Polyoxometalate-Based Radiosensitization ... - ACS Publications

Polyoxometalate-Based Radiosensitization Platform for Treating Hypoxic Tumors by Attenuating Radioresistance and Enhancing Radiation Response Yuan Yong,†,∥,⊥ Chunfang Zhang,‡,⊥ Zhanjun Gu,*,†,‡ Jiangfeng Du,† Zhao Guo,† Xinghua Dong,‡ Jiani Xie,† Guangjin Zhang,*,§ Xiangfeng Liu,*,‡ and Yuliang Zhao*,†,‡ †

CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics and National Center for Nanoscience Technology of China, Chinese Academy of Sciences, Beijing 100049, People’s Republic of China ‡ College of Materials Science and Optoelectronic Technology, University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China § Key Laboratory of Green Process and Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China ∥ College of Chemistry and Environment Protection Engineering, Southwest Minzu University, Chengdu 610041, People’s Republic of China S Supporting Information *

ABSTRACT: Radioresistance is one of the undesirable impediments in hypoxic tumors, which sharply diminishes the therapeutic effectiveness of radiotherapy and eventually results in the failure of their treatments. An attractive strategy for attenuating radioresistance is developing an ideal radiosensitization system with appreciable radiosensitization capacity to attenuate tumor hypoxia and reinforce radiotherapy response in hypoxic tumors. Therefore, we describe the development of Gd-containing polyoxometalates-conjugated chitosan (GdW10@CS nanosphere) as a radiosensitization system for simultaneous extrinsic and intrinsic radiosensitization, by generating an overabundance of cytotoxic reactive oxygen species (ROS) using high-energy X-ray stimulation and mediating the hypoxia-inducible factor-1a (HIF-1a) siRNA to down-regulate HIF-1α expression and suppress broken double-stranded DNA self-healing. Most importantly, the GdW10@CS nanospheres have the capacity to promote the exhaustion of intracellular glutathione (reduced GSH) by synergy W6+-triggered GSH oxidation for sufficient ROS generation, thereby facilitating the therapeutic efficiency of radiotherapy. As a result, the as-synthesized GdW10@CS nanosphere can overcome radioresistance of hypoxic tumors through a simultaneous extrinsic and intrinsic strategy to improve radiosensitivity. We have demonstrated GdW10@CS nanospheres with special radiosensitization behavior, which provides a versatile approach to solve the critical radioresistance issue of hypoxic tumors. KEYWORDS: polyoxometalates, GdW10@CS nanosphere, glutathione, radiosensitization, hypoxic tumor, radiotherapy, gene therapy reconstruction of the broken double-stranded DNA.5−10 However, radiotherapy still suffers from failure to eradicate the hypoxic tumor efficiently. One of the critical obstacles is the substantial presence of intracellular glutathione (GSH), which significantly decreases the effective production of ROS and weakens the therapeutic effect.11−17 Another crucial restriction

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adiotherapy (RT), as an extensively used method for cancer therapy in the clinic, takes advantage of highintensity ionizing radiation to suppress tumor proliferation with no depth restriction, during which it can induce DNA double-strand damage by generating considerable cytotoxic reactive oxygen species (ROS) produced by the ionization of surrounding water.1−4 Therefore, to enhance ionizing radiation-induced cellular damage during radiotherapy, adequate ROS generation is essential to induce DNA doublestrand damage by reacting with DNA and greatly suppressing © XXXX American Chemical Society

Received: May 2, 2017 Accepted: June 22, 2017 Published: June 22, 2017 A

DOI: 10.1021/acsnano.7b03037 ACS Nano XXXX, XXX, XXX−XXX

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Scheme 1. Schematic illustration of the as-prepared GdW10@CS nanosphere as an siRNA delivery platform for efficient radiosensitization efficacy of radiotherapy against hypoxic tumor cells.

irradiation energy within the tumor and effectively enhance the therapeutic effect of RT, they can generate radiochemicals (free radicals and ionizations) under high-energy irradiation by scattering X-rays/photons, Compton electrons, fluorescence photons, electron−positron pairs, and Auger electrons for hypoxic tumor obliteration.44−46 However, the untoward renal clearance and slow biodegradation may cause the accumulation of the radiosensitizers in the body, leading to potential inevitable side effects. Also, these radiosensitizers are incapable of eliminating intracellular GSH, leading to enormous reduction of ROS generation. Furthermore, many other intrinsic optimization radiosensitization strategies from the intrinsic biological nature have also been directed toward the enhancement of radiotherapeutic efficacy in hypoxic tumors. According to the previous report, hypoxia-inducible factor-1a (HIF-1a), as a key transcription factor, interacts with poly(ADP-ribose) polymerase-1 (PARP-1) under hypoxia, which is perceived to be an attractive regulator of major adaptive responses to hypoxia in tumors.47,48 Meanwhile,

of RT is tumor hypoxia, mainly induced by the exhaustion of oxygen with the rapid proliferation of cancer cells,18−26 which leads to the hypoxia-associated resistance of the hypoxic cells, increases the risk of DNA self-repair, and significantly limits the therapeutic efficiency. Moreover, high-energy ionizing radiation (X-ray or γ-ray) applied in radiotherapy may affect normal cells, causing unavoidable damage to normal tissues.27,28 Therefore, to validly improve the efficiency of radiotherapy and maximally enhance tumor eradication, it is highly attractive to develop an ideal radiosensitization system for sufficient ROS generation through simultaneously significantly concentrating a large local radiation dose and greatly consuming GSH, as well as effectively suppressing DNA self-repair to enhance hypoxic tumor therapeutic efficacy. Recently, tremendous extrinsic radiosensitization efforts have been devoted to develop conventional nanomedicine-based radiosensitizers such as metal-based nanoparticles,29−37 quantum dots,38,39 superparamagnetic iron oxides,40−42 non-metalbased nanoparticles,43 and so on. To significantly concentrate B


