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Chemical Engineering Journal

journal homepage: www.elsevier.com/locate/cej

CoWO4-x-based nanoplatform for multimode imaging and enhancedphotothermal/photodynamic therapy

Haixia Liua,1, Qingzhu Yangb,1, Wei Guoa,⁎, Huiming Lina, Feng Zhanga, Jingxiang Zhaoa,Tianyue Maa, Le Zhaoa, Na Xuc, Ruiwen Wangb,⁎, Jingyao Yua, Fengyu Qua,⁎

a Key Laboratory of Photochemical Biomaterials and Energy Storage Materials, Heilongjiang Province and College of Chemistry and Chemical Engineering, Harbin NormalUniversity, Harbin 150025, Chinab School of Life Science and Technology, Harbin Institute of Technology, Harbin, ChinacNanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine, Nanjing, China

H I G H L I G H T S

• A novel nanoplatform based on non-stoichiometric CoWO4-x was fabri-cated.

• The relationship between photo-therapy and immunotherapy was dis-cussed in detail.

• CoWO4-x displayed excellent CT andphotoacoustic imaging.

• Enhanced phototherapy was achievedby weakening the immunoresistanceof HSP60/NRF2.

G R A P H I C A L A B S T R A C T

A R T I C L E I N F O

Keywords:ImmunoresistancePhotothermal therapyPhotodynamic therapyHSP60NRF2

A B S T R A C T

Although phototherapy has received widespread attention in recent years due to its high efficiency, low inva-siveness, and minor side effects, there are still few studies on the relationship between phototherapy and im-munotherapy. Therefore, non-stoichiometric CoWO4-x nanoparticles (NPs) were strategically designed andprepared, 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 photo-dynamic therapy (PDT) on tumors. CoWO4-x NPs not only have strong near-infrared absorption, outstandingbiocompatibility, and excellent photothermal stability but they also can be used as photoacoustic (PA) andComputer Tomography (CT) contrast agents for working with tumors. In particular, the relationships betweenphototherapy, immunogenic cell death (ICD) and immunoresistance are here discussed in depth, and enhancedphototherapy was achieved for the first time by injecting etoposide and ML385, which are two typical inhibitorsfor HSP60 and NRF2, respectively. Furthermore, the biocompatibility of materials in vivo was demonstratedthrough a variety of cell experiments, changes in mouse body weight, and histological analyses. Our workhighlights the great potentials of non-stoichiometric CoWO4-x NPs as a kind of multifunctional therapeutic agentfor implementing enhanced phototherapy in breast cancer by reducing the immunoresistance of HSP60 andNRF2 toward PTT and PDT.

https://doi.org/10.1016/j.cej.2019.123979Received 1 August 2019; Received in revised form 25 December 2019; Accepted 27 December 2019

⁎ Corresponding authors.E-mail addresses: guowei@hrbnu.edu.cn (W. Guo), qufengyu@hrbnu.edu.cn (F. Qu).

1 Equal contribution from Haixia Liu and Qingzhu Yang.

Chemical Engineering Journal 385 (2020) 123979

Available online 28 December 20191385-8947/ © 2019 Elsevier B.V. All rights reserved.

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1. Introduction

Breast cancer is one of the most common malignant tumors inwomen, which has the characteristics of high morbidity, enormousharmfulness, and ease of metastasis [1]. The current standard-of-caretreatment for breast cancer mainly relies on a combination of surgeryand chemotherapy [2]. In theory, surgical treatment can altogetherremove tumor tissue and cure cancer, but it is not effective in cases withdiffuse or multiple tumors [3]. For chemotherapy, most chemother-apeutic drugs often lack specific targeting abilities for tumor cells andhave strong side effects, creating adverse reactions in healthy cells andother organs while eliminating cancer cells [4]. At present, there areother emerging alternative anti-cancer therapies for the treatment ofbreast cancer, such as phototherapy, magnetic hyperthermia, biocata-lytic therapy, immunotherapy, and ultrasonic therapy [5–9]. Amongthese therapeutic techniques, phototherapy (including photothermaltherapy (PTT) and photodynamic therapy (PDT)) has received extensiveattention as a powerful technique for tumor therapy due to its low in-vasiveness, deep tissue penetration, and high efficiency [10–15]. PDT isan oxygen-dependent treatment method, where the therapeutic effectgradually diminishes with increasing consumption of tissue oxygen[16]. In PTT, the process involves a temperature rise in target cells, andthe therapeutic effect increases progressively with time and is not af-fected by oxygen concentration [17]. Therefore, a combination of PDTand PTT should theoretically have synergistic efficacy [18]. To ac-complish this antitumor synergistic impact, most previous studies de-veloped nanoplatforms composed of a photosensitizer (PS) and photo-thermal agent (PTA) for PDT/PTT combination treatment. Thesestudies had some shortcomings, including complex reaction processes,mutual interference, and mismatch of absorption between PTA and PS,for example [19]. Various photothermal agents, including inorganicand organic ones, plasma metals, carbon-based nanomaterials, in-organic semiconductor nanomaterials, organic polymers, or organicdyes, have been used in PTT. As a non-stoichiometric compound,CoWO4-x NPs have the following advantages: (i) Wide optical absorp-tion 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. Notall photothermal materials have both photothermal and photodynamicproperties, and only a few of them have both characteristics. Recently,some novel materials, such as CsxWO3, TiO2-x and Cu2(OH)PO4, havebeen identified as potentially good candidates as both PS and PTAagents, which can be used in PDT/PTT combination treatment becausethey can produce both reactive oxygen species (ROS) and hyperthermiaat the same time under single wavelength laser irradiation [20–23]. (iii)Multimodal imaging abilities. CoWO4-x NPs have excellent near-in-frared absorption properties and contain tungsten elements with highatomic number (Z = 74), making them ideal photoacoustic (PA) andcomputed tomography (CT) contrast agents.

