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X-ray-responsive selenium nanoparticles for enhanced cancer chemo-radiotherapy
Accepted Manuscript Title: X-ray-responsive Selenium Nanoparticles for Enhanced Cancer Chemo-radiotherapy Author: Bo Yu Ting Liu Yanxin Du Zuandi Luo Wenjie Zheng Tianfeng Chen PII: DOI: Reference:
S0927-7765(15)30327-1 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.11.063 COLSUB 7534
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
15-5-2015 5-11-2015 23-11-2015
Please cite this article as: Bo Yu, Ting Liu, Yanxin Du, Zuandi Luo, Wenjie Zheng, Tianfeng Chen, X-ray-responsive Selenium Nanoparticles for Enhanced Cancer Chemo-radiotherapy, Colloids and Surfaces B: Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2015.11.063 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
X-ray-responsive Selenium Nanoparticles for Enhanced Cancer Chemo-radiotherapy Bo Yua#, Ting Liua#, Yanxin Dub, Zuandi Luoa, Wenjie Zhenga, Tianfeng Chena* [email protected] a
Department of Chemistry, Jinan University, Guangzhou 510632, China.
b
Orthopedics Department, Guangdong Provincial Hospital of Traditional Chinese Medicine,
Guangzhou 510120, China *
#
Corresponding author. Tel: +86 20-85225962; Fax: +86 20 85221263.
Authors contributed equally to the work.
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Graphical abstract
X-ray-Responsive Nanomaterials: An X-ray responsive selenium nanoparticles (PEG-SeNPs) were facilely fabricated through PEG-templated synthesis method and identified as synergistic cancer chemoradiation agent for cancers through enhancement of ROS generation, which may provides a novel strategy for design of chemo-radiotherapeutic nanomaterials.
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Highlights
X‐ray responsive PEG‐SeNPs were facilely fabricated through PEG‐templated synthesis method.
The X‐ray responsive property was attributed to its amorphous status.
PEG‐SeNPs sensitizes cancer cells to X‐ray via induction of ROS‐mediated apoptosis.
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Abstract Resistance of cancer to radiotherapy and/or chemotherapy is one of the important reasons of clinical treatment failure and recurrence. Chemoradiation is an optional method to over-coming of radioresistance and chemoresistance. Selenium nanoparticles (SeNPs) with special chemical and physical properties, has been identified as a novel nanocarrier and therapy agent with broad-spectrum anticancer activities due to generate ROS in cells. Herein, X-ray responsive selenium nanoparticles were facilely fabricated by using PEG as surface decorator and template. This nanosystem (PEG-SeNPs) demonstrated X-ray responsive property that was attributed to its amorphous characteristic. Interestingly, the nanosystem demonstrated significant radiosensitization effects with X-ray. Specifically, co-treatment of cancer cells with PEG-SeNPs and X-ray significantly and synergistically enhanced the cells growth inhibition through induction of cell apoptosis, as evidenced by DNA fragmentation and activation of caspase-3. In the cell model, we found that internalized nanoparticles could degrade upon X-ray exposure, which further confirm the X-ray responsive property of the nanoparticles. Moreover, the nanosystem could significantly induced intracellular ROS generation in a time-dependent manner, which peaked at about 40 min and gradually decreased thereafter. As a results, ROS overproduction led to mitochondria fragmentation and the cell apoptosis. Taken together, this study provides a novel strategy for rational design and facile synthesis of chemo-radio therapeutic radiosensitization nanomaterials.
Keywords: X-ray-responsive; selenium nanoparticles; radiosensitization; radiotherapy
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1. Introduction Radiotherapy has been rapidly developed and widely used in clinic in the past decade.[1] Studies have shown that approximately 50% cancer patients receive radiation during the therapy process.[2] Unfortunately, the resistance of cancer cells to radiation has hindered the efficacy of radiotherapy.[3, 4] Chemoradiotherapy, a strategy that combines chemotherapy with radiation therapy, has been increasingly used in cancer treatment.[5] However, chemoradiotherapy is sometimes accompanied with severe side effects together with their limited spectrum of activity.[6] Therefore, the search for cancer-targeting therapeutic agents that could overcome the resistance of cancer cells to chemoradiation has kindled great interest of scientists.[7] The main challenge remains for targeting strategies is to reduce or avoid toxicity towards normal tissues.[8-10] Recent studies have discovered cell receptor targeting molecules, such as folic acid and functional peptides that could improve the selective cellular uptake of drugs in cancer cells.[11] Comparing with other conventional cancer treatment approaches, such as surgery and chemotherapy, radiosensitization therapy has attracted much interest of scientists for its minimally invasive property and its advantage for therapeutic intervention of specific biological targets.[12-14] X-ray is a clinical method of great benefit in terms of local control, and could target different anatomic tumor sites inside human body. Nowadays, X-ray-based radiotherapy represents one of the most effective ways for treatment of cancers. However, radioresistance and severe side effect remain a challenging issue. Cancer nanotechnology is an emerging technique that displays application potential in cancer-targeted chemotherapy, molecular diagnosis and molecular imaging.[15, 16] Nanodrug 5
delivery systems have been found able to improve the cellular targeting of anticancer drugs and exhibited radiosensitization activities.[12, 17] Townley et al. demonstrated intratumoural injection of rare earth elements doped titania nanoparticles can enhance the efficacy of radiotherapy in cancer therapy.[18] And Cook et al. reported that, gold nanoparticle-based X-ray absorbing agents exhibit both chemo-therapeutic potency to cancer cells and Auger-mediated secondary electron emission, showing great potential to improve the therapeutic efficacy of chemo-radiation.[19] Thus, the search for chemoradiation nanomaterials to kill cancer cells but avoid damage of normal tissues has kindled great interest of scientists. Selenium (Se) is a trace element with novel piezoelectricity, photoconductivity, thermoelectricity, and nonlinear optical responses.[20, 21] The role of Se in cancer chemoprevention and chemotherapy has been supported by many epidemiological, preclinical and clinical studies. Se nanomaterials are novel Se species with broad applications in optoelectronics devices and cancer therapy.[22] In medicine, SeNPs attracted more and more attention in the past decade, due to their high bioavailability, low toxicity and novel therapeutic properties.[23] Recently, we reported the use of SeNPs as carriers of pharmaceutical agents such as to enhance their anticancer outcome,[24] and also clarified ROS-mediated apoptosis mechanisms for the action of SeNPs in detail.[23, 25] Recent studies have demonstrated that Se showed promise as an agent to reduce the harmful side-effects of radiation, but not compromise the effectiveness of treatments.[26] Similarly, our studies also found that selenocompounds could effectively enhance the anticancer efficacy of X-ray through activation of diversified ROS-mediated signaling pathways.[27, 28] Base on the 6
interesting physical and biochemical characteristics and therapeutic advantages of SeNPs, we hypothesized that SeNPs under X-ray irradiation could lead to enhanced ROS generation and result in synergistic anticancer efficacy. Therefore, in this study, X-ray responsive selenium nanoparticles were facilely fabricated by using PEG as surface decorator and template. The X-ray responsive property of PEG-SeNPs was attributed to its amorphous characteristics. Interestingly, the nanosystem demonstrated significant radiosensitization effects with X-ray. Specifically, co-treatment of cancer cells with PEG-SeNPs and X-ray significantly and synergistically enhanced the cells growth inhibition through induction of cell apoptosis, as evidenced by DNA fragmentation and activation of caspase-3. In the cell model, we found that internalized nanoparticles could degrade upon X-ray exposure, which further confirm the X-ray responsive property of the nanoparticles. Moreover, the nanosystem could significantly induce intracellular ROS generation in a time-dependent manner, which peaked at about 40 min and gradually decreased thereafter. As a result, ROS overproduction led to mitochondria fragmentation and the cell apoptosis. Taken together, this study provides a novel strategy for rational design and facile synthesis of chemo-radio therapeutic radiosensitization nanomaterials.