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Figure 1. Physicochemical characterizations of GdW10@CS nanospheres. (a) Transmission electron microscopy (TEM) image of the assynthesized GdW10@CS nanospheres. Scale bar: 50 nm. Inset: Size distribution of the as-synthesized GdW10@CS nanospheres in purified water. (b) Fourier transformation infrared spectra of the purified GdW10, chitosan, and the as-fabricated GdW10@CS nanospheres. (c) Raman spectra of the prepared GdW10 as well as GdW10@CS nanospheres. (d) Thermogravimetric analysis of GdW10, bare chitosan, and GdW10@CS nanospheres. (e) Zeta potential analysis of siRNA, GdW10, bare chitosan, GdW10@CS nanospheres, and GdW10@CSsiRNA. (f) Agarose gel retardation assay of GdW10@CSsiRNA complexes with various weight ratios, siRNA release by competitively binding herparin with carriers after degradation, and protection of siRNA from RNase degration by GdW10@CS. Naked siRNA was used as a control.

the remaining GdW10 section could be quickly metabolized through a renal clearance pathway due to its ultrasmall size, thus minimizing its detrimental side effects in vivo.59 Furthermore, tungsten and gadolinium atoms in the GdW10@ CS with high Z and strong X-ray attenuation capacity can also act as a dual-modal contrast agent in magnetic resonance (MR) and computed tomography (CT) imaging, achieving real-time monitoring and precise diagnosis and treatment of cancer. Therefore, the development of GdW10@CS nanospheres may be of great consideration in next-generation radiosensitizers for hypoxic tumor radiotherapy.

previous studies have also provided evidence that knocking out HIF-1α can stimulate the degradation of PARP-1, thereby suppressing reconstruction of the double-stranded DNA break as well as up-regulating the expression of cysteinyl aspartatespecific protease-3 (caspase-3) to promote cell apoptosis.49,50 Therefore, HIF-1α siRNA has been widely applied to targeted hypoxic tumors to down-regulate HIF-1α expression, so as to surmount hypoxia-associated resistance and suppress broken double-stranded DNA self-rehabilitation in radiotherapy.51−56 However, few reports have determined whether nanocarriers can simultaneously overcome the main shortcomings of radioresistant hypoxic cancer cells through an extrinsic strategy of increasing the radiation dose and expending intracellular GSH, as well as the intrinsic method of introducing HIF-1α siRNA to hypoxic cancer cells to interfere with DNA repair and improve radiosensitivity. To substantiate our design, we integrate a radiosensitizerbased Gd-containing polyoxometalates-conjugated chitosan (GdW10@CS nanosphere) and HIF-1α siRNA into one system to enhance radiosensitization (Scheme 1). In the system, the GdW10@CS nanosphere containing high-Z metal elements can serve as an external radiosensitizer to deposit radiation dosage for plentiful ROS generation under X-ray exposure and simultaneously acted as an internal effective HIF-1α siRNA nanocarrier to inhibit broken DNA restoration. Most surprisingly, we find that GdW10@CS nanospheres also have the capacity to deplete the intracellular GSH through a redox reaction for more effective ROS generation by consuming less reduced GSH, which significantly facilitates the therapeutic efficiency of radiotherapy. In addition, the chitosan shell in GdW10@CS nanospheres possesses high biocompatibility and biodegradability, and the strongly condensed positive charge facilitates their delivery of siRNA to the tumor site, thus realizing greatly enhanced transfection efficiency of hypoxic tumors.57,58 Moreover, after the chitosan shell biodegradation,

RESULTS AND DISCUSSION GdW10@CS nanospheres were prepared via a facile and effective procedure according to a simple ionotropic gelation technique. The morphology and typical diameter of the asprepared GdW10@CS nanospheres showed a near-sphere of ∼30 nm in the TEM image (Figure 1a), which is consistent with the dynamic light scattering measurement (∼35 nm). Furthermore, to demonstrate the successful formation of GdW 10 @CS nanospheres, the FT-IR spectrum of the GdW10@CS nanospheres consistently exhibited all the typical characteristic peaks of GdW10 and chitosan corresponding to the literature reports,60 and the energy-dispersive X-ray (EDS) spectra also showed strong tungsten and sodium peaks apart from the carbon peaks (Figure S1), further confirming the existence of the GdW10@CS nanospheres (Figure 1b). Additionally, Raman spectra of the GdW10 before and after chitosan modification are also shown in Figure 1c. The main peaks of GdW10 and GdW10@CS nanospheres had no notable differences, indicating the lack of structure change in GdW10 after chitosan functionization. Furthermore, the thermal stability of the GdW10@CS nanospheres is enhanced in comparison to the bare polymer (Figure 1d), which was ascribed to the successful complexation of chitosan with the C