The phototherapy of tumors can stimulate the immune response ofan organism, so characterization of agents for PTT and PDT is indis-pensable [24]. As a cell death mode, immunogenic cell death (ICD) ischaracterized by the release of high mobility group protein B1(HMGB1), which can serve as the “eat me” signal of the innate immunesystem, as well as the exposure of Calreticulin and secretion of ade-nosine triphosphate (ATP) [25]. According to reports in the literature,both PTT and PDT can induce ICD, but it was difficult to completelyablate solid tumors by relying on such a cell death mode alone, and mayalso lead to the metastasis of cancer cells [26]. We speculate that theimmunoresistance of an organism to phototherapy may be one of themost important aspects for the efficacy of PDT and PDT. The expressionchanges of heat shock protein (HSP) and NF-E2 related factors (NRF2)are critical immune responses of the tumor microenvironment to PTTand PDT, respectively, and both HSP and NRF2 belong to the cell’s“protective switch” [27,28]. PTT and PDT can lead to an increase in theexpression 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 externalenvironment, the organism regulates the expression of HSP, therebyachieving self-protection. Based on the relative molecular weight ofHSPs and their degree of homology, it can be divided into HSP60,HSP70, and HSP90, and so on [30]. NRF2 is an important transcriptionfactor for regulating the oxidative stress response of cells, and it is also acentral regulator for maintaining intracellular redox homeostasis [31].By inducing and regulating the constitutive and inducible expression ofa series of antioxidant proteins, NRF2 can reduce the cell damagecaused by ROS and electrophiles, keeping the cells in a stable state andmaintaining the dynamic redox balance of an organism [32]. However,most studies on the expression of HSP and NRF2 have been carried outat the cellular level, while studies of the changes in HSP and NRF2expression at the tumor level have been relatively rare during photo-therapy. Therefore, it is of considerable significance to monitor changesin the expression levels of HSP and NRF2 at the solid tumor level atvarious stages of phototherapy. Additionally, depending on the trend ofthe expression levels of HSP and NRF2, appropriate molecular drugscan be selected to inhibit their expression, thereby achieving photo-therapy and immune synergistic treatment.

In this work, we developed a novel multifunctional theranosticagent based on Poly (ethylene oxide-b-methacrylic acid) (PEO-b-MAA)functionalized non-stoichiometric CoWO4-x (CoWO4-x@PEO-b-MAA),which showed strong NIR absorbance, for multimodal imaging andhighly effective in vivo enhanced phototherapy of breast tumors for thefirst time. These CoWO4-x NPs can not only function role as excellent CTand PA imaging contrast agents, but they also realize NIR laser-inducedPTT and PDT. To improve the stability and biocompatibility of CoWO4-x

nanoparticles, PEO-b-MAA was modified on its surface through ultra-sonic self-assembly. As shown in Scheme 1, phototherapy can induceICD, which leads to an increase in the expression of HMGB1 and Cal-reticulin, but in general, it was difficult to obtain the desired ther-apeutic effect. Meanwhile, phototherapy can also stimulate the im-munoresistance of an organism to phototherapy, as PTT and PDTinduced an increase in the expression of HSP60 and NRF2, respectively.As specific inhibitors of PTT and PDT respectively, etoposide andML385 were used to weaken the protective effects of the two “protec-tive switches,” thus ultimately achieving phototherapy and synergisticimmune therapy for breast tumors. Consequently, we hypothesize thatthe results presented here will stimulate advances in the use of CoWO4-

x@PEO-b-MAA NPs for highly practical applications in fighting breastcancer tumors using PDT and PTT.

