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Oxygen Production of Modified Core-Shell CuO@ZrO2 Nanocomposites by Microwave Radiation to Alleviate Cancer Hypoxia for Enhanced Chemo-Microwave Thermal Therapy Zengzhen Chen, Meng Niu, Gen Chen, Qiong Wu, Longfei Tan, Changhui Fu, Xiangling Ren, Hongshan Zhong, Ke Xu, and Xianwei Meng ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b07749 • Publication Date (Web): 04 Dec 2018 Downloaded from http://pubs.acs.org on December 4, 2018

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Oxygen Production of Modified Core-Shell CuO@ZrO2 Nanocomposites by Microwave Radiation to Alleviate Cancer Hypoxia for Enhanced ChemoMicrowave Thermal Therapy Zengzhen Chen†,‡, Meng Niu§, Gen Chen‖, Qiong Wu†, Longfei Tan†, Changhui Fu†, Xiangling Ren†, Hongshan Zhong§, Ke Xu§, Xianwei Meng*,† †Laboratory

of Controllable Preparation and Application of Nanomaterials, Key Laboratory of

Cryogenics, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, No.29 East Road Zhongguancun, Beijing 100190, P. R. China §Department

of Radiology, First Hospital of China Medical University Key Laboratory of

Diagnostic Imaging and Interventional Radiology in Liaoning Province, Shenyang 110001, People's Republic of China ‖School

of Materials Science and Engineering, Central South University, Changsha, Hunan

410083, P. R. China ‡ University

of Chinese Academy of Sciences, Beijing 100049, P. R. China

Abstract There are acknowledged risks of metastasis of cancer cells and obstructing cancer treatment from hypoxia. In this work, we design a multifunctional nanocomposite for treating hypoxia based on the oxygen release capability of CuO triggered by microwave (MW). Core-shell CuO@ZrO2 nanocomposites are prepared by confining CuO nanoparticles within the cavities of mesoporous ZrO2 hollow nanospheres. The 1-butyl-3-methylimidazolium hexafluorophosphate (IL) is loaded to the CuO@ZrO2 nanocomposites for improving microwave thermal therapy (MWTT). 1-Tetradecanol (PCM) is introduced to regulate the release of chemotherapeutic drugs of Doxorubicin (DOX). Thus, the IL-DOX-PCM-CuO@ZrO2 multifunctional (IDPC@Zr)

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nanocomposites are obtained. Finally, IDPC@Zr nanocomposites are modified by monomethoxy polyethylene glycol sulfhydryl (mPEG-SH, 5kDa) (IDPC@Zr-PEG nanocomposites). IDPC@ZrPEG nanocomposites can produce oxygen in the tumor microenvironment during the course of tumor treatment, thereby alleviating the hypoxic state and improving the therapeutic effect. In vivo anti-tumor experiments prove that the tumor rate is very high and the inhibition rate is 92.14%. In addition, CT imaging contrast of the nanocomposites can be enhanced due to the high atomic number of Zr. Therefore, IDPC@Zr-PEG nanocomposites can be applied for monitoring the tumor treatment process in real time. The combined therapy offers many opportunities, such as the production of oxygen from CuO nanoparticles by MW to alleviate hypoxia, the enhancement of combined treatment of MWTT and chemotherapy, and the potential application of CT imaging to visualize the treatment process, which therefore provides a promising method for clinical treatment of tumors in the future. Keywords: hypoxia, microwave thermal therapy, chemotherapy, CT imaging, oxygen production, CuO nanoparticles

The ever-increasing metabolic processes of the aggressively proliferating tumor cells lead to inadequate oxygen supply within intratumor microenvironment, termed hypoxia.1-5 It is extensively accepted that hypoxia strengthens not only tumor aggressiveness but also tumor resistance to medical therapies,6-8 such as radiotherapy,9-13 photodynamic therapy,14-17 and chemotherapy.18-23 It can lead to incomplete treatment of the tumor, resulting in a high recurrence state after tumor treatment.24, 25


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To date, a number of strategies have been developed to overcome tumor hypoxia and improve the therapeutic effects of tumors,21, 26, 27 such as perfluorocarbon delivery oxygen therapy,28-30 hyperbaric oxygen therapy,31,

32

catalase or metal catalyzed endogenous peroxides to produce

oxygen therapy,33-36 or oxygen therapy by water-splitting through photosynthesis.37 Among them, the utilization of nanomaterials as catalysts for the production of oxygen from endogenous substances has received increasing attention. For example, taking advantages of instinct reaction within the tumor microenvironment, a number of groups have designed various MnO2 nanocomposites for tumor hypoxia modulating because MnO2 nanocomposites with endogenous H2O2 can sustainably produce oxygen.19,

38-40

Taeghwan Hyeon et al. reported that MnFe2O4

nanoparticles can effectively enhance photodynamic therapy of tumors at hypoxic conditions.41 Despite these encouraging advancements, the oxygen-production strategy suffers from some potential issues, e.g., endogenous dependence.42 The heterogeneity of tumor,43 i.e. the intrinsically uneven endogenous substance distribution within tumor,44, 45 results in the failure of the oxygenproduction strategy.43 Therefore, it is highly desirable to develop a system that can produce oxygen by exogenous stimulation for enhancement of the therapeutic effect of tumors, which is not limited by tumor heterogeneity. Inspired by the chemically decomposition of CuO to oxygen triggered by MW radiation, we designed a multifunctional nanocomposite, exhibiting the capability of oxygen production by MW without limitation from the heterogeneity of the tumor. In order to obtain nanosized CuO, reduce its self-toxicity and collect as-produced oxygen, CuO nanoparticles were confined in the cavities of mesoporous ZrO2 hollow nanospheres to prepare core-shell CuO@ZrO2 nanocomposites. After that, IL was loaded to the CuO@ZrO2 nanocomposites for improving MWTT, and PCM was introduced to control the release of chemotherapeutic drugs of DOX. mPEG-SH modification was