2. Experimental section Reagents Selenium dioxide, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), pro-pidium iodide (PI), solid JC-1, 2’, 7’-dichlorofluorescein diacetate (DCF-DA) and ascorbic acid were purchased from Sigma-Aldrich Chemical Co. Gray selenium powder was 7
purchased from Guangzhou Dinggang Chemical Factory (Guangzhou, China). Reagent grade polyvinyl-pyrrolidone (PVP K-30) and PEG600 were purchased from Guangdong Chemical Technology Research Center (Guangzhou, China). The water used for all experiments was ultrapure, supplied by a Milli-Q water purification system from Millipore. All of the solvents used were of HPLC grade.
Preparation and characterization of SeNPs Firstly, PEG-SeNPs were prepared as previously described with tiny change.[29] Under N2 protection, dissolving 100 mg gray Se powder in 10 mL PEG600 solution at 220 °C–230 °C for 15–20 minutes, with vigorously magnetic stirring. Then, PEG-SeNPs was cooled to -20 °C. PEGW-SeNPs refers to PEG-SeNPs that were prepared in water solution. PVP-SeNPs were prepared as below description, respectively. As a typical procedure, varied volume of PEG600 or PVP water solution was added dropwise into 1 ml of 125 M H2SeO3 solution under magnetic stirring, and then 5 mL of 100 mM ascorbic acid solution was added into the mixture and then it was reconstituted to a final volume of 25 mL with Milli-Q water. The final concentration of Se was 5 mM. Three types of SeNPs were collected by centrifuge at 12000/min for 30 min and were dialyzed against Milli-Q water until no Se was detected in the outer solution by ICP-AES analysis. The shapes of as-prepared products were characterized by using microscopic and spectroscopic methods. Briefly, TEM samples were prepared by dispersing the powder particles onto holey carbon film on copper grids. The micrographs were obtained on Hitachi (H-7650) for TEM operated at an accelerating voltage at 80 kV. And the Philips TECNAI 20 8
high-resolution transmission electron microscopy (HRTEM) worked at 400 kV. SEM-EDX analysis was carried out on an EX-250 system (Horiba) and employed to examine the elemental composition of PEG-SeNPs. The obtained samples were characterized by X-ray diffraction (XRD) performed on a MSAL-XD2 X-ray diffractometer with a Cu target in the 2θ range from 5° to 80° (40 kV, 30 mA).
Determination of Se Se concentration was determined by ICP-AES method.[30] Briefly, the sample was digested with 3 ml concentrated nitric acid and 1 ml H2O2 in a digestive stove (Qian Jian Measuring Instrument Co., Ltd., China) at 180
for 3 h. The digested product was reconstituted to 10 ml
with Milli-Q H2O and used for ICP-AES analysis.
Cell Lines and Cell Culture Human cancer cell lines HeLa cervical cancer cells and mouse embryonic fibroblast NIH3T3 were purchased from American Type Culture Collection (ATCC, Manassas, VA). All cell lines were maintained in DMEM media supplemented with fetal bovine serum (10%), penicillin (100 units/mL) and strep-tomycin (50 units/ml) at 37
in CO2 incubator (95%
relative humidity, 5% CO2).
MTT Assay Cell viability was determined by measuring the ability of cells to transform MTT to a purple formazan dye.[31] Hela and NIH3T3 cells were seeded in 96-well tissue culture plates at 9
2.5×103 cells/well for 24 h. The cells were then incubated with SeNPs at different concentrations for different periods of time. After incubation, 20 μL/well of MTT solution (5 mg/ml in PBS) was added and incubated for 5 h. The medium was aspirated and replaced with DMSO 150 μL/well. The color intensity of the formazan solution, which reflects the cell growth condition, was measured at 570 nm using a microplate spectrophotometer (Versamax).
Flow Cytometric Analysis The cell cycle distribution was analyzed by flow cytometry as previously described..[32] After treatment with PEG-SeNPs, the cells were trypsinized, washed with PBS and fixed with 70% ethanol overnight at -20 ℃. The fixed cells were washed with PBS and stained with PI working solution (1.21 mg/mL Tris, 700 U/ml RNase, 50.1 μg/mL PI, pH 8.0) for 4 h in darkness. The stained cells were analyzed with Epics XL-MCL flow cytometer (Beckman Coulter, Miami, FL). Cell cycle distribution was analyzed using MultiCycle software (Phoenix Flow Systems, San Diego, CA). The proportion of cells in G0/G1, S and G2/M phases was represented as DNA histograms. Apoptotic cells with hypodiploid DNA content were measured by quantifying the sub-G1 peak in the cell cycle pattern. For each experiment, 10,000 events per sample were recorded.
Intracellular Localization of PEG-SeNPs The intracellular localization of PEG-SeNPs in HeLa cells was traced with 6-coumarin-labeled method as previously described.[31] Briefly, the cells cultured on cover 10
glass in 6-well plates till 70% confluence were stained with 1 μg/mL Hochest 33258 for 20 min. After washing with PBS twice, the cells were incubated with different concentrations of 6-coumarin-labeled PEG-SeNPs for various periods of time and examined under a fluorescence microscope (Nikon Eclipse 230 80i).
Mitochondrial mass assay Mitochondrial mass measured by staining with MitoTracker green (Invitrogen, Carlsbad, CA, USA) was performed as described.[33] Cells were incubated with 50 nM MitoTracker Green (30 min) and 10 μM Hoechst 33258 (10 min) to visualize mitochondria and nuclei, respectively. Then, the loading solution was removed with PBS, the cell monolayer were washed three times with ice-cold PBS and examined by confocal laser scanning microscopy (Zeiss LSM 510 Meta laser scanning confocal microscope systems).