2. Results and discussion

2.1. Synthesis and characterization of CoWO4-x@PEO-b-PMAA NPs

Fig. 1a shows the synthesis path for creating CoWO4-x@PEO-b-PMAA NPs and their multi-functional biological applications. Thetransmission electron microscope (TEM) image in Fig. 1b displays thatthe CoWO4 NPs, with a particle size ~ 50 nm, were prepared using apreviously reported hydrothermal method. HR-TEM study showed thatthey exhibited bright lattice fringes, and the lattice parameter wasobserved to be 0.288 nm, which was assigned to the (1 1 1) plane of themonoclinic crystal of the CoWO4 (Fig. S1). The non-stoichiometricCoWO4-x structure was achieved through the reduction of CoWO4 at550 °C for 2 h under a hydrogen/argon atmosphere. After this reductionprocess, the morphology and crystal structure of CoWO4-x NPs werewell preserved based on TEM (Fig. 1c) and X-ray diffraction (XRD)analyses (Fig. 1e). X-ray photoelectron spectroscopy (XPS) analysis(Fig. 1f and g) was further employed to examine the chemical states oftungsten in the CoWO4 and CoWO4-x NPs. For the CoWO4 NPs, therewere two peaks located at 37.3 eV (W4f5/2) and 35.2 eV (W4f7/2),which could be assigned to the spin–orbit coupling of W6+ ions. Incontrast, for the CoWO4-x NPs, except for the W6+ ions at the same

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location, there also existed peaks with lower binding energy at 34.4 eVand 36.5 eV, which could be attributed to the W 4f7/2 and W 4f5/2 corelevels from W5+, implying the successful preparation of non-stoichio-metric CoWO4-x NPs [33].

To improve the dispersity and stability of CoWO4-x NPs in physio-logical conditions, PEO-PMAA was utilized to modify their surfaceusing an ultrasonic self-assembly method to prevent particle aggrega-tion, and TEM of these CoWO4-x@PEO-b-PMAA NPs are shown inFig. 1d [34]. To certify the successful modification of PEO-PMAA on thesurface of CoWO4-x NPs, Fourier transform infrared (FT-IR) spectro-scopy was utilized to explore the chemical compositions of CoWO4-x@PEO-b-PMAA. As displayed in Fig. S2, the absorption bands at 3465 and2923 cm−1 could be assigned to the OeH stretching and CeHstretching vibration modes, respectively, while the bands located at1710 cm−1 belonged to the C]O stretching vibration mode, and all ofthe above three bands resulted from the surface PEO-b-PMAA ligand[35,36].

Additionally, the weight loss variance between CoWO4-x andCoWO4-x@PEO-b-PMAA originated from the PEO-b-PMAA modifica-tion, which indicated that the PEO-b-PMAA layer accounted for 2.3% ofthe weight of the CoWO4-x@PEO-b-PMAA NPs (Fig. S3). As can be seenin Fig. S4, dynamic light scattering (DLS) indicated that the hydro-dynamic size of CoWO4 NPs increased from ~122 nm to ~164 nm afterPEO-PMAA modification. Moreover, the zeta-potential value increasedfrom −23.9 ± 7.36 eV for CoWO4 NPs to −16.3 ± 5.98 eV. Theabove 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 asso-ciated photothermal/photodynamic properties are essential require-ments for the implementation of phototherapy by CoWO4-x@PEO-b-PMAA NPs. As can be seen in Fig. 2a, CoWO4 powders exhibited a highabsorption in the UV–vis spectra, corresponding to a band gap of ap-proximately 2.8 eV. In contrast to CoWO4 powders, an additional ab-sorption band beyond 578 nm extending to the infrared region wasobserved for the CoWO4-x powders. According to the literature, thisphenomenon can be caused for two reasons: (1) oxygen vacanciesproduced by hydrogen reduction and (2) W5+ on the surface of the

CoWO4-x powders [37,38]. These results indicated that CoWO4-x hadbetter application prospects as a NIR phototherapy agent than CoWO4.We further evaluated the absorbance of the CoWO4-x@PEO-b-PMAAdispersions at various concentrations. As illustrated in Fig. 2b, CoWO4-

x@PEO-b-PMAA dispersions showed a concentration-dependent ex-tinction increase from the UV to the NIR region, suggesting their pho-tothermal heating potential for the phototherapy. The photothermalconversion capability of CoWO4 NPs and CoWO4-x@PEO-b-PMAA NPswere evaluated in aqueous dispersions.

As shown in Fig. 2c, under the same laser irradiation (1 W/cm2,10 min), the temperature of the CoWO4-x@PEO-b-PMAA dispersionswith different concentrations increased rapidly compared to purewater, indicating their excellent photothermal properties. As shown inFig. 2d, the temperature increase of CoWO4-x@PEO-b-PMAA disper-sions with the same mass concentration (1 mg/mL) was higher thanthat of CoWO4 dispersions, which demonstrated that CoWO4-x@PEO-b-PMAA dispersions had superior photothermal performance. The pho-tothermal conversion efficiency (ɳ) of CoWO4-x@PEO-b-PMAA NPs wascalculated to be 72.75% by a previously reported method (Fig. S5) [39].To investigate the photothermal stability of the as-prepared photo-therapeutic agent, CoWO4-x@PEO-b-PMAA dispersions were irradiatedby an 808 nm laser for 10 min, and then naturally cooled for 10 min in acycle manner. Based on the recorded temperature values (Fig. 2e), wefound that five cycles showed little significant change in temperature,indicating that CoWO4-x@PEO-b-PMAA had excellent photothermalstability.