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performed for in vitro and in vivo studies to obtain a biocompatible IDPC@Zr-PEG nanocomposites (Scheme 1a). Through dissolved oxygen detection, we found that the IDPC@ZrPEG nanocomposites produced 3.02 times as much oxygen as the blank solution (PBS). Once IDPC@Zr-PEG nanocomposites were indwelling in the tumor microenvironment, it produced highly concentrated dissolved oxygen by the radiation of MW and continuously released chemotherapeutic drugs. Importantly, IDPC@Zr-PEG nanocomposites produced persistent dissolved oxygen in the tumor microenvironment by MW radiation, which can improve the effect of combined treatment of MWTT and chemotherapy. And the results of in vivo anti-tumor experiments also show that IDPC@Zr-PEG nanocomposites have a significant effect on inhibiting tumor growth. Compared with the non-oxygen group IL-DOX-PCM@ZrO2-PEG nanocomposites (only the combined treatment of MWTT and chemotherapy), the tumor inhibition rate of the IDPC@Zr-PEG nanocomposites group (experimental group) increases from 51.11% to 92.14%. Therefore, as-designed IDPC@Zr-PEG nanocomposites are promising tumor hypoxia regulator, which can also significantly improve the effect of combined tumor treatment of CT imagingguided MWTT and chemotherapy.


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Scheme 1. a) Schematic diagram of the preparation of IDPC@Zr-PEG nanocomposites, which is not drawn to scale. b) Schematic diagram of IDPC@Zr-PEG nanocomposites with oxygen production regulating cancer hypoxia for enhanced combination of chemo-microwave thermal therapy under MW radiation.

Results and Discussion In this experiment, SiO2 nanoparticles were used as templates to synthesize mesoporous ZrO2 hollow nanospheres. Then CuO nanoparticles was loaded into mesoporous ZrO2 hollow nanospheres to obtain the CuO@ZrO2 nanocomposites. There are many advantages: i) hollow structure of ZrO2 are more profit for collecting oxygen produced by the radiation of MW, ii) to improve the CuO nanoparticles biocompatibility, iii) to increase the CuO nanoparticles stability, iv) microwave radiation has a deeper penetration depth, and v) allowing the materials to better enrich into the tumor area. After that the IDPC@Zr nanocomposites were obtained by physical aspiration. And finally the nanomaterials were modified with mPEG-SH yielding IDPC@Zr-PEG nanocomposites to improve the biocompatibility and reduce the cytotoxicity.


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Figure 1. Characterization of the IDPC@Zr-PEG nanocomposites. (a) SEM images and TEM images of SiO2 nanoparticles. (b) SEM images and TEM images of ZrO2 nanoparticles. (c) SEM images and TEM images of CuO@ZrO2 nanocomposites. (d) Dark-field TEM image of CuO@ZrO2 nanocomposites. (e) Bright-field TEM images of CuO@ZrO2 nanocomposites. (f) Cu. (g) HRTEM picture of CuO lattice in CuO@ZrO2 nanocomposites. (h) Zr. (i) O. (j) FTIR spectra of IDPC@Zr-PEG nanocomposites. (k) The EDS of IDPC@ZrPEG nanocomposites.


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Characterization

of

IDPC@Zr-PEG

Nanocomposites.

The

IDPC@Zr-PEG

nanocomposites were prepared according to the method described in the experiment. The morphology and structure of SiO2 nanoparticles, mesoporous ZrO2 hollow nanospheres, and CuO@ZrO2 nanocomposites were characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) (Figure 1a-c). It can be seen from Figure 1a that SiO2 nanoparticles show solid structure and the particle size is 134.69±10 nm (Supporting Information, Figure S1a). Figure 1b shows the hollow nanosphere structure of ZrO2 and the particle size is 173.69±5 nm (Supporting Information, Figure S1b). Surface area and porosimetry analyzer was used to measure the specific surface area and pore diameter of the mesoporous ZrO2 hollow nanospheres (Supporting Information, Figure S2a, b), the specific surface area and pore diameter were 440.884 m2/g and 2.583 nm, respectively. We used TGA (NETZSCH STA 409 PC/PG, Germany) to determine IL and PCM amount in mesoporous ZrO2 hollow nanospheres (Supporting Information, Figure S2c), the loading efficiencies of PCM and IL were 4.98% and 17.27%, respectively. It can be clearly seen from Figure 1c that CuO nanoparticles are encapsulated in the cavity of mesoporous ZrO2 hollow nanospheres and the CuO@ZrO2 nanocomposites size is 174.05±5 nm (Supporting Information, Figure S1c).The high-resolution TEM image (HRTEM) of CuO@ZrO2 nanocomposites are shown in Figure 1. Figure 1d and 1e are TEM images of CuO@ZrO2 nanocomposites in the dark-fields and bright-field, respectively. By comparing the two diagrams, the presence of CuO nanoparticles can be clearly observed and the CuO grows in the hollow space of the mesoporous ZrO2 hollow nanospheres. The X-ray diffraction (XRD) tests to further verify the successful preparation of CuO@ZrO2 (Supporting Information, Figure S3). As shown in Figure S3, we find that there is no obvious peak in mesoporous ZrO2 hollow nanospheres, indicating that it is an amorphous structure. The crystal


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morphology was observed in CuO@ZrO2 nanocomposites. Moreover, by comparing with the XRD peak of the standard, the XRD peaks of CuO@ZrO2 can be well matched.46-48 The results show that the CuO nanoparticles are successfully loaded into the cavity of mesoporous ZrO2 hollow nanospheres. The mapping from CuO@ZrO2 nanocomposites show the elemental distribution of Cu, Zr, and O in the nanocomposites (Figure 1f, 1h-i). HRTEM image (Figure 1g) shows that CuO@ZrO2 nanocomposites have high crystallinity with a layer spacing of 0.25 nm due to the lattice spacing of CuO nanoparticles. In order to further verify the successful loading of DOX, PCM, IL and the successful modification of mPEG-SH, the functional groups of IDPC@Zr-PEG nanocomposites were determined by fourier transform infrared spectroscopy (FT-IR, Varian, model 3100 Excalibur, Figure 1j). The wavenumber in 1440, 1495, 1580, and 1621 cm-1 are the vibrational peak of the aromatic ring framework, which confirms the presence of DOX.49 The OH stretching vibration peak appears at 3450 cm-1.50 C-O single bond has a characteristic peak at 1050 cm-1.51 And 687 cm-1 is the plane bending peak of O-H. The above characteristic peaks prove that PEG and PCM have been successfully loaded into CuO@ZrO2 nanocomposites. P-F has a characteristic absorption peak at 850 cm-1. The vibration of imidazole ring stretching is 1169 cm1.