3. Results and Discussion 3.1 Rational design, preparation and characterization of SeNPs Three different types of selenium nanoparticles used in this study were prepared by liquid phase reaction methods. Representative TEM images of the as-made PEG-SeNPs, PEGW-SeNPs and PVP-SeNPs were shown in Fig. 1a-c. It was clearly revealed that the overall morphology of SeNPs adopted a ball-like shape with mean size was at about 500, 500 and 200 nm, respectively. We also characterized the structure of PEG-SeNPs, PEGW-SeNPs and PVP-SeNPs by FT-IR. As shown in Fig. S1a, the peak at 1110 cm-1 in PEG-SeNPs and PEGW-SeNPs could be attributed to the bending vibration of C-O-C of PEG, while the peak 11
at 1740 cm-1 in PVP-SeNPs was assigned to the stretching vibration of O=C-O of PVP. Moreover, the nanomaterials were further characterized by energy-dispersive X-ray (EDX) elemental-mapping analysis. As shown in Fig. S1b, the element mapping of Se and oxygen highly was corresponded to the shape of PEG-SeNPs with some cavities found in the element mapping of Se. The elemental-mapping analysis revealed that element signals of Se, C and O were homogeneously distributed throughout the as-prepared nanoparticles indicating that PEG molecule was incorporated into selenium nanoparticle. The incorporation of PEG eventually leaded to amorphous selenium nano-structure. Our previous work had demonstrated that the preparation of SeNPs in the water solution system could produce the crystal structure.[23] Herein, we observed the same phenomenon that the crystalline core of Se was enclosed by an amorphous coating layer in PEGW-SeNPs and PVP-SeNPs systems (inset images in Fig. 1b-c). Importantly, nanomaterials surfaces can be readily modified with decoration molecular containing functional groups such as thiols, hydroxy, and amines and resulted in special physical/chemical properties[27]. It was reported that hydroxyl group was able to conjugate to Se atom and resulted in surface amorphous structure.[30] Herein, PEG, a polyether compound with hydroxyl groups, was used as the decoration agent. Therefore, possibly, the amorphous core status of PEG-SeNPs could be due to the surface decoration and incorporation of PEG into the nanoparticle. In contrast to PEG-SeNPs, the data analysis confirmed crystalline core structure of PEGW-SeNPs and PVP-SeNPs. Furthermore, TEM was used to characterize the morphological change of different types of SeNPs due to X-ray radiation in cell culture medium. As shown in Fig. 1d-f, smooth and 12
spherical shape of PEG-SeNPs turned to rough and smaller nanoparticles with mean diameter less than 40 nm. In contrast, under the same treatment conditions, PEGW-SeNPs and PVP-SeNPs were not observed this phenomenon. Thus, the structural sensitivity of PEG-SeNPs to X-ray stimulation may be explained by the PEG incorporation in SeNPs. It was also interesting to find that the morphology of PEG-SeNPs changed from a black and round shape to white and empty one in a time-dependent manner under the TEM electron beam stimulation, which suggested that Se atoms in the nanoparticles could evaporate gradually after adsorbing high-energy X-ray (Fig. 2). Unlike PEG-SeNPs showed obvious shape change, PEGW-SeNPs and PVP-SeNPs didn’t display this phenomenon, which was consistent with the result shown in Fig. 1. Taken together, these results indicate the radiation-responsive property of PEG-SeNPs.
3.2 Anticancer efficacy of different nanosystems Studies were also carried out to examine the application potentials of PEG-SeNPs as radiosensitizer. As illustrated in Fig. 3a, X-ray irradiation (8 Gy) or PEG-SeNPs (10 μM) alone induced only slight growth inhibition on HeLa cells, where the cell viability was found at 79%, 71% and 54% in cells treated with 20, 40 and 80 μM PEG-SeNP, respectively. In contrast, co-treatment of the cells with PEG-SeNPs and X-ray significantly enhanced the cells growth inhibition at the same concentration of PEG-SeNP. Especially, co-treatment of 20 μM PEG-SeNP and X-ray significantly suppressed the cell viability to 39%. However, this synergistic effect was not observed in cells which were exposed to PEGW-SeNPs and PVP-SeNPs in combination with X-ray irradiation (Fig. 3b and c). The results of microscopic 13
examination of cells also demonstrated consistent morphological changes after treatment with PEG-SeNPs and X-ray in combination, such as cell shrinkage, rounding, and the appearance of apoptotic bodies (Fig. S2). We have also examined the cytotoxicity of PEG-SeNPs and PEG-SeNPs with X-ray in normal cell lines NIH3T3 by MTT assay. As shown in the Fig 3d, the cell viability still keep high at 85 % after treatment of 10-80 μM PEG-SeNPs with or without X-ray. The combined anticancer effects of them were much lower than those in HeLa cancer cells (Fig. 3a), and no synergistic effects were observed in this normal cell line. Collectively, these results demonstrate the important roles of surface decorators on the anticancer efficacy, radio-sensitization effects and toxicity of SeNPs. The possible reasons for inhibition of cancer cells proliferation could be induction of apoptosis, cell cycle arrest, or both modes.[34, 35] In order to confirm the action mechanisms of cell death induced by X-ray and PEG-SeNPs, we performed DNA flow cytometry analysis to examine the effects of them on the cell cycle distribution. As shown in Fig 4a and b, X-ray alone caused slight G2/M phase arrest from 7.11% (control) to 11.7%. 20 μΜ PEG-SeNPs in combination with X-ray induced an obvious increase in Sub-G1 apoptotic fraction (41.6%) and G2/M phase arrest (21.1%). As shown in Fig S4, 10 μΜ PEG-SeNPs in combination with X-ray induced an obvious increase in Sub-G1 apoptotic fraction (36.8%). The apoptotic cell death was further confirmed by morphological changes, such as cell shrinkage, nuclear condensation and formation of apoptotic bodies (Fig. S3). These results indicate the more important role of cell apoptosis in this anticancer action. Furthermore, we also examined caspase-3 activity induced by PEG-SeNPs with X-ray to verify the induction of apoptosis. As shown in the Fig 5, the effector caspase (caspase-3), was significantly triggered by 14
PEG-SeNPs with X-ray in HeLa cells to 148% of control. These results indicate that apoptosis was the major mode of cell death induced by PEG-SeNPs and X-ray in cancer cells. ICP-AES was used compare the internalization of PEG-SeNPs, PEGW-SeNPs and PVP-SeNPs by cancer cells. As shown in Fig. S5a, the Se concentration in cells treated with PVP-SeNPs was 3 times that of PEG-SeNPs and PEGW-SeNPs groups. These results indicate the important contribution of the surface decorators to the cellular uptake of the nanoparticles in the cancer cells. However, the higher cellular uptake of PVP-SeNPs was not associated with more intensive radiosensitization, which indicate the important roles of decorators in the action of SeNPs. This result suggested that the radiosensitization effects of SeNPs are independent of intracellular Se contents but dependent on SeNPs structure. Further studies were also carried out to elucidate the interaction mechanisms between X-ray and SeNPs. Studies have showed that the photoelectric absorption and secondary electron caused by gamma or X-ray irradiation can produce ROS.[36] ROS plays an important role in regulating cancer cell fate during chemotherapy and radiotherapy. In theory, the photoelectric absorption cross sections of radiosensitive materials directly depend on atom radius.[37, 38] This physical principle determines that Se (atomic-number Z = 34) could be a candidate for radiosensitizer by photoelectric effect. Herein, as shown in Fig. 6a, in cell-free model, pure PEG-SeNPs treated with X-ray irradiation resulted in significant increase in ROS generation, which was much higher than those of PEGW-SeNPs and PVP-SeNPs. Thus, these results indicate that the structural sensitivity of PEG-SeNPs to X-ray stimulation was the fatal fator and led to generate significant amounts of ROS more than those of PEGW-SeNPs and PVP-SeNPs. Future, in cell model, treatment of HeLa cells with PEG-SeNPs under X-ray 15
irradiation also resulted in significant increase in intracellular ROS generation in a time-dependent manner and significantly higher than that of X-ray alone (Fig. S5b). And, the combined treatment induced significant mitochondria fragmentation as probed by MitoTracker Red (Fig. 6b). Thus it was shown that, in cancer cells treated with X-ray alone, the mitochondria recovered after 48 h, while the mitochondrial damage caused by the combined treatment was irreversible (Fig. S5c).
