washed three times with PBS. Then, the 4T1 cells were irradiated with an 808

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nm laser at a power density of 1.0 W/cm2 for different time intervals (2, 6, and 10 min). Subsequently, the cells were stained with calcein-AM and PI for 20 min to distinguish living and dead cells after being rinsed with PBS. Finally, the stained cells were immediately observed with a fluorescent microscope. 4.9. Cell membrane permeability study 4T1 cells at a density of 5 × 103 were seeded into a 35-mm culture dish and cultivated with RPMI culture medium at 37 °C in a humidified 5% CO2 incubator. After incubation for 12 h, the culture medium was replaced with fresh media containing CoWO4-x@PEO-b-PMAA and EB at concentrations of 250 μg/mL and 10 μg/mL, respectively. Untreated cells served as a control group. After 6 h incubation at 37 °C, the cells were washed with PBS and irradiated with a NIR laser at a power density of 1.0 W/cm2. After that, the medium was removed, and the 4T1 cells were rinsed with PBS three times. Then, 200 µL of Calcein-AM (1 μg/mL) was added to each dish, and the cells were incubated for another 20 min in the dark at 37 °C. After being washed with PBS three times, the stained cells were immediately visualized using a fluorescent microscope. 4.10. In vivo antitumor therapy For the in vivo experiments, all the animal experiments were performed according to the criteria of the National Regulation of China for Care and Use of Laboratory Animals. All healthy female BALB/c mice (4-5 weeks old) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing). To establish the tumor model, 1 × 106 4T1 cells suspended in DMEM and Matrigel were subcutaneously injected into the left front leg of each mouse. When the tumor volume reached ≈200 mm3, mice were randomly divided into five groups (n = 5 for each group): (i) PBS as control; (ii) 808 nm laser irradiation for 10 min; (iii) CoWO4-x@PEO-b-PMAA NPs injection; (iv) CoWO4-x@PEO-b-PMAA NPs injection + 808 nm laser irradiation for 10 min; (v)

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pre-injection of specific inhibitor + CoWO4-x@PEO-b-PMAA NPs injection + 808 nm laser irradiation for 10 min. For group i and ii, mice were intratumorally injected with 100 μL of PBS, while for the group iii and iv, mice were intratumorally injected with 100 μL of CoWO4-x@PEOb-PMAA NPs (1.0 mg/mL). The mice were irradiated by 808 nm laser (1.0 W/cm2) for 10 min at 1 h post injection of CoWO4-x@PEO-b-PMAA NPs. The tumor volumes and body weights of mice were monitored every day and normalized in comparison with their initial values. The tumor volume was evaluated using a caliper according to the formula: volume = (tumor length) × (tumor width)2/2. Relative tumor volume is defined as V/V0, while relative body weight is defined as W/W0. V0 and W0 are the initial tumor volume and initial body weight of mice, respectively. 4.11. Western blotting assay Tissues were harvested and then lysed in RIPA buffer (50 nM Tris-HCl pH 8.0, 150 mM sodium chloride, 1.0% NP-40, 0.1% sodium dodecyl sulfate) with 1% protease inhibitor cocktail (MCE). And then centrifuged at 12000 rpm for 10 min. Total proteins were resolved by 8%-12% SDSPAGE and transferred on PVDF (Millipore, Germany). Membranes were blocked with 10% nonfat milk powder for 2 h at room temperature, and incubated overnight at 4 °C with primary antibodies: anti-GAPDH antibody (1:2000; Proteintech), anti-NRF2 antibody (1:1000; Proteintech), anti-HSP60 antibody (1:1000; Proteintech), anti-HMGB1 antibody (1:1000; Abclonal), anti-CALR antibody (1:1000; Abclonal). After washed in triethanolamine-buffered saline solution with 1‰ Tween-20 (TBST), membranes were incubated with horseradish peroxidase secondary antibody (1:5000) for 1 h at room temperature. After washed in TBST, visualized with an enhanced chemi-luminescence kit (ECL-kit, ThermoFisher). 4.12. Histology analysis

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The mice were sacrificed to collect the tumors and main organs (heart, liver, spleen, lung, and kidney), which were fixed in 4% paraformaldehyde and then embedded with paraffin. The slices of the major organs and tumors of the mice were stained with hematoxylin and eosin (H&E) for histological analysis.

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1. A novel nanoplatform based on non-stoichiometric CoWO4-x was fabricated. 2. The relationship between phototherapy and immunotherapy was discussed in detail. 3. CoWO4-x displayed excellent CT and photoacoustic imaging. 4. Enhanced phototherapy was achieved by weakening the immunoresistance of HSP60 /NRF2.

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