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Figure 2. GdW10-enhanced generation of reactive oxygen species with the depletion of GSH. (a) Fluorescence spectra of DCFH mixed with GdW10@CS (100 μg/mL) in the presence or absence of GSH (1 mM) exposed to X-rays. (b) Relative GSH after different treatments. (c) GSH/GSSG ratios of a solution containing GSH and GdW10@CS in the presence or absence of X-rays in an external analogue and intracellularly. (d) Photographs of glutathione oxidation by recording the color change of the solution mixture. A GSH solution in the absence of GdW10@CS nanospheres was used as a negative control. H2O2 (1 mM) was used to oxidize GSH (0.4 mM) and as a positive control. (e) Mechanism diagram of the reaction. (f) ROS generation of BEL-7402 cells with different treatments. Error bars were calculated by SD of three parallel samples. The scale bar is 100 μm. P values were based on the Student’s test: *P < 0.05, **P < 0.01.

considerably, and the GdW10@CS can also protect siRNA from degradation induced by RNase, manifesting that GdW10@ CS nanospheres can function as an efficient HIF-1α siRNA nanocarrier to reduce hypoxia-associated resistance and enhance radiosensitization of hypoxic tumors. Apart from GdW10@CS nanospheres being a gene nanocarrier, we further demonstrate whether they can be used as a favorable radiosensitizer for enhanced radiotherapy through Xray-triggered ROS generation as well as an electron acceptor or oxidant for proton conduction by exhaustion of the GSH level. First, we employed 2′,7′-dichlorofluorescein (DCF) as a probe to monitor the generation of ROS from GdW10@CS nanospheres under X-ray irradiation. After the phosphate buffer exposure to X-ray, weak fluorescence of the DCF solution was detected. However, the fluorescence intensity of the DCF solution containing the GdW10 was sharply increased

thermally stable GdW10. Next, to be compatible with biomedical applications, we simulated the stability of GdW10@CS nanospheres dispersed in Dulbecco’s modified Eagle’s medium (DMEM). The result showed that the hydrodynamic size of GdW10@CS nanospheres does not obviously increase, indicating their favorable stability in cell medium (Figure S2). As shown in Figure 1e, the ζ potential of GdW10 changed from −21.9 to +42.8 mV after conjugation with positively charged chitosan, which suggested that the asprepared GdW10@CS nanosphere had great potential to be an optimal gene nanocarrier by an electrostatic interaction with the negatively charged siRNA. A gel retardation assay further demonstrated that GdW10@CS nanospheres have an efficient siRNA loading efficacy at a molar ratio of nitrogen/phosphate (N/P ratio) of 7:1 (Figure 1f). After introducing heparin, siRNA can release from GdW 10@CS siRNA nanospheres D


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Figure 3. In vitro cytotoxicity evaluation of GdW10@CS nanospheres under hypoxia. Cytotoxicity of different concentrations of GdW10@CS nanospheres to (a) HeLa and (b) BEL-7402 cells under both normoxia and hypoxia. (c) Flow cytometric analysis of HeLa and BEL-7402 cell death with different treatments under hypoxia. (d) Statistical data analysis of the percentage of the corresponding apoptotic, dead, and late apoptotic/necrotic cells under different treatments.

by 10 times after irradiation with the same X-ray irradiation, indicating the generation of efficient ROS caused by the reaction between GdW10 and the X-rays (Figure 2a). This may be ascribed to the large amount of ROS generation from the interaction of Compton and Auger electrons produced by GdW10@CS with the surrounding water or oxygen molecules. Moreover, we also found that the GdW10@CS has the ability to decrease the GSH level. GSH is the most abundant reducing agent in cells, which can protect the cells from the damage of ROS, while W6+ often acts as an electron acceptor or oxidant and may react with GSH by the redox reaction to eliminate GSH. Thus, we next utilized Ellman’s assay to estimate the oxidative stress level by confirming the ratio of reduced (GSH) and oxidized (GSSG) glutathione in an external analogue and intracellularly. As expected, the relative GSH level and the ratio of GSH to GSSG were obviously reduced after incubation with

GdW10@CS nanospheres (Figure 2b and c), which was further intuitively demonstrated by tracing the color variance of the solution mixture (Figure 2d). Therefore, these results effectively validated that GdW 10 @CS nanospheres can effectively deplete GSH through redox reaction. For a more vivid illustration of our result, a brief enhancement mechanism of GdW10@CS nanospheres to produce ROS can be described as follows (Figure 2e): as we all know, due to the existence of GSH, the produced ROS could be efficiently diminished so as to attenuate the cytotoxicity of cancer cells. Surprisingly, we found that the GdW10@CS nanospheres can consume GSH through the redox reaction between W6+ and GSH,61,62 thereby drastically reducing the removal of the generated ROS and effectively enhancing cytotoxicity. Furthermore, we further employed the 2′,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe to monitor the formation of ROS induced E


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Figure 4. In vitro colony formation assay and DNA double-strand damage of GdW10@CS nanospheres in BEL-7402 cells under hypoxia. (a) GdW10@CS enhances the inhibitory effects of BEL-7402 cell proliferation in vitro under X-ray radiation (6 Gy) during colony formation. (b) Responding surviving fraction of BEL-7402 cells with various treatments. (c) Colony formation assay of BEL-7402 cells incubated with GdW10@CS and GdW10@CSsiRNA in a dose-dependent X-ray manner under hypoxia. (d) Corresponding surviving fraction of cells after different treatments. (e) Representative γ-H2AX immufluorescence images of DNA double-strand damage under hypoxia induced by GdW10@ CS (100 μg mL−1, 2 mL) or/and GdW10@CSsiRNA (100 μg mL−1, 2 mL) or/and X-ray radiation (6 Gy), stained with Hoechst and γ-H2AX for nuclear visualization and DNA fragmentation. The scale bar is 50 μm. (f) Corresponding normalized number of γ-H2AX under hypoxia after different treatments. Error bars were calculated by SD of three parallel samples. P values were based on the Student’s test: *P < 0.05, **P < 0.01, ***P < 0.001.

by GdW10@CS nanospheres under X-ray irradiation in hypoxic cells. As shown in Figure 2f, qualitative analysis showed that significantly enhanced levels of ROS were observed in cells treated with GdW10@CS nanospheres after exposure to X-rays, compared with the control group, indicating the prominent radiosensitization ability of GdW10@CS by producing ROS under X-ray treatment in hypoxic cancer cells. Therefore, all these results indicated that GdW10@CS nanospheres have the capacity to enhance radiotherapy through X-ray-triggered ROS generation and the depletion of GSH levels by synergy W6+triggered GSH oxidation.