3.3 Intracellular fate of SeNPs in cancer cells The cellular uptake and localization PEG-SeNPs in cancer cells was investigated by live cell fluorescence imaging. As shown in Fig. 7a, PEG-SeNPs entered and accumulated in the cells in a time-dependent manner, which became saturated after 6 h. Interestingly, PEG-SeNPs cluster were also found in the cells (Fig. 7b). Quantitatively, the results of ICP-AES revealed that, intracellular Se contents increased in a time-dependent manner, from 0.03 (control) to 0.33, 0.43 and 0.46 μM/106 cells (Fig. 7c). Taken together, these results demonstrate that PEG-SeNPs enhance the ROS generation induced by X-ray, which may contribute to the synergistic anticancer efficacy through induction of mitochondria fragmentation. Based on above discussion, X-ray irradiation enhances in vitro anticancer activity of PEG-SeNPs but that doesn't depend on cellular uptake of SeNPs. We hypothesized the enhanced anticancer activity of PEG-SeNPs could be due to the X-ray responsive property of the nanoparticles. To confirm this hypothesis, we examined the morphological changes of the nanoparticles inside the cells. As show in Fig. 8, the green fluorescence of PEG-SeNPs 16
decreased drastically after X-ray irradiation. Only weak fluorescence could be observed from the enlarge picture (Fig. S6). It may be due to the fragmentation of PEG-SeNPs in response to X-ray. However, the green fluorescence of PEGW-SeNPs and PVP-SeNPs kept constant after X-ray irradiation (Fig. 9). Moreover, no significant morphological changes were observed in cells treated with PEG-SeNPs and X-ray in combination (Fig. S7). These results further confirmed the X-ray responsive property of PEG-SeNPs.
4. Conclusions Herein, X-ray responsive Se nanoparticles were facilely fabricated by using PEG as surface decorator and template. This nanosystem (PEG-SeNPs) demonstrated X-ray responsive property that was attributed to its amorphous characteristic. Interestingly, the nanosystem demonstrated significant radiosensitization effects with X-ray. Specifically, co-treatment of cancer cells with PEG-SeNPs and X-ray significantly and synergistically enhanced the cells growth inhibition through induction of cell apoptosis, as evidenced by DNA fragmentation and activation of caspase-3. In the cell model, we found that internalized nanoparticles could degrade upon X-ray exposure, which further confirm the X-ray responsive property of the nanoparticles. Moreover, the nanosystem could significantly induced intracellular ROS generation in a time-dependent manner, which peaked at about 40 min and gradually decreased thereafter. As a result, ROS overproduction led to mitochondria fragmentation and the cell apoptosis. Taken together, this study provides a novel strategy for rational design and facile synthesis of chemo-radio therapeutic radiosensitization nanomaterials. 17
Acknowledgements This work was supported by National High Technology Research and Development Program of China (863 Program, SS2014AA020538), Science Foundation for Distinguished Young Scholars (S2013050014667) of Guangdong Province, Natural Science Foundation of China and Guangdong (S2013010012218), Foundation for High-level Talents in Higher Education of Guangdong, YangFan Innovative & Entepreneurial Research Team Project (201312H05), Guangdong Special Support Program and Guangdong Frontier and Key Technological Innovation Special Funds.
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[30] B. Yu, Y. Zhang, W. Zheng, C. Fan, T. Chen, Positive surface charge enhances selective cellular uptake and anticancer efficacy of selenium nanoparticles. Inorg Chem, 51 (2012) 8956-8963. [31] H. Wu, X. Li, W. Liu, T. Chen, Y. Li, W. Zheng, C.W.-Y. Man, M.-K. Wong, K.-H. Wong, Surface decoration of selenium nanoparticles by mushroom polysaccharides-protein complexes to achieve enhanced cellular uptake and antiproliferative activity. J. Mater. Chem., 22 (2012) 9602-9610. [32] Y. Zhang, X. Li, Z. Huang, W. Zheng, C. Fan, T. Chen, Enhancement of cell permeabilization apoptosis-inducing activity of selenium nanoparticles by ATP surface decoration. Nanomedicine, 9 (2013) 74-84. [33] W. Jiang, Y. Fu, F. Yang, Y. Yang, T. Liu, W. Zheng, L. Zeng, T. Chen, Gracilaria lemaneiformis Polysaccharide as Integrin-Targeting Surface Decorator of Selenium Nanoparticles to Achieve Enhanced Anticancer Efficacy. ACS Applied Materials & Interfaces, 6 (2014) 13738-13748. [34] J.A. Kim, C. Åberg, G. de Cárcer, M. Malumbres, A. Salvati, K.A. Dawson, Low Dose of Amino-Modified Nanoparticles Induces Cell Cycle Arrest. ACS Nano, 7 (2013) 7483-7494. [35] A.J. Wirth, M. Platkov, M. Gruebele, Temporal Variation of a Protein Folding Energy Landscape in the Cell. Journal of the American Chemical Society, 135 (2013) 19215-19221. [36] X.D. Zhang, D. Wu, X. Shen, J. Chen, Y.M. Sun, P.X. Liu, X.J. Liang, Size-dependent radiosensitization of PEG-coated gold nanoparticles for cancer radiation therapy. Biomaterials, 33 (2012) 6408-6419. [37] P.D. Liu, Z.H. Huang, Z.W. Chen, R.Z. Xu, H. Wu, F.C. Zang, C.L. Wang, N. Gu, Silver 23
nanoparticles: a novel radiation sensitizer for glioma. Nanoscale, 5 (2013) 11829-11836. [38] X.D. Zhang, J. Chen, Z.T. Luo, D. Wu, X. Shen, S.S. Song, Y.M. Sun, P.X. Liu, J. Zhao, S.D. Huo, S.J. Fan, F.Y. Fan, X.J. Liang, J.P. Xie, Enhanced Tumor Accumulation of Sub-2 nm Gold Nanoclusters for Cancer Radiation Therapy. Adv. Healthc. Mater., 3 (2014) 133-141.
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Figure Captions
Fig. 1 TEM images of (a) PEG-SeNPs, (b) PEGW-SeNPs and (c) PVP-SeNPs. The inset images in b and c demonstrate the lattice fringes of an individual particle by using HRTEM (scar bar, 5 nm). The morphological changes of (d) PEG-SeNPs, (e) PEGW-SeNPs and (f) PVP-SeNPs after X-ray treatment (8 Gy) in cell culture medium.
25
Fig. 2 Morphology change of three different SeNPs under TEM electronic beam stimulation. (a) TEM images of PEG-SeNPs exposed to electron beam irradiation for 5 and 60 s. (b) PEGW-SeNPs. (c) PVP-SeNPs.
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Cell Viability % 25 50 75 100
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Fig. 3 Viability of cells treated with X-ray and (a) PEG-SeNPs, (b) PEGW-SeNPs and (c) PVP-SeNPs in HeLa cells, (d) PEG-SeNPs in NIH3T3 cells as examined by MTT assay. The cells were pretreated with SeNPs for 6 h and then treated with X-ray at 8 Gy, followed by a 12 h-incubation. Each value represents means ± SD (n=3).