To clarify whether CoWO4-x@PEO-b-PMAA NPs had sound photo-dynamic effects in aqueous solution, we applied a 1,3-diphenyliso-benzofuran (DPBF)-based spectroscopic method to measure the for-mation ability of ROS in CoWO4-x@PEO-b-PMAA dispersions duringlaser irradiation. The DPBF probe reacts with ROS to be decomposedinto 1,2-dibenzoylbenzene, thus leading to a decrease in optical ab-sorption intensity at 410 nm [40]. As shown in Fig. 2f, pure water re-sulted 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 ad-dition, the absorbance of DPBF dispersed in a CoWO4 solution with thesame mass concentration was lower than that of CoWO4-x@PEO-b-PMAA dispersions, indicating that CoWO4-x@PEO-b-PMAA dispersions

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

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had enhanced photodynamic property. As can be seen in Fig. S6,CoWO4-x@PEO-b-PMAA NPs possessed a very similar hydrodynamicdiameter after dispersion in PBS for different time (~164 nm), whichindicated 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 firstexamined the cytotoxicity of the as-prepared CoWO4-x@PEO-b-PMAANPs using a standard MTT assay. As shown in Fig. 3a, our resultsshowed that with an increase in the concentration of CoWO4 andCoWO4-x@PEO-b-PMAA NPs, the survival rate of the cells graduallydecreased, which demonstrated a dose-dependent effect in vitro.

The cell viabilities of 4T1 cells were 82.2% and 85.1%, respectively,when the concentrations of CoWO4 and CoWO4-x@PEO-b-PMAA were1000 μg/mL, suggesting that these compounds had low cytotoxicity. Inthis work, CoWO4-x@PEO-b-PMAA NPs could simultaneously producehyperthermia and ROS under a NIR laser irradiation and lead to thesynergistic effect of PDT/PTT in 4T1 cells. The cellular uptakes of

CoWO4-x@PEO-b-PMAA NPs were investigated by inductively coupledplasma optical emission spectrometry (ICP-OES) analysis. As shown inFig. S7, as the incubation time increased, the cellular uptake of CoWO4-

x@PEO-b-PMAA NPs gradually increased, and there was no significantdifference between uptake by 12 and 24 h, which proved that thematerial might have reached its maximum cellular uptake by this time.To distinguish the contribution of individual PDT or PTT to the overalltherapeutic effect, we divided the experimental groups into a controlgroup, a PTT only group, a PDT only group, and a PTT combined withPDT group. In the PTT only group, the photodynamic properties of thematerial were eliminated by adding the ROS quenching agent sodiumazide [20]. For the PDT only group, the photothermal properties of thematerial were eliminated by placing the cells on an ice cube. As dis-played in Fig. 3b, for a single therapeutic effect in the PDT or PTTgroup, the inhibition rates of 4T1 cells were only 31.2% and 59.8%,respectively, under an 808 nm laser irradiation. In contrast, the in-hibition rate of 4T1 cells in the PTT combined with PDT experimentalgroup was 88.2%, which proved that CoWO4-x@PEO-b-PMAA NPscould simultaneously produce photothermal and photodynamic therapy

Fig. 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) TEMimage of CoWO4-x@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.

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for 4T1 cells under near-infrared laser irradiation. Next, we evaluatedthe laser-activated therapeutic effect of CoWO4-x@PEO-b-PMAA NPsusing a live/dead double-staining method. Briefly, Calcein-AM, whichhad green emission, and propidium (PI), which had red emission wereemployed to stain living and dead 4T1 cells, respectively. As shown inFig. 3c, the cells of the control group, NIR laser group, and CoWO4-x@PEO-b-PMAA group showed strong green fluorescence, implying eithera NIR laser or CoWO4-x@PEO-b-PMAA alone did not cause cell death. Incontrast, in the CoWO4-x@PEO-b-PMAA combined with laser irradia-tion group, we observed a large number of cell deaths, and as theduration of irradiation increased, the number of cell deaths increasedsignificantly. The PDT performance of CoWO4-x@PEO-b-PMAA in 4T1cells was evaluated using a dichlorofluorescein diacetate (DCFH-DA)probe, which was commonly used to detect intracellular ROS produc-tion. 4T1 cells without any treatment were used as a negative controland 4T1 cells treated with H2O2 (50 mM) were used as a positivecontrol. As shown in Fig. 3d, the negative control and CoWO4-x@PEO-b-PMAA treated cells had no observable green fluorescence, while theNIR irradiated group had only a weak fluorescent signal. In contrast,H2O2 and CoWO4-x@PEO-b-PMAA combined with laser irradiationgroups emitted strong green fluorescence, suggesting that a largenumber of ROS were produced. When the ROS quencher sodium azidewas added to the 4T1 cells in which CoWO4-x@PEO-b-PMAA nano-particles were dispersed, the cells did not show green fluorescenceunder NIR irradiation, which was due to the ROS produced during ir-radiation having been quenched. The above results clearly indicatedthat the CoWO4-x@PEO-b-PMAA NPs were a promising candidate forboth realizing combined PTT/PDT treatments. We hypothesized thatcell membrane permeability and mitochondrial membrane potentialmight change during photothermal therapy. To test our hypothesis, we