To further substantiate the aforementioned results at the cellular level, the relative viabilities of HeLa and BEL-7402 cells treated with GdW10@CS nanospheres under normoxia and hypoxia were assessed by using the Cell Counting Kit (CCK-8) assay. After incubation with large concentrations of GdW10@ CS nanospheres for 24 h, no obvious cytotoxicity was induced in both cells, even at high concentrations up to 100 μg mL−1 (Figure 3a and b), which was further confirmed in vivo by H&E staining of main organs, blood chemistry, and hematological analysis as shown in Figures S3−S5. Next, we explored them as radiosensitizers and HIF-1α siRNA nanocarriers for synergistic F


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Figure 5. In vivo antitumor efficacy of BEL-7402 tumor bearing nude mice after sample administrations. (a) Relative tumor volume curves of different groups of mice after different treatments: (i) PBS injection; (ii) GdW10@CS solution i.t. injection; (iii) X-ray only; (iv) GdW10@ CSsiRNA solution; (v) GdW10@CS+X-ray; (vi) GdW10@CSsiRNA+X-ray. Tumor weights (b) and photos (c) of different groups of mice after different treatments. (d) Body weights of different groups of BEL-7402 tumor bearing nude mice after various administrations. (e) Representative H&E stained images and immunohistochemical analysis of tumor slices collected from various groups of mice. Error bars were calculated by SD of three parallel samples. P values were based on the Student’s test: *P < 0.05, **P < 0.01. G


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Figure 6. Western blot analysis of various proteins in tumors after different treatments. (a) HIF-1α level in tumor after different treatments: (i) PBS injection; (ii) GdW10@CS solution i.t. injection; (iii) X-ray only; (iv) GdW10@CSsiRNA solution; (v) GdW10@CS+X-ray; (vi) GdW10@ CSsiRNA+X-ray. (b) γ-H2AX expression in tumor after different treatments. (c) VEGF expression level in tumor following different treatments. (d) c-Met expression levels in tumor as determined by Western blot analysis.

regulating HIF-1α expression to inhibit broken DNA selfhealing, the clonogenic survival assay was conducted to evaluate the radiosensitization efficacy of GdW10@CS nanospheres under hypoxia. As described in Figure 4a and b, the colony formation assay indicated that hypoxic BEL-7402 cells treated with GdW10@CS nanospheres could significantly enhance the RT efficacy, where the enhanced efficacy was more obvious after introducing HIF-1α siRNA at the same GdW10@CS concentration (100 μg mL−1). Additionally, we found that the radiosensitization performance of GdW10@CS nanospheres was dose-dependent (Figure 4c and d) and the sensitizer enhancement ratio, as a method to measure the capacity of a radiosensitizer to inhibit cancer cell proliferation, was also calculated (Figure S7). Next, we continued to explore the

radiotherapy and gene therapy (GT) under hypoxia with an annexin V-FITC/PI method. As shown in Figures 3c and S6, after simultaneous treatment with siRNA and X-rays, both cell types under hypoxic conditions exhibited more significant cell late apoptosis/necrosis (33.58%, 28.83%) in comparison to GT alone (10.92%, 14.37%) or RT alone (14.37%, 15.91%), respectively, indicating the obvious synergistic efficacy of GT and RT under hypoxia in vitro (Figure 3d). Therefore, it is suggested that GdW10@CS nanospheres have a tremendous potential to be a prominent radiosensitizer for synergistic GT and RT. Meanwhile, to substantiate whether GdW10@CS nanospheres can induce DNA double-strand damage by producing ROS and delivering HIF-1α siRNA to hypoxic cells for downH


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and CT imaging (Figures S12 and S13), for real-time monitoring and precise diagnosis and treatment of cancer. Finally, in order to correlate the observable therapeutic efficacy in vivo with these proteins’ relative expression, Western blot analysis was performed to quantify these protein expression levels. As indicated in Figure 6a and b, the expression levels of HIF-1α and γ-H2AX were similar to the immunohistochemical analysis in vivo, indicating the efficient transfection, splendid silencing effect of HIF-1α siRNA, and the efficiently enhanced radiosensitization of GdW10@CS nanospheres to radiotherapy. Furthermore, the expression levels of VEGF and c-Met, as the two main hallmarks in tumor proliferation responsible for angiogenesis and invasion, are also discussed. Similarly, the expression levels of VEGF and cMet treated with GdW10@CS+X-ray were significantly upregulated but were remarkably decreased after loading HIF-1α siRNA (Figure 6c and d), demonstrating the hypothesis that the inhibition of tumor angiogenesis and metastasis was related to HIF-1α silencing. All these results suggested that GdW10@ CS nanospheres can enhance the antitumor effect of radiotherapy.