27
Fig. 4 (a) The effects of PEG-SeNPs and X-ray on the cell cycle distribution of HeLa cells were examined by flow cytometry analysis. (b) The percent of the cell cycle of HeLa cells treated with 20 μM PEG-SeNPs and X-ray. 28
Caspase activity (% of control)
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Fig. 5 Analysis of caspase-3 activity treated with 20 μM PEG-SeNPs and X-ray-induced apoptosis in HeLa cells. Caspase activities were determined by syntheticfluorogenic substrate. Bars with different characters are statistically different at P<0.05 level. Each value represents means ± SD (n=3). 29
Fig. 6 Induction of ROS-mediated mitochondrial dysfunction. (a) The levels of ROS generated by 20 μM SeNPs and X-ray irradiation were analyzed by DCF assay. (b) The morphological change of mitochondria in cells after treatments with PEG-SeNPs/X-ray irradiation for 12 h. The cells were pre-stained with Hoechst 33342 and Mito-Tracker Red, and observed by laser scanning confocal microscopy (magnification, 400 X).
30
Fig. 7 (a) Time-course internalization of PEG–SeNPs (20 μM, green fluorescence) in HeLa cells. (b) Changes in the intracellular morphology of PEG-SeNPs. (c) ICP-AES quantitative analysis of cellular uptake efficiency of PEG-SeNPs in HeLa cells after different time incubation. 31
Fig. 8 (a) Illustration of the changes of PEG-SeNPs inside the cells, (b) Changes of PEG-SeNPs fluorescence (20 μM, green fluorescence) in HeLa cells after exposure to X-ray (8 Gy) and incubation for another 4 h.
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Fig. 9 Changes of PEGW-SeNPs (a) and PEGW-SeNPs (b) (20 μM, green fluorescence) in HeLa cells after exposure to X-ray (8 Gy) and incubation for another 4 h.
33
S0927-7765(15)30327-1 http://dx.doi.org/doi:10.1016/j.colsurfb.2015.11.063 COLSUB 7534
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
15-5-2015 5-11-2015 23-11-2015
Please cite this article as: Bo Yu, Ting Liu, Yanxin Du, Zuandi Luo, Wenjie Zheng, Tianfeng Chen, X-ray-responsive Selenium Nanoparticles for Enhanced Cancer Chemo-radiotherapy, Colloids and Surfaces B: Biointerfaces http://dx.doi.org/10.1016/j.colsurfb.2015.11.063 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
X-ray-responsive Selenium Nanoparticles for Enhanced Cancer Chemo-radiotherapy Bo Yua#, Ting Liua#, Yanxin Dub, Zuandi Luoa, Wenjie Zhenga, Tianfeng Chena* [email protected] a
Department of Chemistry, Jinan University, Guangzhou 510632, China.
b
Orthopedics Department, Guangdong Provincial Hospital of Traditional Chinese Medicine,
Guangzhou 510120, China *
#
Corresponding author. Tel: +86 20-85225962; Fax: +86 20 85221263.
Authors contributed equally to the work.
1
Graphical abstract
X-ray-Responsive Nanomaterials: An X-ray responsive selenium nanoparticles (PEG-SeNPs) were facilely fabricated through PEG-templated synthesis method and identified as synergistic cancer chemoradiation agent for cancers through enhancement of ROS generation, which may provides a novel strategy for design of chemo-radiotherapeutic nanomaterials.
2
Highlights
X‐ray responsive PEG‐SeNPs were facilely fabricated through PEG‐templated synthesis method.
The X‐ray responsive property was attributed to its amorphous status.
PEG‐SeNPs sensitizes cancer cells to X‐ray via induction of ROS‐mediated apoptosis.
3
Abstract Resistance of cancer to radiotherapy and/or chemotherapy is one of the important reasons of clinical treatment failure and recurrence. Chemoradiation is an optional method to over-coming of radioresistance and chemoresistance. Selenium nanoparticles (SeNPs) with special chemical and physical properties, has been identified as a novel nanocarrier and therapy agent with broad-spectrum anticancer activities due to generate ROS in cells. Herein, X-ray responsive selenium nanoparticles were facilely fabricated by using PEG as surface decorator and template. This nanosystem (PEG-SeNPs) demonstrated X-ray responsive property that was attributed to its amorphous characteristic. Interestingly, the nanosystem demonstrated significant radiosensitization effects with X-ray. Specifically, co-treatment of cancer cells with PEG-SeNPs and X-ray significantly and synergistically enhanced the cells growth inhibition through induction of cell apoptosis, as evidenced by DNA fragmentation and activation of caspase-3. In the cell model, we found that internalized nanoparticles could degrade upon X-ray exposure, which further confirm the X-ray responsive property of the nanoparticles. Moreover, the nanosystem could significantly induced intracellular ROS generation in a time-dependent manner, which peaked at about 40 min and gradually decreased thereafter. As a results, ROS overproduction led to mitochondria fragmentation and the cell apoptosis. Taken together, this study provides a novel strategy for rational design and facile synthesis of chemo-radio therapeutic radiosensitization nanomaterials.
Keywords: X-ray-responsive; selenium nanoparticles; radiosensitization; radiotherapy
4
1. Introduction Radiotherapy has been rapidly developed and widely used in clinic in the past decade.[1] Studies have shown that approximately 50% cancer patients receive radiation during the therapy process.[2] Unfortunately, the resistance of cancer cells to radiation has hindered the efficacy of radiotherapy.[3, 4] Chemoradiotherapy, a strategy that combines chemotherapy with radiation therapy, has been increasingly used in cancer treatment.[5] However, chemoradiotherapy is sometimes accompanied with severe side effects together with their limited spectrum of activity.[6] Therefore, the search for cancer-targeting therapeutic agents that could overcome the resistance of cancer cells to chemoradiation has kindled great interest of scientists.[7] The main challenge remains for targeting strategies is to reduce or avoid toxicity towards normal tissues.[8-10] Recent studies have discovered cell receptor targeting molecules, such as folic acid and functional peptides that could improve the selective cellular uptake of drugs in cancer cells.[11] Comparing with other conventional cancer treatment approaches, such as surgery and chemotherapy, radiosensitization therapy has attracted much interest of scientists for its minimally invasive property and its advantage for therapeutic intervention of specific biological targets.[12-14] X-ray is a clinical method of great benefit in terms of local control, and could target different anatomic tumor sites inside human body. Nowadays, X-ray-based radiotherapy represents one of the most effective ways for treatment of cancers. However, radioresistance and severe side effect remain a challenging issue. Cancer nanotechnology is an emerging technique that displays application potential in cancer-targeted chemotherapy, molecular diagnosis and molecular imaging.[15, 16] Nanodrug 5
delivery systems have been found able to improve the cellular targeting of anticancer drugs and exhibited radiosensitization activities.[12, 17] Townley et al. demonstrated intratumoural injection of rare earth elements doped titania nanoparticles can enhance the efficacy of radiotherapy in cancer therapy.[18] And Cook et al. reported that, gold nanoparticle-based X-ray absorbing agents exhibit both chemo-therapeutic potency to cancer cells and Auger-mediated secondary electron emission, showing great potential to improve the therapeutic efficacy of chemo-radiation.[19] Thus, the search for chemoradiation nanomaterials to kill cancer cells but avoid damage of normal tissues has kindled great interest of scientists. Selenium (Se) is a trace element with novel piezoelectricity, photoconductivity, thermoelectricity, and nonlinear optical responses.[20, 21] The role of Se in cancer chemoprevention and chemotherapy has been supported by many epidemiological, preclinical and clinical studies. Se nanomaterials are novel Se species with broad applications in optoelectronics devices and cancer therapy.[22] In medicine, SeNPs attracted more and more attention in the past decade, due to their high bioavailability, low toxicity and novel therapeutic properties.[23] Recently, we reported the use of SeNPs as carriers of pharmaceutical agents such as to enhance their anticancer outcome,[24] and also clarified ROS-mediated apoptosis mechanisms for the action of SeNPs in detail.[23, 25] Recent studies have demonstrated that Se showed promise as an agent to reduce the harmful side-effects of radiation, but not compromise the effectiveness of treatments.[26] Similarly, our studies also found that selenocompounds could effectively enhance the anticancer efficacy of X-ray through activation of diversified ROS-mediated signaling pathways.[27, 28] Base on the 6
interesting physical and biochemical characteristics and therapeutic advantages of SeNPs, we hypothesized that SeNPs under X-ray irradiation could lead to enhanced ROS generation and result in synergistic anticancer efficacy. Therefore, in this study, X-ray responsive selenium nanoparticles were facilely fabricated by using PEG as surface decorator and template. The X-ray responsive property of PEG-SeNPs was attributed to its amorphous characteristics. Interestingly, the nanosystem demonstrated significant radiosensitization effects with X-ray. Specifically, co-treatment of cancer cells with PEG-SeNPs and X-ray significantly and synergistically enhanced the cells growth inhibition through induction of cell apoptosis, as evidenced by DNA fragmentation and activation of caspase-3. In the cell model, we found that internalized nanoparticles could degrade upon X-ray exposure, which further confirm the X-ray responsive property of the nanoparticles. Moreover, the nanosystem could significantly induce intracellular ROS generation in a time-dependent manner, which peaked at about 40 min and gradually decreased thereafter. As a result, ROS overproduction led to mitochondria fragmentation and the cell apoptosis. Taken together, this study provides a novel strategy for rational design and facile synthesis of chemo-radio therapeutic radiosensitization nanomaterials.