The vibrational peaks of the imidazole skeleton are at 1470 and 1575cm-1, this confirms that IL

is successfully loaded into the IDPC@Zr-PEG nanocomposites.52, 53 Figure 1k shows the content of characteristic elements and characteristic elements of IDPC@Zr-PEG nanocomposites. It can be seen that the ratio of the Cu element to the Zr element is 8.68% and 20.16%, respectively. The zeta of CuO@ZrO2 nanocomposites, IDPC@Zr nanocomposites and IDPC@Zr-PEG nanocomposites are 23 mV, -1.1 mV and -17.9 mV, respectively, further validating the successful synthesis of IDPC@Zr-PEG nanocomposites.


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Evaluation of Microwave-produced Dissolved Oxygen. Microwave radiation of CuO causes it to undergo electron transfer and produce oxygen. The reaction mechanism is as follows: 𝐌𝐖

𝐂𝐮𝐎

(1)

𝐂𝐮𝟐𝐎 + 𝐎𝟐↑

The oxygen production from CuO@ZrO2 nanocomposites can be qualitatively detected by dissolved oxygen reagents. Figure 2a can be observed blank solution control group dH2O and PBS, no MW radiation group dH2O+CuO@ZrO2 nanocomposites and PBS+CuO@ZrO2 nanocomposites, dH2O solvent MW radiation group dH2O+CuO@ZrO2 nanocomposites+MW and PBS as solvent MW radiation group PBS+CuO@ZrO2 nanocomposites+MW (PBS pH=5.5, the concentration of the CuO@ZrO2 nanocomposites were 2 mg/mL) in the MW power of 0.9 W (5 min) radiation produced changes in dissolved oxygen value. It can be seen that pure dH2O and PBS itself have a very small amount of dissolved oxygen (Figure 2a). With the addition of CuO@ZrO2 nanocomposites, there was a small increase in the dissolved oxygen value in the solution. When the CuO@ZrO2 nanocomposites were added at the same time as the MW radiation, the amount of dissolved oxygen in the solution was significantly increased. The effect of using PBS as a solvent in this process is better than that of dH2O. As can be seen from Figure 2b, very little dissolved oxygen (2.41 mol/L and 1.13 mol/L) is present in pure PBS and dH2O under MW radiation. The dissolved oxygen of CuO@ZrO2 nanocomposites produced in dH2O is 4.38 mol/L. The dissolved oxygen of CuO@ZrO2 nanocomposites produced in PBS at pH=5.5 is as high as 7.28 mol/L. From the data we can see that the oxygen produced by the materials under MW radiation is 3.02 times that of the blank solution (PBS). In the tumor microenvironment, the asprepared CuO@ZrO2 nanocomposites can release dissolved oxygen better under MW radiation. Therefore, CuO@ZrO2 nanocomposites can provide more oxygen for tumor hypoxia therapy.


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Figure 2. Oxygen production capability of CuO@ZrO2 nanocomposites under microwave radiation. (a) Qualitative analysis of dH2O, PBS (pH=5.5), dH2O+CuO@ZrO2 nanocomposites, PBS+CuO@ZrO2 nanocomposites, dH2O+CuO@ZrO2 nanocomposites+MW, and PBS+CuO@ZrO2 nanocomposites+MW in the production of oxygen by dissolved oxygen indicator. (b) Micro-computer DO-BOD dissolved oxygen meter for measuring dissolved oxygen.

Performance Evaluation of Microwave Heating Experiments in Vitro. IDPC@Zr nanocomposites had MW sensitization due to the confinement effect of IL in closed spaces.52 In this work, we used a near-infrared (FLIR) imager to monitor the MW heating effect of IDPC@Zr nanocomposites in real time (Figure 3). Under the MW radiation of 0.9 W (450 MHz) for 5 minutes, the temperature of the control group (the saline solution) increased by 18.2 °C. The saline solution of IDPC@Zr nanocomposites rise by 34.0 °C at 10 mg/mL under the same conditions (Figure 3a, b). Moreover, we also founded that with the increase of IDPC@Zr nanocomposites concentration, the effect of MW heating is better. The temperature of 2.5, 5, 7.5, and 10 mg/mL IDPC@Zr nanocomposites saline solution rise to 25.4, 27.1, 29.6, and 34.0 °C after a 5-minute MW heating (Figure 3a, b). From these results, the temperature changes of the saline solution at different concentrations are 7.2, 8.9, 11.4, and 15.8 °C higher than the saline solution in the control group, respectively. Figure 3c is the FLIR thermal image of IDPC@Zr nanocomposites. The Figure shows that as the concentration of IDPC@Zr nanocomposites increased, the color of the image became more vivid, and it also represents the better temperature heating effect of IDPC@Zr


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nanocomposites. The results of MW heating experiments in vitro show that the as-made IDPC@Zr nanocomposites were appropriate for tumor therapy in vivo.