damage of double-stranded DNA immunofluorescently labeled by γ-H2AX, a marker of double-stranded DNA breakage. The results showed that obviously detectable γ-H2AX immunofluorescent spots were discovered in the GdW10@CS group in the presence of X-rays (6 Gy), especially the cells incubated with GdW10@CSsiRNA (Figure 4e and f), and similar results were also found in hypoxic HeLa cells (Figure S8). Therefore, all these results demonstrated the radiosensitization capacity of GdW10@CS nanospheres through depositing a massive radiation dose and effectively loading HIF-1α siRNA for down-regulating HIF-1α expression to inhibit DNA self-repair, so as to generate more DNA damage and inhibit cell proliferation. Inspired by the encouraging results in vitro, we further investigated the potential enhanced radiosensitization of GdW10@CS nanospheres for radiotherapy in vivo. GdW10@ CS with or without HIF-1α siRNA was intratumorally injected every 2 days for 25 days. The kinetics of the tumor growth was monitored using a caliper. As indicated in Figures 5a and S9, sharp tumor growth suppression was observed and no relapse was found in the GdW10@CSsiRNA+X-ray group during the experimental period, demonstrating the prominently enhanced radiosensitization of GdW10@CS nanospheres to radiotherapy in our predicted mouse model. Meanwhile, we found the GdW10@CS nanospheres could be gradually cleared out from the body (Figure S10), thereby sharply reducing the potential side effects. The tumor weight and sizes were also measured at day 25. As shown in Figure 5b and c, the tumor weight and sizes were in accordance with the tumor volumes, and no obvious difference in weight was discovered in each group of mice (Figure 5d), validating no significant systemic toxicity of the GdW10@CS nanospheres. Next, to further explore the reason for the suppression of tumor growth, representative hematoxylin and eosin (H&E) staining and immunohistochemical analysis were carried out (Figure 5e). H&E staining images showed that the GdW10@CSsiRNA+X-ray group had the most necrosis of tumors cells, and no metastasis in the liver and lung tissues was discovered (Figure S11), suggesting the proliferative activity of tumors was massively reduced by the enhanced radiosensitization performance of GdW10@CS nanospheres upon radiotherapy. In addition, representative immunohistochemistry photomicrographs were employed to evaluate the expression levels of γ-H2AX, HIF-1α, VEGF, and CD31. As indicated in Figure 5e, the highest expression level of γ-H2AX was observed in the GdW10@CSsiRNA+X-ray group, which was consistent with the in vitro results. Next, the HIF-1α staining assay showed that the brown color of the tumor cells was sharply reduced after being treated with HIF-1α siRNA, especially in the GdW10@CSsiRNA+X-ray group, suggesting the effective knockdown of HIF-1α expression by HIF-1α siRNA. Moreover, it is worth noting that the expression level of HIF-1α significantly increased after GdW10@CS+X-ray treatment. This phenomenon may be ascribed to the nuclear accumulation of HIF-1α under a decrease of the oxygen pressure after X-ray illumination. Accordingly, the quantitative analysis of VEGF and CD31, the two downstream molecules of HIF-1α, was also in accordance with HIF-1α expression. Therefore, GdW10@CS nanospheres could greatly improve radiotherapy efficiency by incorporating GdW10@CS as an external radiosensitizer and loading HIF-1α siRNA as an internal sensitization strategy to simultaneously overcome radioresistance. In addition to the treatment efficacy, GdW10@CS nanospheres with high-Z metals could also act as a dual-modal imaging contrast agent for MR

CONCLUSION In conclusion, we successfully designed and synthesized a GdW10@CS nanosphere via a simple ionotropic gelation technique for enhanced radiosensitization to radiotherapy in hypoxic tumors. GdW10@CS nanospheres can be a radiosensitizer as an external radiosensitization way to deposit radiation dosage and obliterate the intracellular GSH for more effective ROS generation. Moreover, the GdW10@CS nanospheres also have the capacity to enhance radiosensitization and therapeutic efficacy of radiotherapy through an internal radiosensitization strategy by delivering HIF-1α siRNA into hypoxic tumors to enable significant HIF-1α silencing, so as to reduce radioresistance and inhibit broken DNA restoration. Therefore, GdW10@CS nanospheres can synergistically enhance radiosensitization efficacy of radiotherapy by integrating the X-ray-triggered ROS generation and the depletion of the GSH level by synergy W6+-triggered GSH oxidation, as well as suppressing the double-stranded DNA repair. In brief, the GdW10@CS nanospheres provide a promising radiosensitizer strategy by simultaneously taking advantage of GdW10@CS as an external radiosensitization means and HIF-1α siRNA as an internal stimulation method to inhibit double-stranded DNA repair to realize a radiosensitization effect of radiotherapy. METHODS Materials. All reagents were employed as received without any modification. Na2WO4·2H2O and GdCl6·6H2O were supplied by Alfa Aesar Reagent Company. Chitosan was purchased from J&K Chemical Ltd. Acetic acid was obtained from Beijing Chemical Works. Glutathione was obtained from Beyotime Company. Ellman’s reagent, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), was supplied by Alfa Aesar Company. DMEM and Roswell Park Memorial Institute 1640 (RPMI-1640) were obtained from HyClone, USA. CCK-8 was supplied by Boster Co., Ltd. (Wuhan, China). Fetal bovine serum was purchased from Gibco BRL (Grand Island, NY, USA). DCFH-DA and the GSH/GSSH assay kit were obtained from Beyotime Biotechnology. Annexin V-FITC apoptosis detection kit and whole cell lysis assay kit were purchased KeyGEN BioTECH Co., Ltd. (Nanjing, China). The siRNA duplexes of HIF-1α (siRNA HIF-1α) (sense: 5′-UUCUCCGAACGUGUCACGUTT-3; antisense: 5′ACGUGACACGUUCGGAGAATT-3′) were provided by GenePharm Co. Ltd. (Shanghai, China). BeyoECL Plus, VEGF antibody, I