2. Experimental section Reagents Selenium dioxide, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT), pro-pidium iodide (PI), solid JC-1, 2’, 7’-dichlorofluorescein diacetate (DCF-DA) and ascorbic acid were purchased from Sigma-Aldrich Chemical Co. Gray selenium powder was 7
purchased from Guangzhou Dinggang Chemical Factory (Guangzhou, China). Reagent grade polyvinyl-pyrrolidone (PVP K-30) and PEG600 were purchased from Guangdong Chemical Technology Research Center (Guangzhou, China). The water used for all experiments was ultrapure, supplied by a Milli-Q water purification system from Millipore. All of the solvents used were of HPLC grade.
Preparation and characterization of SeNPs Firstly, PEG-SeNPs were prepared as previously described with tiny change.[29] Under N2 protection, dissolving 100 mg gray Se powder in 10 mL PEG600 solution at 220 °C–230 °C for 15–20 minutes, with vigorously magnetic stirring. Then, PEG-SeNPs was cooled to -20 °C. PEGW-SeNPs refers to PEG-SeNPs that were prepared in water solution. PVP-SeNPs were prepared as below description, respectively. As a typical procedure, varied volume of PEG600 or PVP water solution was added dropwise into 1 ml of 125 M H2SeO3 solution under magnetic stirring, and then 5 mL of 100 mM ascorbic acid solution was added into the mixture and then it was reconstituted to a final volume of 25 mL with Milli-Q water. The final concentration of Se was 5 mM. Three types of SeNPs were collected by centrifuge at 12000/min for 30 min and were dialyzed against Milli-Q water until no Se was detected in the outer solution by ICP-AES analysis. The shapes of as-prepared products were characterized by using microscopic and spectroscopic methods. Briefly, TEM samples were prepared by dispersing the powder particles onto holey carbon film on copper grids. The micrographs were obtained on Hitachi (H-7650) for TEM operated at an accelerating voltage at 80 kV. And the Philips TECNAI 20 8
high-resolution transmission electron microscopy (HRTEM) worked at 400 kV. SEM-EDX analysis was carried out on an EX-250 system (Horiba) and employed to examine the elemental composition of PEG-SeNPs. The obtained samples were characterized by X-ray diffraction (XRD) performed on a MSAL-XD2 X-ray diffractometer with a Cu target in the 2θ range from 5° to 80° (40 kV, 30 mA).
Determination of Se Se concentration was determined by ICP-AES method.[30] Briefly, the sample was digested with 3 ml concentrated nitric acid and 1 ml H2O2 in a digestive stove (Qian Jian Measuring Instrument Co., Ltd., China) at 180
for 3 h. The digested product was reconstituted to 10 ml
with Milli-Q H2O and used for ICP-AES analysis.
Cell Lines and Cell Culture Human cancer cell lines HeLa cervical cancer cells and mouse embryonic fibroblast NIH3T3 were purchased from American Type Culture Collection (ATCC, Manassas, VA). All cell lines were maintained in DMEM media supplemented with fetal bovine serum (10%), penicillin (100 units/mL) and strep-tomycin (50 units/ml) at 37
in CO2 incubator (95%
relative humidity, 5% CO2).
MTT Assay Cell viability was determined by measuring the ability of cells to transform MTT to a purple formazan dye.[31] Hela and NIH3T3 cells were seeded in 96-well tissue culture plates at 9
2.5×103 cells/well for 24 h. The cells were then incubated with SeNPs at different concentrations for different periods of time. After incubation, 20 μL/well of MTT solution (5 mg/ml in PBS) was added and incubated for 5 h. The medium was aspirated and replaced with DMSO 150 μL/well. The color intensity of the formazan solution, which reflects the cell growth condition, was measured at 570 nm using a microplate spectrophotometer (Versamax).
Flow Cytometric Analysis The cell cycle distribution was analyzed by flow cytometry as previously described..[32] After treatment with PEG-SeNPs, the cells were trypsinized, washed with PBS and fixed with 70% ethanol overnight at -20 ℃. The fixed cells were washed with PBS and stained with PI working solution (1.21 mg/mL Tris, 700 U/ml RNase, 50.1 μg/mL PI, pH 8.0) for 4 h in darkness. The stained cells were analyzed with Epics XL-MCL flow cytometer (Beckman Coulter, Miami, FL). Cell cycle distribution was analyzed using MultiCycle software (Phoenix Flow Systems, San Diego, CA). The proportion of cells in G0/G1, S and G2/M phases was represented as DNA histograms. Apoptotic cells with hypodiploid DNA content were measured by quantifying the sub-G1 peak in the cell cycle pattern. For each experiment, 10,000 events per sample were recorded.
Intracellular Localization of PEG-SeNPs The intracellular localization of PEG-SeNPs in HeLa cells was traced with 6-coumarin-labeled method as previously described.[31] Briefly, the cells cultured on cover 10
glass in 6-well plates till 70% confluence were stained with 1 μg/mL Hochest 33258 for 20 min. After washing with PBS twice, the cells were incubated with different concentrations of 6-coumarin-labeled PEG-SeNPs for various periods of time and examined under a fluorescence microscope (Nikon Eclipse 230 80i).