Figure 3. Evaluation of the in vitro microwave heating, drug release results and cell experiment results of the IDPC@Zr-PEG nanocomposites. (a) Temperature-raising effect of different concentrations of IDPC@Zr nanocomposites saline solution under MW radiation. (b) Corresponding to the highest temperature rise of (a), (c) FLIR thermal image corresponding to (a) image. (d) Results of drug release of IDPC@Zr-PEG nanocomposites under different conditions. (e) Results of drug release of IDPC@Zr-PEG nanocomposites at 55 ℃. (f-h) The viability of L929, HepG2, H22 cells measured by MTT assay with different concentration of the IDPC@Zr-PEG nanocomposites for 24 h. (i) H22 Cells viability of different materials under the same treatment method.

Drug Release Results. Owing to the porous structure, the mesoporous ZrO2 hollow nanospheres could be acted as the role of perfect drug carrier. DOX as a commonly used anti-tumor drug was also widely used in studies of drug loading and release. With a hydrophobic tail and a hydrophilic head structure, PCM could be easily compatible with hydrophobic and hydrophilic chemicals. PCM are used as a thermal response switch, due to the proper melting point in the range of 38-40 °C, which could stable in solid under normal physiological environments. When the temperature


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reached over 38 °C, the thermal switch opened and released the drug fast. The loading efficiency of the drug in this paper is 16.51%. The reason may be that the hollow part is occupied by CuO nanoparticles, leading to space shrinkage. IDPC@Zr-PEG nanocomposites released to 12.13% of the drug at 37 °C. When IDPC@Zr-PEG nanocomposites were radiated with MW at 37 °C, the drug released to 21.15%. It can be found that the release of MW-radiated drugs increase by nearly 9.02% (Figure 3d). Under MW radiation, the temperature of the IDPC@Zr-PEG nanocomposites increased to approximately 55 °C. Figure 3e shows the drug release of IDPC@Zr-PEG nanocomposites directly at 55 °C. The final result of the release is 21.97% which was similar to the MW radiation at 37 °C. The experimental results show that the temperature-responsive switch can achieve controlled release of the drug, and it also proves that the PCM are successfully loaded into the CuO@ZrO2 nanocomposites. In Vitro Tumor Cell Inhibition. In our work, we used mouse normal fibroblast L929, human hepatoma cells HepG2 and mouse hepatoma cells H22 to evaluate the cytotoxicity of IDPC@ZrPEG nanocomposites, and H22 to evaluate the inhibitory effect of IDPC@Zr-PEG nanocomposites on tumor cells in vitro. The cytotoxicity of IDPC@Zr-PEG nanocomposites were first evaluated by MTT assay. When the concentration of IDPC@Zr-PEG nanocomposites reach 100 μg/mL, the viability of L929 and HepG2 cells was 88.52% and 86.32%, respectively. When the concentration of IDPC@Zr-PEG nanocomposites reach 200 μg/mL, the cell activity can reach as high as 65.50% (Figure 3f-h). It is proved that the relatively low toxicity of IDPC@Zr-PEG nanocomposites. In addition, we also performed mouse acute toxicity experiments (Support Information, Figure S4a), blood biochemistry experiments (Support Information, Figure S4b), blood routine experiments (Support Information, Figure S4c) and H&E staining experiments of major organs (Support Information, Figure S5) demonstrates the low biotoxicity of IDPC@Zr-


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PEG nanocomposites in vivo. Figure 3i shows the viability of H22 cells of the control, free DOX, IL-DOX-PCM@ZrO2-PEG nanocomposites, and IDPC@Zr-PEG nanocomposites after the same treatment method. It can be seen from the Figure 3i the superior tumor killing capability to other groups of IDPC@Zr-PEG nanocomposites at different times under MW. It shows that the formation of dissolved oxygen can improve the treatment efficiency. The cell survival rates of the corresponding tumor cells after treating with IDPC@Zr-PEG nanocomposites by 0, 3, 5 and 7 min is 90.84%, 75.68%, 52.78%, and 35.36%, respectively. From the side, it is proved that as the MW time increases, the release of oxygen from the IDPC@Zr-PEG nanocomposites increase, thereby improving the therapeutic effect. Therefore, when IDPC@Zr-PEG nanocomposites are enriched in tumor cells, it can produce a large amount of in situ oxygen under the radiation of MW, thereby improving the combination treatment effect of MWTT and chemotherapy. Evaluation of Experimental Results of Microwave Thermal Therapy Combined with Chemotherapy in Vivo. According to the above experimental results, the IDPC@Zr nanocomposites have good heating effect and antitumor effect. We further studied the synergistic effect of IDPC@Zr-PEG nanocomposites in vivo. In order to further found the optimal treatment time, the distribution of IDPC@Zr-PEG nanocomposites in various organs was detected by ICPMS. Figure 4a shows the Zr content distribution in different tissues and organs at 6 h and 24 h after injection of IDPC@Zr-PEG nanocomposites into the tail vein. Figure 4b shows the distribution of Zr content in tumor tissues and organs at 6 h and 24 h after injection of IDPC@ZrPEG nanocomposites into the tail vein. The results of the two images show that the IDPC@ZrPEG nanocomposites content in the tumor area is the highest at 6 h after injection, which is 5.46% significantly higher than 1.89% at 24 h. Therefore, we chose the tail vein injection for 6 h as the treatment time of the tumor (Scheme 1b). Figure 4c is a FLIR image per minute of MW heating in


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mice with simple MW, MW radiation of IL-DOX-PCM@ZrO2-PEG nanocomposites without oxygen production, and the oxygen-producing group of the MW-radiated IDPC@Zr-PEG nanocomposites. It can be seen from the Figure 4c that the radiation area produced by our experimental group is wider under the same time and the same MW power. Figures 4d shows that compared with the MW control group, the IL-DOX-PCM@ZrO2-PEG nanocomposites group and the experimental groups have better MW heating effect than the MW treatment group. The temperature change in the tumor area is 18.3 ℃, 26.4 ℃ and 26.8 ℃, respectively. Local heating can cause local hyperthermia and achieve the purpose of tumor ablation. It can be seen from Figure 4e that there are no significant decrease in body weight of mice, indicating that IDPC@Zr-PEG nanocomposites have good biocompatibility in mice. Figure 4f shows the functional relationship between tumor volume and practice. As can be seen from Figure 4f, the MWTT group, the chemotherapeutic drug DOX treatment group, and the IDPC@Zr-PEG nanocomposites without MW radiation group (with IDPC@Zr-PEG nanocomposites but without MW radiation, can't produce oxygen) are better than those of the control group. However, the effect is far lower than that of the experimental group, which may be due to hypoxia in the tumor area, resulting in poor therapeutic effect. The therapeutic effect of IL-DOX-PCM@ZrO2-PEG nanocomposites+MW treatment group was better than that of the previous four groups. But hypoxia in tumor tissue area limits the therapeutic effect. From the Figure 4f, we can see that the experimental group has the best

inhibitory

effect

on

tumor.