used ethidium bromide (EB) and JC-1 probes to detect the cell mem-brane permeability and mitochondrial membrane potential of theCoWO4-x@PEO-b-PMAA treated 4T1 cells under irradiation, respec-tively [41]. As illustrated in Fig. 3e, the cells of the control group, NIRlaser group, and CoWO4-x@PEO-b-PMAA group showed little redfluorescence. For the CoWO4-x@PEO-b-PMAA combined with the laserirradiation group, 4T1 cells showed strong red fluorescence, mainly dueto the damage of the cell membrane, and then EB entered the cellsthrough the damaged cell membrane and bound to nucleic acids. Basedon previous literature reports, the JC-1 probe can be used to assess thefunction of mitochondria, as it can stain normal mitochondria and da-maged mitochondria with red and green fluorescence, respectively.Among the four groups, a large number of damaged mitochondria wereobserved in the CoWO4-x@PEO-b-PMAA combined with the laser irra-diation group (Fig. 3f). The above two experimental results clearlydemonstrated that photothermal therapy can kill cancer cells bychanging the membrane permeability of cancer cells and damagingmitochondria.

2.4. PA and CT imaging

Among the numerous extant multi-mode imaging techniques, thedual-imaging modalities of PA and CT imaging have critical applica-tions in the field of accurate disease diagnosis [42]. The main reason isthat CT imaging has the advantages in creating 3D visual images anddeep tissue penetration, while PA imaging can provide increased detailsabout the microstructure of a disease site [43]. Considering thatCoWO4-x@PEO-b-PMAA NPs have excellent near-infrared absorptionproperties and contain tungsten elements, which has a high atomicnumber (Z = 74), they should theoretically be ideal PA and CT contrast

Fig. 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 differentconcentrations. (c) Photothermal heating curves of CoWO4-x@PEO-b-PMAA dispersions under laser irradiation (808 nm, 1 W/cm2). (d) Comparison of the photo-thermal performance of CoWO4 and CoWO4-x@PEO-b-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.

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agents. As expected, the PA signal value of CoWO4-x@PEO-b-PMAA waspositively correlated with increased concentration, indicating thatCoWO4-x@PEO-b-PMAA should be a promising PA imaging candidate(Fig. 4a).

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-bearingmice by intratumoral injection and then imaged using a PA imagingsystem. As shown in Fig. 4c, almost no PA signal was detected in thetumor site before the injection of the CoWO4-x@PEO-b-PMAA disper-sion, and a relatively clear PA signal could be detected one hour afterdosing with CoWO4-x@PEO-b-PMAA. The intensity of the PA signal atthe 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.Fig. 4b displays the Hounsfield unit (HU) values of CoWO4-x@PEO-b-PMAA solutions at different concentration, which showed a good linearrelationship with CoWO4-x@PEO-b-PMAA concentration increase. Wethen injected a CoWO4-x@PEO-b-PMAA dispersion into the tumor of amouse and detected the changes in CT signal at the tumor site of mice atdifferent time (Fig. 4d). The results showed that the strongest CT signalin a tumor appeared after 6 h of material injection, and this trend wasconsistent with the results of PA imaging, which proved the potential ofCoWO4-x@PEO-b-PMAA NPs for use in CT imaging. The changes of CTand PA signals may have been mainly related to the diffusion of the NPsafter intratumoral administration. After intratumoral injection, the NPs

Fig. 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 differenttreatments, including the control group, PDT group, PTT group, and PTT + PDT group. (c) Fluorescence microscope images of Calcein-AM and PI co-stained 4T1 cellsafter receiving different treatments (Scale bars = 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 potentialchanges in different groups by JC-1 staining (Scale bars = 50 µm).

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reached their maximum diffusion level in the tumor around 6 h, afterwhich they may have spread through the blood vessels inside the tumorto other locations.

2.5. In vivo enhanced phototherapeutic and immunological investigation

Encouraged by the results of CoWO4-x@PEO-b-PMAA in vitrotherapy, we further assessed its therapeutic effects on 4T1-tumor miceusing in vivo experiments. After intratumoral injection of 100 μL of PBSor CoWO4-x@PEO-b-PMAA into a mouse, the tumor site was irradiatedusing an 808 nm laser with a power density of 1 W/cm2, and the surfacetemperature of the tumor was monitored with an IR photothermalcamera. As displayed in Fig. 5a and b, the tumor of CoWO4-x@PEO-b-PMAA injected mice exhibited a significant increase in temperaturewithin 10 min after exposure to an 808 nm laser, which increasing from35 °C to 61.7 °C, while the tumor of PBS injected mice showed only amild temperature change from 33.6 °C to 42.9 °C. When the tumorvolume of mice reached 200–300 mm3, the tumor-bearing mice werefirst randomly divided into four groups: (i) control group, (ii) NIR lasergroup, (iii) CoWO4-x@PEO-b-PMAA group, and (iv) NIRlaser + CoWO4-x@PEO-b-PMAA group (five mice per group). Weevaluated the inhibitory effect of each group on tumors by calculatingthe relevant volume of the tumors in each group of mice at differenttreatment time.