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ACS Nano and β-actin antibody were obtained from Beyotine Institute of Biotechnology Company (Nanjing, China). HIF-1a antibody and γH2AX antibody were purchased from Proteintech Group Inc. Co., Ltd. (Wuhan, China). C-met antibody and anti-CD31 antibody were supplied by Cell Signaling Technology Company (USA) and Abcam Company (USA), respectively. Purified water was used throughout. Synthesis of GdW10O36. Na9[Gd(W5O18)2]·xH2O was prepared via a previously described method.63 Briefly, Na2WO4·2H2O (8.3 g, 25 mmol) was dissolved in 20 mL of purified water set in a 50 mL beaker to form a uniform and transparent solution. Then, acetic acid was added to the aforementioned solution dropwise to adjust the pH to 7.4−7.5 under vigorous magnetic stirring at room temperature. Subsequently, 2 mL of GdCl6·6H2O (477.95 g/mol, 2.5 mmol) aqueous solution was added to the aforementioned solution dropwise, and a white precipitate was generated immediately. Then, the beaker with the white precipitate was set on the heater and heated to 85 °C to form a homogeneous solution under continuous stirring. Finally, crude crystals were separated out after cooling at room temperature, and the unreactive ions were dialyzed with a dialysis tube (molecular cutoff: 2.0 kDa). Preparation of Chitosan/GdW10O36 (GdW10@CS) Nanospheres. To make the GdW10O36 a gene delivery nanocarrier for the nanomedicine system, chitosan was dissolved in 1% acetic acid and GdW10O36 was dispersed in 500 μL of purified water. Then the GdW10O36 solution was added to the chitosan solution dropwise to form a stable colloidal suspension under continuous stirring, in which the mass ratio of chitosan to GdW10O36 was calculated to be 10. Subsequently, chitosan-GdW10O36 nanospheres (GdW10@CS) were separated by centrifugation for 30 min at 12 000 rpm. The obtained GdW10@CS nanospheres were redispersed in purified water and dialyzed (MWCO: 2.0 kDa) against water for 24 h to remove free ions. Finally the GdW10@CS nanosphere solution was set at 4 °C for further experiment. Agarose Gel Electrophoresis of GdW10@CSsiRNA Nanospheres. To form GdW10@CSsiRNA nanospheres, different concentrations of GdW10@CS nanospheres and amounts of siRNA were mixed in nuclease-free water at the designated N/P ratios (N/P = 0.7, 1.4, 2.8, 5.6, 7.0, 14.0) and then gently shaken and maintained still for 30 min at room temperature. To assess the release capacity of siRNA from GdW10@CSsiRNA nanospheres, heparin was co-incubated with GdW10@CSsiRNA nanospheres for 20 min at room temperature with the weight ratio 1:5 of siRNA/heparin. To evaluate the protection of siRNA by GdW10@CS, 2 μL of RNase (1 μg/μL) was applied to digest 1 μg of siRNA formulated in the GdW10@CS nanospheres at 37 °C for 1 h. Free siRNA was used as a control group. Finally, a 1% agarose gel electrophoresis assay was used to determine the loading efficiency, release of siRNA, and protection of siRNA by GdW10@CS. The release rate of siRNA was calculated to be 84.93%. Glutathione (γ-L-Glutamyl-L-Cysteinyl-Glycine) Oxidation Examination by Ellman’s Assay. First, 225 μL of GdW10@CS nanospheres (100 μg/mL) in 50 mM bicarbonate buffer (pH 8.6) was incubated with 225 μL of GSH (0.8 mM in the bicarbonate buffer) to incur an oxidation reaction in a microcentrifugal tube with an alumina foil covering to prevent light interference. Then the mixture was set in a shaker with a speed of 150 rpm at room temperature for a 4 h incubation. After the reaction, 785 μL of 0.05 M Tris-HCl and 15 μL of 100 mM DTNB were added into the mixture, respectively, and shaken at the same speed for 5 min. After that, a microplate reader (Thermo Scientific, Multiscan MNK3) was employed to measure their absorbance at 412 nm. Bicarbonate buffer (50.0 mM at pH 8.6) and H2O2 (1.0 mM) in the absence of GdW10@CS nanospheres were used as negative and positive controls in the GSH oxidation experiments, respectively. Detection of Reactive Oxygen Species Produced by GdW10@CS Nanospheres under X-ray Irradiation. First, 0.5 mL of DCFH-DA in DMSO was mixed with 2 mL of NaOH (0.01 M) in the dark at room temperature to chemically hydrolyze to DCFH. After 30 min, 10 mL of the phosphate buffer (PBS, 25 mM, pH 7.2) was added to stop the reaction, and the formed DCFH solution was covered by aluminum foil and set on ice for subsequent experiment.