Compared

with

the

IL-DOX-PCM@ZrO2-PEG

nanocomposites+MW treatment group, with the presence of CuO nanoparticles resulted in increased tumor inhibition. The results show that oxygen production reduced the resistance to treatment induced by hypoxia, improving the effect of combined treatment of MWTT and chemotherapy. In our work, we determined that the mouse was dead when the tumor volume


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exceeded 2000 mm3. Figure 4g is a survival rate curve. The survival rate of the mice in the experimental group (group IDPC@Zr-PEG nanocomposites) is 100% significantly higher than that of the IL-DOX-PCM@ZrO2-PEG nanocomposites+MW treatment group (33%), the control group (0%), the free DOX treatment group (0%), the simple MW treatment group (0%), and the IDPC@Zr-PEG nanocomposites treatment group (0%). From Figure 4h and 4i, we can see that after fifteenth days of treatment, the tumor picture in the treatment group is the smallest one. By analyzing the results of H&E staining of the organs of mice including tumor, liver, lung, kidney, heart and spleen, IDPC@Zr-PEG nanocomposites show no obvious damage in all kinds of organs. This result indicates that IDPC@Zr-PEG nanocomposites have good biocompatibility (Supporting Information, Figure S6). Moreover, from the data of the tumor tissue section of Figure 4j, it is apparent that the untreated tumor cells have almost no damage. Only part of the tumor cells died in the MWTT or chemotherapy group. Only half of the cancer cells died in the MWTT combined with chemotherapy group, possibly due to poor treatment effect caused by hypoxia. The tumor cells in the experimental group is almost completely destroyed. Compared with MWTT and chemotherapy combined treatment group, the results show that the oxygen produced by CuO nanoparticles under MW radiation could improve the effect of combined treatment of MWTT and chemotherapy.


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Figure 4. Characterization of IDPC@Zr-PEG nanocomposites in in vivo treatment experiments. (a) Distribution of Zr in different tissues at 6 h and 24 h after injection of IDPC@Zr-PEG nanocomposites in tail vein of mice. (b) Distribution of Zr in tumor tissues at 6 h and 24 h after injection of IDPC@Zr-PEG nanocomposites in tail vein of mice. (c) FLIR images of MW heating per minute for MW, IL-DOX-PCM@ZrO2-PEG nanocomposites, IDPC@Zr-PEG nanocomposites mice, and (d) Comparison of the final temperature difference corresponding to Figure a. (e) Body weight changes in each experimental mouse within 14 days of treatment. (f) Tumor volume curve of each experimental mouse after 14 days of treatment. (g) Survival curves during the 14-day treatment period of each experimental mouse. (h) Photographs of tumor tissues treated on the 14th day of each experimental mouse, (i) Tumor weight charts on the 14th day of each experimental mouse. (j) H&E staining sections of tumor tissues after treatment for 14 days in each experimental mouse. The scale bar in the Figure is 50 μm.

Evaluation of CT Imaging Results. Due to the high atomic number and atomic mass of Zr, mesoporous ZrO2 hollow nanospheres has the property of enhancing CT imaging. In contrast, Cu also has a high atomic number and atomic mass. Therefore, CuO also has the property of enhancing


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CT imaging. We have studied the CT imaging function of IDPC@Zr-PEG nanocomposites. First, we studied the CT imaging effect of mesoporous ZrO2 hollow nanospheres and CuO@ZrO2 nanocomposites at different concentrations (1-10 mg/mL). As shown in Figure 5a and 5b, we can clearly observe that both mesoporous ZrO2 hollow nanospheres and CuO@ZrO2 nanocomposites have good CT imaging capabilities. At certain concentration, the imaging ability of CT increases with the increase of material concentration. At the same concentration, the imaging ability of CuO@ZrO2 nanocomposites are better than that of mesoporous ZrO2 hollow nanospheres, which is due to the introduction of CuO nanoparticles. Because of the strong X-ray contrast of our IDPC@Zr-PEG nanocomposites, the therapeutic effect in vivo can be visualized and monitored in real time. In our work, we used H22 tumor bearing mice to study the CT imaging of IDPC@ZrPEG nanocomposites in vivo. By changing the CT imaging values before and after the injection of IDPC@Zr-PEG nanocomposites in the tail vein of mice, it can be clearly seen that the imaging ability of IDPC@Zr-PEG nanocomposites after injection of 24 h increase from the original 24 HU to 53 HU (Figure 5c and 5d). Figure 5e-g show the CT imaging effect (5 mg/mL) obtained by intratumoral injection of IDPC@Zr-PEG nanocomposites with different doses. It can be seen from the graph that CT imaging function is enhanced with the increase of injection dose. The result of the appeal indicates that IDPC@Zr-PEG nanocomposites can be used as potential CT contrast agents for visualized real-time monitoring of the treatment effect of tumors.