Mitochondrial mass assay Mitochondrial mass measured by staining with MitoTracker green (Invitrogen, Carlsbad, CA, USA) was performed as described.[33] Cells were incubated with 50 nM MitoTracker Green (30 min) and 10 μM Hoechst 33258 (10 min) to visualize mitochondria and nuclei, respectively. Then, the loading solution was removed with PBS, the cell monolayer were washed three times with ice-cold PBS and examined by confocal laser scanning microscopy (Zeiss LSM 510 Meta laser scanning confocal microscope systems).
3. Results and Discussion 3.1 Rational design, preparation and characterization of SeNPs Three different types of selenium nanoparticles used in this study were prepared by liquid phase reaction methods. Representative TEM images of the as-made PEG-SeNPs, PEGW-SeNPs and PVP-SeNPs were shown in Fig. 1a-c. It was clearly revealed that the overall morphology of SeNPs adopted a ball-like shape with mean size was at about 500, 500 and 200 nm, respectively. We also characterized the structure of PEG-SeNPs, PEGW-SeNPs and PVP-SeNPs by FT-IR. As shown in Fig. S1a, the peak at 1110 cm-1 in PEG-SeNPs and PEGW-SeNPs could be attributed to the bending vibration of C-O-C of PEG, while the peak 11
at 1740 cm-1 in PVP-SeNPs was assigned to the stretching vibration of O=C-O of PVP. Moreover, the nanomaterials were further characterized by energy-dispersive X-ray (EDX) elemental-mapping analysis. As shown in Fig. S1b, the element mapping of Se and oxygen highly was corresponded to the shape of PEG-SeNPs with some cavities found in the element mapping of Se. The elemental-mapping analysis revealed that element signals of Se, C and O were homogeneously distributed throughout the as-prepared nanoparticles indicating that PEG molecule was incorporated into selenium nanoparticle. The incorporation of PEG eventually leaded to amorphous selenium nano-structure. Our previous work had demonstrated that the preparation of SeNPs in the water solution system could produce the crystal structure.[23] Herein, we observed the same phenomenon that the crystalline core of Se was enclosed by an amorphous coating layer in PEGW-SeNPs and PVP-SeNPs systems (inset images in Fig. 1b-c). Importantly, nanomaterials surfaces can be readily modified with decoration molecular containing functional groups such as thiols, hydroxy, and amines and resulted in special physical/chemical properties[27]. It was reported that hydroxyl group was able to conjugate to Se atom and resulted in surface amorphous structure.[30] Herein, PEG, a polyether compound with hydroxyl groups, was used as the decoration agent. Therefore, possibly, the amorphous core status of PEG-SeNPs could be due to the surface decoration and incorporation of PEG into the nanoparticle. In contrast to PEG-SeNPs, the data analysis confirmed crystalline core structure of PEGW-SeNPs and PVP-SeNPs. Furthermore, TEM was used to characterize the morphological change of different types of SeNPs due to X-ray radiation in cell culture medium. As shown in Fig. 1d-f, smooth and 12
spherical shape of PEG-SeNPs turned to rough and smaller nanoparticles with mean diameter less than 40 nm. In contrast, under the same treatment conditions, PEGW-SeNPs and PVP-SeNPs were not observed this phenomenon. Thus, the structural sensitivity of PEG-SeNPs to X-ray stimulation may be explained by the PEG incorporation in SeNPs. It was also interesting to find that the morphology of PEG-SeNPs changed from a black and round shape to white and empty one in a time-dependent manner under the TEM electron beam stimulation, which suggested that Se atoms in the nanoparticles could evaporate gradually after adsorbing high-energy X-ray (Fig. 2). Unlike PEG-SeNPs showed obvious shape change, PEGW-SeNPs and PVP-SeNPs didn’t display this phenomenon, which was consistent with the result shown in Fig. 1. Taken together, these results indicate the radiation-responsive property of PEG-SeNPs.
3.2 Anticancer efficacy of different nanosystems Studies were also carried out to examine the application potentials of PEG-SeNPs as radiosensitizer. As illustrated in Fig. 3a, X-ray irradiation (8 Gy) or PEG-SeNPs (10 μM) alone induced only slight growth inhibition on HeLa cells, where the cell viability was found at 79%, 71% and 54% in cells treated with 20, 40 and 80 μM PEG-SeNP, respectively. In contrast, co-treatment of the cells with PEG-SeNPs and X-ray significantly enhanced the cells growth inhibition at the same concentration of PEG-SeNP. Especially, co-treatment of 20 μM PEG-SeNP and X-ray significantly suppressed the cell viability to 39%. However, this synergistic effect was not observed in cells which were exposed to PEGW-SeNPs and PVP-SeNPs in combination with X-ray irradiation (Fig. 3b and c). The results of microscopic 13
examination of cells also demonstrated consistent morphological changes after treatment with PEG-SeNPs and X-ray in combination, such as cell shrinkage, rounding, and the appearance of apoptotic bodies (Fig. S2). We have also examined the cytotoxicity of PEG-SeNPs and PEG-SeNPs with X-ray in normal cell lines NIH3T3 by MTT assay. As shown in the Fig 3d, the cell viability still keep high at 85 % after treatment of 10-80 μM PEG-SeNPs with or without X-ray. The combined anticancer effects of them were much lower than those in HeLa cancer cells (Fig. 3a), and no synergistic effects were observed in this normal cell line. Collectively, these results demonstrate the important roles of surface decorators on the anticancer efficacy, radio-sensitization effects and toxicity of SeNPs. The possible reasons for inhibition of cancer cells proliferation could be induction of apoptosis, cell cycle arrest, or both modes.[34, 35] In order to confirm the action mechanisms of cell death induced by X-ray and PEG-SeNPs, we performed DNA flow cytometry analysis to examine the effects of them on the cell cycle distribution. As shown in Fig 4a and b, X-ray alone caused slight G2/M phase arrest from 7.11% (control) to 11.7%. 20 μΜ PEG-SeNPs in combination with X-ray induced an obvious increase in Sub-G1 apoptotic fraction (41.6%) and G2/M phase arrest (21.1%). As shown in Fig S4, 10 μΜ PEG-SeNPs in combination with X-ray induced an obvious increase in Sub-G1 apoptotic fraction (36.8%). The apoptotic cell death was further confirmed by morphological changes, such as cell shrinkage, nuclear condensation and formation of apoptotic bodies (Fig. S3). These results indicate the more important role of cell apoptosis in this anticancer action. Furthermore, we also examined caspase-3 activity induced by PEG-SeNPs with X-ray to verify the induction of apoptosis. As shown in the Fig 5, the effector caspase (caspase-3), was significantly triggered by 14
PEG-SeNPs with X-ray in HeLa cells to 148% of control. These results indicate that apoptosis was the major mode of cell death induced by PEG-SeNPs and X-ray in cancer cells. ICP-AES was used compare the internalization of PEG-SeNPs, PEGW-SeNPs and PVP-SeNPs by cancer cells. As shown in Fig. S5a, the Se concentration in cells treated with PVP-SeNPs was 3 times that of PEG-SeNPs and PEGW-SeNPs groups. These results indicate the important contribution of the surface decorators to the cellular uptake of the nanoparticles in the cancer cells. However, the higher cellular uptake of PVP-SeNPs was not associated with more intensive radiosensitization, which indicate the important roles of decorators in the action of SeNPs. This result suggested that the radiosensitization effects of SeNPs are independent of intracellular Se contents but dependent on SeNPs structure. Further studies were also carried out to elucidate the interaction mechanisms between X-ray and SeNPs. Studies have showed that the photoelectric absorption and secondary electron caused by gamma or X-ray irradiation can produce ROS.[36] ROS plays an important role in regulating cancer cell fate during chemotherapy and radiotherapy. In theory, the photoelectric absorption cross sections of radiosensitive materials directly depend on atom radius.[37, 38] This physical principle determines that Se (atomic-number Z = 34) could be a candidate for radiosensitizer by photoelectric effect. Herein, as shown in Fig. 6a, in cell-free model, pure PEG-SeNPs treated with X-ray irradiation resulted in significant increase in ROS generation, which was much higher than those of PEGW-SeNPs and PVP-SeNPs. Thus, these results indicate that the structural sensitivity of PEG-SeNPs to X-ray stimulation was the fatal fator and led to generate significant amounts of ROS more than those of PEGW-SeNPs and PVP-SeNPs. Future, in cell model, treatment of HeLa cells with PEG-SeNPs under X-ray 15
irradiation also resulted in significant increase in intracellular ROS generation in a time-dependent manner and significantly higher than that of X-ray alone (Fig. S5b). And, the combined treatment induced significant mitochondria fragmentation as probed by MitoTracker Red (Fig. 6b). Thus it was shown that, in cancer cells treated with X-ray alone, the mitochondria recovered after 48 h, while the mitochondrial damage caused by the combined treatment was irreversible (Fig. S5c).