As shown in Fig. 5c, d and S8, tumors in the control group, NIR lasergroup, and CoWO4-x@PEO-b-PMAA group were continuously in-creasing, indicating that NIR laser or CoWO4-x@PEO-b-PMAA treat-ment alone did not effectively inhibit tumor growth. For the NIRlaser + CoWO4-x@PEO-b-PMAA group, we found that the 4T1 tumorswere significantly inhibited during the first week, but curiously, thetumors recurred after this time. It is well known that solid tumors areless able to withstand heat than healthy tissues, and when tumor cellsare exposed to a temperature above 48 °C, they can undergo irreversibledamage within 4–6 min. Moreover, if a material can produce ROS underNIR irradiation, the resulting ROS can penetrate tumor cells to achievesynergistic therapeutic goals with PTT. Additionally, both PTT and PDTcan induce ICD, which further caused a large amount of HMGB1 andcalreticulin (CALR) release around cancer cells. As shown in Fig. 5e, weexamined 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 thesetwo proteins increased over treatment time, suggesting that the innateimmune system of the mouse had released an “eat me” signal in thetumors. The above three conditions were beneficial for the ablation ofsolid tumors, but why did tumors recur after one week of treatment?According to reports in the literature, this might be mainly related tothe immunoresistance of HSP60 and NRF2 toward phototherapy [44].PTT and PDT can respectively stimulate the tumor cell microenviron-ment to overexpress HSP60 and NRF2 to achieve self-protection againstexternal aggression, which was very unfavorable for treatment and maybe the leading cause of cancer recurrence. Therefore, we examined theexpression of HSP60 and NRF2 at tumor sites at different treatmenttime. From Fig. 5e, we found a positive correlation between their ex-pression and treatment time. Moreover, compared with the group be-fore treatment, the expression levels of HSP60 and NRF2 were 1.9 timesand 4.4 times of their initial values, respectively (Fig. 5f). To test ourabove hypothesis, we used etoposide and ML385 to reduce the im-munoresistance of the organism to phototherapy, thus achieving en-hanced phototherapy. As a specific inhibitor of HSP60 and NRF2, re-spectively, ML385, with a concentration of 30 mg/kg, and etoposidewith a level of 10 mg/kg, were intravenously injected into mice the daybefore treatment, and phototherapy was performed next day (Fig. 5g).The inhibition effects were firstly evaluated by measuring the averagetumor size after treatments with etoposide, ML385 or etopo-side + ML385. As shown in Fig. S9, tumors in the etoposide, ML385 oretoposide + ML385 treatment group continued to grow during the14 days of treatment compared with control group, indicating thatthere was no significant inhibition of tumor growth through in-travenous injection of etoposide, ML385 or etoposide + ML385. Next,we examined the expression levels of HMGB1, CALR, HSP 60 and NRF2in tumors with etoposide, ML385 or etoposide + ML385 treatments. Asshown in Fig. S10, the etoposide only, ML385 only or etopo-side + ML385 displayed nearly negligible influence on the expressionof HMBG1 and CALR in tumors on 1, 7, and 14 days of treatment.Moreover, the expression of HSP60 or NRF2 in the tumor site sig-nificantly decreased after one day of injection of etoposide or ML385through the tail vein. As time increases, the downward trend of ex-pression of HSP or NRF2 was gradually reduced, which may be due tothe progressive metabolism of etoposide or ML385 in mice. As

Fig. 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 of4T1 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.

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expected, the expression of both HSP60 and NRF2 in the tumor siteremarkablely decreased after one day of intravenous injection of eto-poside + ML385. And the downward trend of expression of HSP orNRF2 was also gradually decreased from the first day to the 14th day.

As shown in Figs. 5g, h, and 6, compared with the phototreatmentgroup without inhibitor injection, the treatment effect in the inhibitorsinjected group was significantly enhanced, and no tumor recurrenceoccurred after one week of treatment. Then, the expression changes ofthe four different proteins during the first two days. As shown in Fig.S11, the expression levels of HMGB 1 and CALR were continually in-creased from the first day to the second day, which may be due to theimmune response of the innate immune system. And the expressionlevels of HSP60 and NRF2 showed a significant decrease on the first dayand 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 ofthe organism to phototherapy was an essential cause of tumor recur-rence, and the reduction of immunoresistance can achieve enhancedtumor phototherapy outcomes.