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Figure 5. In vivo and in vitro CT imaging results of IDPC@Zr-PEG nanocomposites. (a, b) In vitro CT results of the mesoporous ZrO2 hollow nanospheres and CuO@ZrO2 nanocomposites in different concentrations aqueous solution. (c) CT images of mice before injection of IDPC@Zr-PEG nanocomposites. (d) CT imaging pictures of IDPC@Zr-PEG nanocomposites injected into caudal vein at 24 h. (e-g) CT Imaging of Different doses of IDPC@Zr-PEG nanocomposites (5 mg/mL).

Conclusion In conclusion, we have successfully prepared the IDPC@Zr-PEG nanocomposites. The asmade nanocomposites can continuously produce oxygen under the radiation of MW, which can improve the effect of combined treatment of MWTT and chemotherapy. This multifunctional


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nanocomposites were obtained by loading CuO nanoparticles into the cavities of the mesoporous ZrO2 hollow nanospheres. In addition, due to the high atomic number and atomic mass of Zr, mesoporous ZrO2 hollow nanospheres have the ability to enhance CT imaging. Therefore, we can use mesopore ZrO2 hollow nanospheres to guide the treatment of tumors. Microwave sensitizer IL was loaded in the CuO@ZrO2 nanocomposites for enhanced MWTT. PCM was loaded to control the release of chemotherapeutic drugs of DOX. Finally, IDPC@Zr-PEG nanocomposites were prepared by surface modification with mPEG-SH. The MW heating experiment in vivo and in vitro proved that IDPC@Zr-PEG nanocomposites were suitable for tumor therapy. Good biocompatibility through cytotoxicity and acute experiments toxicity in vivo of the IDPC@Zr-PEG nanocomposites have been verified. Through the dissolved oxygen test, the CuO@ZrO2 nanocomposites produced 3.02 times more oxygen than the blank solution under MW radiation. IDPC@Zr-PEG nanocomposites can produce oxygen to regulate the tumor microenvironment under the radiation of MW, thereby improving the effect of combined treatment of MWTT and chemotherapy. In vivo antitumor experiments proved that IDPC@Zr-PEG nanocomposites inhibited tumor growth effectively with a tumor inhibition rate of 92.14%. Therefore, we have realized the multifunctional microwave treatment of gas therapy, chemotherapy, MWTT and CT imaging, which provided a feasible method for the treatment of tumors.

Experimental section Materials. Zirconium (IV) propoxide was provided by Tokyo Chemical Industry Co., Ltd. Ethanol, acetonitrile, 1, 4-dioxane and NaOH were purchased from Beijing Chemical Factory. Purchased the IL from Shanghai Chengjie Chemical Co., Ltd. DOX was obtained from Beijing Huafeng Chemical Reagent Co., Ltd. Copper sulfate pentahydrate (CuSO4•5H2O) was purchased from Xiqiao Chemical Co., Ltd. Ammonia and PCM were provided by Sinopharm Group


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Chemical Reagent Co., Ltd. The reagents used are analytical and without further purification required Characterization and Testing. The size and morphology of the nanocomposites were observed and measured with a biological transmission microscope (HT7700, Nisshin Japan) and a scanning electron microscope (HITACHI SEM-4800, Nisshin Japan). Cell viability was detected by the enzyme labelling apparatus (Thermo Fisher Instruments, Inc.). An FLIR was used to observe the temperature changes in the MW heating both in vivo and in vitro. The surface functional groups of IDPC@Zr-PEG nanocomposites were characterized using FT-IR. The concentration of oxygen produced by CuO@ZrO2 nanocomposites under MW radiation was quantified using a microcomputer-based dissolved oxygen DO-biochemical oxygen demand (BOD) detector (DOBOD, HT98193, Hanaward Beijing Instrument Co., Ltd.). Paraffin-embedded tissue sections and H&E-stained cells were further characterized by confocal fluorescence microscopy (Olympus X71, Japan). Animals. We strictly implement the 8th Edition (International Publication No: 978-0-309-15400-0) Guide For The Care And Use Of Laboratory Animals to conduct animal experiments, which were approved by the Use Committee and the Institutional Animal Care of the First Hospital of China Medical University (CMU). We raised the female mice at the Experimental Animal Center of China Medical University (at a temperature of 25 ±3 °C and a humidity of 50-55%). The weight of the mice were 33±3 g. We subcutaneously injected 200 mL DMEM (containing 2×107 H22 cells) into the right subthoracic or abdominal region of each mouse to establish a tumor model. Preparation of IDPC@Zr-PEG Nanocomposites. The IDPC@Zr-PEG nanocomposites were synthesized in 4 steps processes. First, we used SiO2 nanoparticles to synthesize mesoporous ZrO2 hollow nanospheres. Next, CuO@ZrO2 nanocomposites were synthesized on the basis of


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SiO2@ZrO2 nanocomposites. Then IDPC@Zr nanocomposites were obtained by loading the IL and the chemotherapy drugs of DOX. Finally, the biocompatible IDPC@Zr-PEG nanocomposites were obtained by surface modification with mPEG-SH. The detailed synthesis processes were as follows; Preparation of mesoporous ZrO2 hollow nanospheres: In this experiment, mesoporous ZrO2 hollow nanospheres were prepared by a template method. Since the system was anhydrous during the reaction, SiO2 nanoparticles (2 mL, approximately 240 mg) were first dehydrated with ethanol. All SiO2 nanoparticles were appealed uniformly dispersed in a mixture of 1.2 mL ammonia (NH3•H2O, 25-28%), 120 mL ethanol, and 40 mL acetonitrile (ethanol: acetonitrile=3:1). Then 0.5 mL of zirconium (IV) propoxide was dispersed in a mixed solution of 10 mL of acetonitrile and 30 mL of ethanol (acetonitrile: ethanol=1:3). Next, we quickly pour the zirconium (IV) propoxide solution into the silica nanoparticles solution. After magnetic stirring for 6 h at room temperature, zirconium (IV) propoxide will be hydrolyzed in a weakly alkaline solution to form ZrO2 and adhere to the surface of SiO2 nanoparticles. After the reaction was completed, the mixed solution was centrifuged to obtain ZrO2@SiO2 nanocomposites. To remove the SiO2 nanoparticles, the resulting ZrO2@SiO2 nanocomposites were dispersed in 110 ml of 5 mL of NaOH (1M) deionized water (dH2O) and stirred at 80 ℃ for 4 h. Mesoporous ZrO2 hollow nanospheres were obtained by centrifugation and deionized water washing 3 times. After that, surface area and porosimetry analyzer was used to determine the specific surface area and pore diameter of the ZrO2 nanospheres. The contents of IL and PCM loaded on ZrO2 nanospheres were detected by thermogravimetric analysis. Preparation

of

CuO@ZrO2

nanocomposites:

In

this

experiment,

CuO@ZrO2

nanocomposites were synthesized by using the method of anterior shell nucleus. 3 mL of dH2O,


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30 mg of mesoporous ZrO2 hollow nanospheres and 0.5 g of CuSO4•5H2O were uniformly dispersed in the sonicator. The mixed solution was kept under vacuum suction until the solvent was discharged. After the solid powder was dispersed in ethanol (20 mL), 2 mL of concentrated ammonia was added to form a basic system.54, 55 The reaction system was refluxed at 80 °C for 2 h. The reaction was washed 3 times by centrifugation and dH2O. The obtained precipitates were treated with 0.25% HCl to remove CuO nanoparticles from the outer shell of the mesoporous ZrO2 hollow nanospheres, thereby obtaining the CuO@ZrO2 nanocomposites. After that, we used X-ray diffraction (XRD) to further verify the synthesis of CuO nanoparticles, and it was successfully loaded into the cavity of mesoporous ZrO2 hollow nanospheres. Preparation of IL-CuO@ZrO2 nanocomposites: In a 50 mL conical flask, 1 mL of IL, 5 mL of ethanol, 5 mL dH2O, 2 mL of 1, 4-dioxane and 20 mg of CuO@ZrO2 nanocomposites were added. After 10 min of ultrasonic dispersion, the mixed solution was maintained under vacuum suction until it became a viscous liquid. Finally, the precipitate were washed 3 times with dH2O to obtain IL-CuO@ZrO2 nanocomposites. Preparation of IDPC@Zr nanocomposites: The DOX was used as an antitumor drug in the experiment, and PCM (above 38 °C) was used as a drug release trigger. In a 250 mL conical flask, 20 mg DOX, 10 mg PCM, 5 mL ethanol, 5 mL dH2O, and IL-CuO@ZrO2 were added. The DOX and PCM were loaded into IL-CuO@ZrO2 by vacuum suction. Finally, the precipitates were washed 3 times with dH2O to obtain IDPC@Zr nanocomposites. The washing supernatant was collected to detect the loading capacity of DOX. Preparation of IDPC@Zr-PEG nanocomposites: PEG modification can be widely used in nanocarriers modification. The 50 mL erlenmeyer flask was filled with 5 mg mPEG-SH, 10 ml Tris-HCl bufter (pH=8.0) and the IDPC@Zr nanocomposites obtained in the previous step. After


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stirring at room temperature for 4 h, the precipitates were washed by PBS (pH=7.4) for 3 times to obtain IDPC@Zr-PEG nanocomposites. Determination of Dissolved Oxygen. The Oxygen production from CuO@ZrO2 nanocomposites can be qualitatively detected by dissolved oxygen reagents. The experiment was divided into 6 groups: the blank solution control group of dH2O and PBS, no MW radiation group of dH2O+CuO@ZrO2 nanocomposites and PBS+CuO@ZrO2 nanocomposites, dH2O as solvent MW radiation group of dH2O+CuO@ZrO2 nanocomposites+MW and PBS as solvent MW radiation group of PBS+CuO@ZrO2 nanocomposites+MW (wherein the pH of PBS was 5.5, and the concentration of CuO@ZrO2 nanocomposites were 2 mg/mL). The MW power was 0.9 W (5 min). Two drops of dissolved oxygen reagent i and ii were added after the end of the MW radiation. After standing for 5 min, added 2 drops of dissolved oxygen reagent iii. It can be qualitatively detected by CuO@ZrO2 nanocomposites to produce oxygen. The oxygen concentration of CuO@ZrO2 nanocomposites under MW radiation can be quantitatively detected by the micro-computer DO-BOD detector. The experiment was divided into four groups: dH2O+MW group, dH2O+CuO@ZrO2 nanocomposites+MW, PBS+MW (pH=5.5) group and CuO@ZrO2 nanocomposites+PBS+MW (pH=5.5) group, in which the concentration of CuO@ZrO2 nanocomposites were 2 mg/L. Under the radiation of MW, the concentration of dissolved oxygen produced by the CuO@ZrO2 nanocomposites were quantitatively detected using a micro-computer DO-BOD detector meter. In Vitro Microwave Heating Experiment. The MW sensitivity of IDPC@Zr nanocomposites were evaluated by an in vitro MW heating ramp experiment. Different masses of IDPC@Zr nanocomposites (0, 2.5, 5, 7.5, 10 mg) were dispersed into 1 mL of saline solution and then added the solution into the reactor. We used 1 mL saline solution as a control group and radiated with