Subsequently, 100 μg/mL of GdW10@CS nanospheres was mixed with the DCFH (10 μM) solution in the absence or presence of GSH (1 mM) and then immediately irradiated with X-rays for 10 min. After that, the fluorescence of the solution was measured for the evaluation of the generated ROS. Determination of the GSH/GSSG Ratio. Colorimetric microplate assay kits were used to measure the total glutathione and oxidative glutathione. For a simple reaction, GdW10@CS nanospheres (100 μg/mL) were mixed with GSH (1 mM) for 10 min under shaking. Then, the solution mixture was employed for GSH and GSSG assays according to the instructions. Reduced GSH was calculated as the difference between total GSH and GSSG, and GSH/GSSG was determined. In Vitro Cytotoxicity Assay under Hypoxia. The cytotoxicity of GdW10@CS nanospheres to BEL-7402 cells and HeLa cells under normoxia and hypoxia was assessed by using the CCK-8 assay. BEL7402 cells and HeLa cells were maintained in a 96-well plate for 24 h at a density of 4 × 103 cells per well, respectively. Then the cells were further incubated with 100 μL of medium or medium containing 100 μM of the hypoxia-mimic cobalt chloride (CoCl2) with different concentrations of GdW10@CS nanospheres ranging from 6.25 to 100 μg/mL. After incubation for 24 h, 10 μL of CCK-8 was added and further incubated for 1 h. After that, the absorbance of the sample solution was measured at 450 nm using a microplate reader (Thermo Scientific, Multiscan MNK3). Intracellular Internalization of HIF-1α siRNA Delivered by GdW10@CS Nanospheres and Sensitization to X-rays after Inhibition of HIF-1α Expression. Before the experiment, BEL-7402 and HeLa cells were cultured in a 24-well plate at a density of 5 × 104 cells per well. Each well was treated differently (DMEM, GdW10@CS nanospheres, X-ray, GdW10@CSsiRNA, GdW10@CS+X-ray, GdW10@ CSsiRNA+X-ray). After incubation for 24 h, cells were washed three times with PBS (pH 7.4) and then collected by trypsinization and centrifugation. Finally, cells were resuspended in 200 μL of PBS for flow cytometric analysis using a Becton Dickinson FACSCalibur flow cytometer (Bedford, MA, USA). For each experiment, 10 000 cells were employed and analyzed using WinMDI 2.9 software. In Vitro Colony Formation Assays and DNA Double-Strand Breaks under Hypoxia. For clonogenic assays, hypoxic BEL-7402 and HeLa cells of different numbers (125, 250, 500, 1000, 2000 cells) were seeded in a 24-well plate with complete medium containing 100 μM of the hypoxia-mimic CoCl2 for 48 h. After attachment, the hypoxic cells were treated with or without siRNA or/and X-ray irradiation. The plates with different numbers of cells were treated with different doses of radiation, respectively (0, 2, 4, 6, and 8 Gy). After different treatments, the cells were further cultured for another 10 days and stained with Giemsa dye. To further test DNA double-strand breaks, hypoxic BEL-7402 and HeLa cells were incubated in a 35 mm confocal dish at a density of 3 × 104 cells per well for 24 h and divided into six groups (control, GdW10@CS nanospheres, X-ray, GdW10@ CSsiRNA, GdW10@CS+X-ray, and GdW10@CSsiRNA+X-ray). When BEL-7402 and HeLa cells had grown to 30% in the plates, different approaches were employed with or without siRNA and X-ray (6 Gy), respectively. After treatment for 24 h, the cells were fixed with 4% paraformaldehyde for 10 min. Then Triton X-100 was used to penetrate the cells, and bovine serum albumin was used to prevent other nonspecific protein interactions. Finally, the cells were incubated with γ-H2AX antibody overnight at 4 °C, and the secondary antibody Cy3 tag goat anti-rabbit IgG (H+L) was added for 1 h. Hoechst was used to stain the cell nuclei. 2′,7′-Dichlorofluorescin Diacetate Microscopy. To test the reactive oxygen species induced by GdW10@CS after illuminating the X-ray tube, DCFH-DA (Beyotine Institute of Biotechnology Company) was used to detect the oxidative species in BEL-7402 cells under hypoxia. After reacting with oxidizing species, DCFH-DA is oxidized to fluorescent 2′,7′-dichlorofluorescin (DCFH) to generate the green fluorescence. For fluorescence imaging, cells were seeded in a confocal dish with complete medium containing 100 μM of the hypoxia-mimic CoCl2 for 24 h. After attachment, the cells were treated with or without GdW10 or/and X-ray exposure. The concentration of J