We also monitored the weight changes of mice in each experimentalgroup during the 14 days of treatment, and our results demonstratedthat the weight of mice in each experimental group increased slightly,which proved that the CoWO4-x@PEO-b-PMAA had excellent biose-curity (Fig. 5i). As shown in Fig. 6, the H&E staining of organs collectedfrom different groups showed no visible damage, suggesting that thetherapeutic method and materials in this work had little toxicity inmice.

Fig. 5. (a) Infrared thermal images of 4T1 tumor-bearing mice and (b) corresponding temperature curves. (c) Relative tumor volumes of mice after differenttreatments. (d) Representative photographs of mice after the various treatments on the 14th day. (e) Western blotting analysis of tumors from the treatmentprocedure and (f) corresponding quantitative analysis of the Western blotting data. (g) Schematic diagram of immunoadjuvant therapy and obtained correspondingexperimental results. (h) Quantitative measurement of tumor volume in mice with different treatments. (i) Body weight changes of mice during the treatmentprocedure.

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3. Conclusions

This work constructed and applied a novel non-stoichiometricCoWO4-x@PEO-b-PMAA with excellent NIR absorption for enhancedphototherapy outcome under an 808 nm laser irradiation. On the basisof its excellent NIR absorption and high atomic number (Z = 74) ele-ment content, these CoWO4-x@PEO-b-PMAA NPs can be employed forPA imaging and CT imaging. Moreover, Western blotting was utilized toexplore the mechanisms of enhanced phototherapy. The results showedthat although phototherapy led to enhanced expression of ICD-relatedproteins during a 14-day treatment, tumor recurrence still occurred,mainly due to overexpression of HSP60 and NRF2, which facilitatedtumor immunoresistance toward to PTT and PDT. Enhanced photo-therapy can be achieved if the expression of HSP60 and NRF2 was re-duced using specific HSP60 and NRF2 inhibitors. Therefore, this workdemonstrated a method for improving the efficacy of phototherapy, andwe speculate that it will be promising if better specific inhibitors aredeveloped in the future.

4. Experimental

4.1. Materials

All of the reagents were used without further purification unlessotherwise indicated. Sodium tungstate (Na2WO4·2H2O), cobalt chloride(CoCl2·6H2O), ethidium bromide (EB) and 1,3-diphenylisobenzofuranwere obtained from Aladdin. 2′,7′-dichlorodihydrofluorescein diacetate(DCFH-DA), calcein acetoxymethyl ester (Calcein-AM), propidium io-dide (PI), 3-(4,5-dimethylthialzol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT) and etoposide were purchased from Sigma-Aldrich. Poly(ethylene oxide-b-methacrylic acid) (PEO-PMAA, Mw = 7500-b-15,000) 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 deionizedwater 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, theobtained cobalt chloride solution was added dropwise to the sodiumtungstate solution under magnetic stirring for another 0.5 h. After that,the resulting solution was transferred into a 100 mL Teflon-lined au-toclave, and the hydrothermal reaction was maintained at 160 °C in anelectric oven for 24 h. The obtained blue products were collected bycentrifuge, further alternately washed with water and ethanol threetimes, 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 °Cfor 2 h under a hydrogen/argon atmosphere. Then, 18 mg of the as-prepared CoWO4-x nanoparticles and 60 mg of PEO-PMAA were addedto 25 mL of deionized water under continuous ultrasonic agitation for30 min. Finally, the resulting dispersed solution was stored at 4 °C forfuture use.

4.4. Characterization

Transmission electron microscopy (TEM) was performed on a FEITecnai G2 F20 microscope under 200 kV acceleration voltage. Thephase composition of the sample was recorded by X-ray diffractionanalysis (XRD, Bruker AXS D8 Advance). The chemical valence of W

Fig. 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 daysof treatment.

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ions was determined by X-ray photoelectron spectroscopy (XPS, PerkinElmer PHI 5600). The optical properties were recorded on a U-4100spectrophotometer (Hitachi, Japan). The temperature changes of tu-mors were obtained using an infrared camera (FLIR E6). MTT experi-ments 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 ahumidified atmosphere with 5% CO2.

4.6. In vitro cell cytotoxicity assay

The viability of 4T1 cells in the presence of nanoparticles wasevaluated 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 mediumand grown overnight. Then, cells were sequentially incubated withvarious concentrations of nano-materials for another 24 h.Subsequently, 20 µL of MTT solution (5 mg/mL) was added to each welland incubated with the 4T1 cells for 4 h. When the media with MTT wasremoved, 150 µL of DMSO was added to each well for dissolving theformazan crystals at room temperature for 30 min, and the absorbancewas then measured at 490 nm using a multi-detection microplatereader.