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MW (0.9 W) for 5 minutes. During MW radiation, the FLIR system was used for real-time monitoring and temperature and thermal imaging were recorded in each 10 s. Drug Release of IDPC@Zr-PEG Nanocomposites. The controlled release characteristics of DOX in IDPC@Zr-PEG nanocomposites were studied. During the experiment, there were three groups (three parallel samples in each group): Group one was to shake the IDPC@Zr-PEG nanocomposites in a 37 °C constant temperature water bath oscillation box, the second group was to shake the IDPC@Zr-PEG nanocomposites in a 37 °C constant temperature water bath oscillation box and then radiated by MW (5 min, 0.9 W); the third group was to shake the IDPC@Zr-PEG nanocomposites in a 55 °C constant temperature water bath oscillation box. First, the same amount of IDPC@Zr-PEG nanocomposites were dispersed in a sample tube filled with 1 mL of PBS (pH=5.5). The DPC@Zr-PEG nanocomposites were then homogeneously mixed in PBS by continuously shaking the sample tube. The concentration of DOX in supernatant of nanocomposites (482 nm) was determined by UV-Vis spectrophotometer. The DOX release at different time points was calculated from the standard curve. The standard curve is y=0.01855c+0.0014 (R2=0.99963), here the y represents the absorbance of DOX at 482 nm and the c represents the corresponding calculated DOX concentration (μg/mL). Cytotoxicity Test. The cytotoxicity of IDPC@Zr-PEG nanocomposites were evaluated by MTT assay. Mouse normal fibroblast L929, human hepatoma cells HepG2 and mouse hepatoma cells H22 were plated in 96 well plates and cultured under suitable conditions for 24 h (37 ℃, 5% CO2). Then, different concentrations (0, 6.25, 12.5, 25, 50, 100, 200 μg/mL) of IDPC@Zr-PEG nanocomposites were further co-cultured with the cells for 24h. Then MTT (0.5 mg/mL) was added to the DMEM. They were incubated for another 4 h. After the MTT was removed, 150 μL of dimethyl sulfoxide (DMSO) was added. The absorbance of each well in a 96-well plate was


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detected by the enzyme labelling apparatus (absorption peak at 492). All experiments were repeated 5 times. In Vitro Inhibition of Tumor Cells. The H22 cells was first plated in 6-well plates and cultured for 24 h under suitable conditions. The experiment was divided into 8 groups: the blank control group, MW group (MWTT group), free DOX group (chemotherapy group), DOX+MW group (chemotherapy combined with MWTT group), IL-DOX-PCM@ZrO2-PEG nanocomposites group (control materials without MW group), IL-DOX-PCM@ZrO2-PEG nanocomposites+MW group (control materials+MW group), IDPC@Zr-PEG nanocomposites group (no MW test group), IDPC@Zr-PEG nanocomposites+MW Group (experimental group). The nanocomposites concentration were 25 μg/mL, which contained the same DOX content. The MW power was 0.9 W and the MW time was 3, 5, and 7 min, respectively. After MW radiation, the cells were transferred to 96-well plates for 24 h. Finally, we used the MTT assay to detect cell viability and inhibit tumor size. Acute Toxicity Test. Acute toxicity experiments were used to evaluate the effect of IDPC@ZrPEG nanocomposites on the health of mice. Different concentrations of IDPC@Zr-PEG nanocomposites (3 in each group) were injected into healthy mice via tail veins. The IDPC@ZrPEG nanocomposites were dispersed into PBS (pH=7.4) and the injection doses were 0, 25, 50, 75, and 100 mg/kg, respectively. During the experiment, the mice were observed daily for growth status and body weight was weighed. Groups of mice were sacrificed 18 days later. Blood from mice was collected for blood tests and biochemical tests. The kidney, heart, spleen, lungs, and liver of the mice were fixed with 4% neutral formaldehyde and used for tissue studies. In Vivo Microwave Thermal Therapy Combined with Chemotherapy Experiment. Female mice with 33±3 g weight and H22 cells were selected as experimental mice to assess the tumor


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inhibitory effect of IDPC@Zr-PEG nanocomposites. The mice bearing the tumor cells in the experiment were divided into 6 groups (Three mice per group and the tumor volume was 150±30 mm3). Control group (without any treatment), free DOX group (chemotherapy group), blank MW group (MWTT group. Microwave power and time were 0.9 W and 5 min, respectively. Herein after the same), IDPC@Zr-PEG nanocomposites (experimental materials without MW radiation group), IL-DOX-PCM@ZrO2-PEG nanocomposites+MW (without CuO producing an oxygen group under MW radiation), IDPC@Zr-PEG nanocomposites+MW (experimental group). The amount of DOX contained in the materials used in the above experiment was the same. Treatment was initiated 6 h after tail vein injection of the mouse and temperature and thermal imaging were monitored and recorded using the FLIR system. The formula for calculating tumor volume in mice is: W2H/2 (where the H is the length of the tumor and the W is the width of the tumor). During the experiment, mice were recorded daily for changes in tumor volume and photographed. Groups of mice were sacrificed 14 days later and liver, lung, heart, kidney, spleen, and tumors of the mice were collected and fixed in 4% neutral formaldehyde for histological study. CT Imaging Experiment. The CT imaging ability of CuO@ZrO2 nanocomposites was evaluated by CT value. In the experiment, different concentrations of CuO@ZrO2 nanocomposites were dispersed in an in vitro CT imaging test tube (materials concentration 1-10 mg/mL). CT scans were performed and CT values were calculated in vitro. In vivo CT values were measured by injecting 50 mg/kg of IDPC@Zr-PEG nanocomposites into the tail vein of mice and testing it in vivo CT values at 24 h and comparing them with those when they were not injected.

Supporting Information


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The supporting Information is available free of charge via the Internet at http://pubs.acs.org. The supporting information of the article includes: particle size distribution of IDPC@Zr-PEG nanocomposites; biocompatibility evaluation of IDPC@Zr-PEG nanocomposites (blood routine, blood biochemistry, mouse acute toxicity); the image of animal tissue slices; XRD data of mesoporous ZrO2 hollow nanospheres and CuO@ZrO2 nanocomposites; TGA data of mesoporous ZrO2 hollow nanospheres, PCM@ZrO2 nanocomposites and IL@ZrO2 nanocomposites; N2 adsorption−desorption analysis of mesoporous ZrO2 hollow nanospheres. Corresponding Author *E-mail: [email protected] (X. Meng).

Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 81630053, 61671435), and CAS-DOE program (No. GJHZ1705).

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Black

Phosphorus

Nanosheet-Based

Drug

Delivery

System

for

Synergistic

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