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ACS Nano GdW10 was 100 μM, and the dose of X-rays was 2 Gy. After treatment for 4 h, the cells were washed with PBS and then stained with the solution mixture of Hoechst 33342 and DCFH-DA for 15 min without light interference. After staining, PBS was replaced and then exposed to an X-ray tube for 10 min. Finally, the confocal dishes with cells were immediately imaged under an inverted luminescence microscope (Olympus X-73, Japan). MR and CT Imaging in Vitro. To detect the linearity of the MRI signal as a function of GdW10@CS nanosphere concentration, different concentrations (0, 6.25, 12.5, 25.0, and 50 mg/mL) of GdW10@CS nanospheres were dispersed in purified water and imaged on the 4.7 T MR imaging instrument (Biospec; Bruker; Ettlingen, Germany) with the following parameters: matrix size, 128 × 128; field of view, 40 × 40 mm; and slice thickness, 1.20 mm. In addition, to test the CT imaging capacity of GdW10@CS nanospheres, a series of sample solution concentrations and commercially used iopromide (0, 6.25, 12.5, 25.0, and 50 mg/mL) were set in the 1.5 mL microcentrifugal tubes for detecting the CT signal in vitro, respectively. Hounsfield unit values and CT images were obtained by the Triumph TM X-O TM CT system and Gamma Medica-Ideas instrument with the following parameters: effective field of view, 1024 pixels × 1024 pixels; effective pixel size, 80 kV, 50 μm, 270 μA. MR and CT Imaging in Vivo. To obtain MR imaging in vivo, BEL7402 tumor-bearing BALB/c nude mice were intravenously (i.v.) injected with GdW10@CS nanospheres (15 mg/mL, 200 μL) and then imaged on the 4.7 T MR imaging instrument (Biospec; Bruker; Ettlingen, Germany). The MR images were acquired with a microgel system (America). To obtain CT images, CT imaging in vivo was immediately accomplished on the animal X-ray CT instrument (Gamma Medica-Ideas) after BEL-7402 tumor-bearing BALB/c nude mice were i.v. injected with GdW10@CS nanospheres (25 mg/ mL, 200 μL). Finally a Triumph X-O CT system was used and threedimensional CT images were obtained. BEL-7402 Tumor Model and GT/RT Synergistic Treatment in Vivo. Thirty BALB/c nude male mice were supplied by Beijing Vital River Laboratory Animal Technology Co., Ltd. and were subcutaneously inoculated with 1.0 × 106 BEL-7402 cells suspended in 100 μL of DMEM. After 10 days, the BEL-7402 tumors reached approximately 100 mm3, and then these mice were randomly divided into six groups: (i) PBS only; (ii) GdW10@CS nanospheres only; (iii) X-ray only; (iv) GdW10@CSsiRNA only; (v) GdW10@CS + RT; (vi) GdW10@ CSsiRNA+RT. For group i, the mice were only intratumorally (i.t.) injected with 20 μL of PBS. Then the mice in groups ii and v were i.t. injected with 20 μL of GdW10@CS nanospheres, and the mice in groups iv and vi were i.t. injected with 20 μL of GdW10@CSsiRNA, respectively. After administration, the BEL-7402 tumor-bearing nude mice in groups iii, v, and vi received X-ray administration (10 Gy) under hypoxic conditions, and 20 μL of GdW10@CSsiRNA was i.t. injected every other 2 days to keep the siRNA activity in the tumor to achieve better therapy efficacy. Subsequently, tumor growth was recorded by measuring the tumor’s perpendicular diameter using a caliper estimated by employing the following equation: tumor volume V = ab2/2, where a is length and b is width; body weight was also monitored. After 1 day of treatment, one mouse in each group was sacrificed and tumors were collected for protein extraction and immunohistochemistry analysis. The tumors in the remaining four mice in each group continually grew until the end of the experiment. Western Blot Analysis and Immunohistochemical Staining Analyses. All proteins were collected from the tumors after different treatments and were then analyzed by a Western blot apparatus (Azure Biosystems, C300, USA). To obtain the immunohistochemical staining images, the collected tumors were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. For antigen retrieval, the obtained sections after deparaffinizion were set in a pressure cooker containing 10 mM citrate buffer (pH 6.0) for pretration for 5 min. Subsequently, after blocking in 10% serum of the secondary antibody species, 3% H2O2 in PBS was used to quench the tissue peroxidase activity for 10 min. Finally, immunohistochemistry was accomplished with γ-H2AX, HIF-1α, VEGF, and CD31, respectively. All the staining

images were observed by an inverted luminescence microscope (Olympus X73, Japan) in randomly selected fields (40× objective). Characterizations. TEM (Tecnai G2 20 S-TWIN, FEI, USA) was used to acquire the size and morphology of the samples. The size distribution and ζ-potential of the as-prepared samples were measured by a Nicomp380 ZLS Plus ZETADi Fourier transform Bruker EQUINOX55 spectrometer (EQUINOX55, Bruker, Genmany), and the KBr pellet technique was employed to obtain the FT-IR spectra. The thermogravimetry spectra were acquired on a Diamond TG/DTA (Perkin Elmer). Agarose gel electrophoresis and Western blot analysis of samples were performed by an Azure Biosystems apparatus (C300, USA). To assess the cell cytotoxicity, a microplate reader (Thermo Multiskan MK3, Molecular Devices, Sunnyvale, CA, USA) was adopted at an optical absorbance of 450 nm. The flow cytometry assay was accomplished on a Becton Dickinson FACSCalibur flow cytometer (Bedford, MA, USA). The luminescence microscopy images were accomplished with an inverted luminescence microscope (Olympus X-73, Japan).

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.7b03037. EDS spectra of GdW10@CS nanospheres, DLS assessments of GdW10@CS nanospheres dispersed in DMEM, H&E-stained images of main organs, blood chemistry analysis and hematological parameters of mice as a function of time, hematological parameters of mice after various administrations, in vitro cytotoxicity assay of GdW10@CSsiRNA and CoCl2 under hypoxia, sensitizer enhancement ratio of GdW10@CS and GdW10@CSsiRNA, in vitro colony formation assay of GdW10@CS nanospheres in HeLa cells under hypoxia, tumor images of different mice, biodistribution of GdW10@CS nanospheres in vivo, H&E-stained images of main organs under different treatments, MR and CT imaging in vitro, MR and CT imaging in vivo (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: *E-mail: *E-mail: *E-mail:

[email protected]. [email protected]. [email protected]. [email protected].

ORCID

Zhanjun Gu: 0000-0003-3717-2423 Xiangfeng Liu: 0000-0001-9633-7721 Yuliang Zhao: 0000-0002-9586-9360 Author Contributions ⊥

Y. Yong and C. Zhang contributed equally.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Basic Research Programs of China (973 Programs, Nos. 2016YFA0201600, 2014CB931900, and 2015CB932104), National Natural Science Foundation of China (Nos. 31571015, 11621505, 11435002, and 21320102003), and Youth Innovation Promotion Association CAS (2013007). K


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