The phototherapeutic effect of different treatment was also mea-sured using the MTT method. For the in vitro PTT group, 4T1 cells wereincubated with CoWO4-x@PEO-b-PMAA (250 µg/mL) in a 96-well platefor 24 h. Then, 50 μL of sodium azide (10 μM) was added to the cellsand cells were irradiated with an 808 nm laser at a power density of1.0 W/cm2 for 2, 6 and 10 min. For in vitro PDT experiments, 4T1 cellscultured in 96-well plates were incubated with CoWO4-x@PEO-b-PMAA(250 µg mL) for 24 h and then irradiated with an 808 nm laser at apower density of 1.0 W/cm2 on an ice box for 2, 6 and 10 min. For thein vitro PDT/PTT combined experiment, experimental conditions suchas cell density, the concentration of CoWO4-x@PEO-b-PMAA, irradia-tion time and power density of laser remain unchanged, but neithersodium azide nor ice box was applied.

4.7. Detection of ROS

The ROS detection in solution consisted of two groups, which in-cluded 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 mLof above three solutions. Then, they were irradiated with a NIR laser fordifferent time. After centrifugation, the collected supernatant was as-sayed using a spectrophotometer. The DCFH-DA probe was used toevaluate the intracellular ROS generation in 4T1 cells. Briefly, 4T1 cellswith a density of 5 × 103 per dish were seeded into a 35-mm culturedish and incubated at 37 °C overnight. Subsequently, the medium wasdiscarded, and cells were incubated with fresh medium containingCoWO4-x@PEO-b-PMAA (250 μg mL) for 4 h. After being washed withthe PBS, the cells were stained with DCFH-DA (50 μL, 10 mM) for an-other 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 afluorescent microscope. Untreated cells were used as a negative controlgroup, while the cells incubated with H2O2 (200 μL, 50 mM) at 37 °C for1 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 mediumcontaining the CoWO4-x@PEO-b-PMAA (250 μg/mL) was added to theculture dish to replace the previous culture medium. After incubationfor another 6 h, the cells were washed three times with PBS. Then, the4T1 cells were irradiated with an 808 nm laser at a power density of1.0 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 distinguishliving and dead cells after being rinsed with PBS. Finally, the stainedcells 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 culturedish and cultivated with RPMI culture medium at 37 °C in a humidified5% CO2 incubator. After incubation for 12 h, the culture medium wasreplaced with fresh media containing CoWO4-x@PEO-b-PMAA and EBat concentrations of 250 μg/mL and 10 μg/mL, respectively. Untreatedcells served as a control group. After 6 h incubation at 37 °C, the cellswere washed with PBS and irradiated with a NIR laser at a powerdensity of 1.0 W/cm2. After that, the medium was removed, and the4T1 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 foranother 20 min in the dark at 37 °C. After being washed with PBS threetimes, the stained cells were immediately visualized using a fluorescentmicroscope.

4.10. In vivo antitumor therapy

For the in vivo experiments, all the animal experiments were per-formed according to the criteria of the National Regulation of China forCare and Use of Laboratory Animals. All healthy female BALB/c mice(4–5 weeks old) were purchased from Vital River Laboratory AnimalTechnology Co., Ltd. (Beijing). To establish the tumor model, 1 × 106

4T1 cells suspended in DMEM and Matrigel were subcutaneously in-jected into the left front leg of each mouse. When the tumor volumereached ~200 mm3, mice were randomly divided into five groups(n = 5 for each group): (i) PBS as control; (ii) 808 nm laser irradiationfor 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)pre-injection of specific inhibitor + CoWO4-x@PEO-b-PMAA NPs in-jection + 808 nm laser irradiation for 10 min. For group i and ii, micewere intratumorally injected with 100 μL of PBS, while for the group iiiand iv, mice were intratumorally injected with 100 μL of CoWO4-x@PEO-b-PMAA NPs (1.0 mg/mL). The mice were irradiated by 808 nmlaser (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 mon-itored every day and normalized in comparison with their initial values.The tumor volume was evaluated using a caliper according to the for-mula: volume = (tumor length) × (tumor width)2/2. Relative tumorvolume 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 ofmice, 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 do-decyl sulfate) with 1% protease inhibitor cocktail (MCE). And thencentrifuged at 12,000 rpm for 10 min. Total proteins were resolved by8-12% SDS-PAGE and transferred on PVDF (Millipore, Germany).Membranes were blocked with 10% non-fat milk powder for 2 h atroom temperature, and incubated overnight at 4 °C with primary an-tibodies: anti-GAPDH antibody (1:2000; Proteintech), anti-NRF2 anti-body (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

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with 1‰ Tween-20 (TBST), membranes were incubated with horse-radish peroxidase secondary antibody (1:5000) for 1 h at room tem-perature. After washed in TBST, visualized with an enhanced chemi-luminescence kit (ECL-kit, ThermoFisher).

4.12. Histology analysis

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

Declaration of Competing Interest

The authors declare that they have no known competing financialinterests or personal relationships that could have appeared to influ-ence the work reported in this paper.

Acknowledgement

This work was supported by National Natural Science Foundation ofChina (No. 21771047).

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.cej.2019.123979.

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