3.3 Intracellular fate of SeNPs in cancer cells The cellular uptake and localization PEG-SeNPs in cancer cells was investigated by live cell fluorescence imaging. As shown in Fig. 7a, PEG-SeNPs entered and accumulated in the cells in a time-dependent manner, which became saturated after 6 h. Interestingly, PEG-SeNPs cluster were also found in the cells (Fig. 7b). Quantitatively, the results of ICP-AES revealed that, intracellular Se contents increased in a time-dependent manner, from 0.03 (control) to 0.33, 0.43 and 0.46 μM/106 cells (Fig. 7c). Taken together, these results demonstrate that PEG-SeNPs enhance the ROS generation induced by X-ray, which may contribute to the synergistic anticancer efficacy through induction of mitochondria fragmentation. Based on above discussion, X-ray irradiation enhances in vitro anticancer activity of PEG-SeNPs but that doesn't depend on cellular uptake of SeNPs. We hypothesized the enhanced anticancer activity of PEG-SeNPs could be due to the X-ray responsive property of the nanoparticles. To confirm this hypothesis, we examined the morphological changes of the nanoparticles inside the cells. As show in Fig. 8, the green fluorescence of PEG-SeNPs 16
decreased drastically after X-ray irradiation. Only weak fluorescence could be observed from the enlarge picture (Fig. S6). It may be due to the fragmentation of PEG-SeNPs in response to X-ray. However, the green fluorescence of PEGW-SeNPs and PVP-SeNPs kept constant after X-ray irradiation (Fig. 9). Moreover, no significant morphological changes were observed in cells treated with PEG-SeNPs and X-ray in combination (Fig. S7). These results further confirmed the X-ray responsive property of PEG-SeNPs.
4. Conclusions Herein, X-ray responsive Se nanoparticles were facilely fabricated by using PEG as surface decorator and template. This nanosystem (PEG-SeNPs) demonstrated X-ray responsive property that was attributed to its amorphous characteristic. Interestingly, the nanosystem demonstrated significant radiosensitization effects with X-ray. Specifically, co-treatment of cancer cells with PEG-SeNPs and X-ray significantly and synergistically enhanced the cells growth inhibition through induction of cell apoptosis, as evidenced by DNA fragmentation and activation of caspase-3. In the cell model, we found that internalized nanoparticles could degrade upon X-ray exposure, which further confirm the X-ray responsive property of the nanoparticles. Moreover, the nanosystem could significantly induced intracellular ROS generation in a time-dependent manner, which peaked at about 40 min and gradually decreased thereafter. As a result, ROS overproduction led to mitochondria fragmentation and the cell apoptosis. Taken together, this study provides a novel strategy for rational design and facile synthesis of chemo-radio therapeutic radiosensitization nanomaterials. 17
Acknowledgements This work was supported by National High Technology Research and Development Program of China (863 Program, SS2014AA020538), Science Foundation for Distinguished Young Scholars (S2013050014667) of Guangdong Province, Natural Science Foundation of China and Guangdong (S2013010012218), Foundation for High-level Talents in Higher Education of Guangdong, YangFan Innovative & Entepreneurial Research Team Project (201312H05), Guangdong Special Support Program and Guangdong Frontier and Key Technological Innovation Special Funds.
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Figure Captions
Fig. 1 TEM images of (a) PEG-SeNPs, (b) PEGW-SeNPs and (c) PVP-SeNPs. The inset images in b and c demonstrate the lattice fringes of an individual particle by using HRTEM (scar bar, 5 nm). The morphological changes of (d) PEG-SeNPs, (e) PEGW-SeNPs and (f) PVP-SeNPs after X-ray treatment (8 Gy) in cell culture medium.
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Fig. 2 Morphology change of three different SeNPs under TEM electronic beam stimulation. (a) TEM images of PEG-SeNPs exposed to electron beam irradiation for 5 and 60 s. (b) PEGW-SeNPs. (c) PVP-SeNPs.
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Fig. 3 Viability of cells treated with X-ray and (a) PEG-SeNPs, (b) PEGW-SeNPs and (c) PVP-SeNPs in HeLa cells, (d) PEG-SeNPs in NIH3T3 cells as examined by MTT assay. The cells were pretreated with SeNPs for 6 h and then treated with X-ray at 8 Gy, followed by a 12 h-incubation. Each value represents means ± SD (n=3).
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Fig. 4 (a) The effects of PEG-SeNPs and X-ray on the cell cycle distribution of HeLa cells were examined by flow cytometry analysis. (b) The percent of the cell cycle of HeLa cells treated with 20 μM PEG-SeNPs and X-ray. 28
Caspase activity (% of control)
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Fig. 5 Analysis of caspase-3 activity treated with 20 μM PEG-SeNPs and X-ray-induced apoptosis in HeLa cells. Caspase activities were determined by syntheticfluorogenic substrate. Bars with different characters are statistically different at P<0.05 level. Each value represents means ± SD (n=3). 29
Fig. 6 Induction of ROS-mediated mitochondrial dysfunction. (a) The levels of ROS generated by 20 μM SeNPs and X-ray irradiation were analyzed by DCF assay. (b) The morphological change of mitochondria in cells after treatments with PEG-SeNPs/X-ray irradiation for 12 h. The cells were pre-stained with Hoechst 33342 and Mito-Tracker Red, and observed by laser scanning confocal microscopy (magnification, 400 X).
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Fig. 7 (a) Time-course internalization of PEG–SeNPs (20 μM, green fluorescence) in HeLa cells. (b) Changes in the intracellular morphology of PEG-SeNPs. (c) ICP-AES quantitative analysis of cellular uptake efficiency of PEG-SeNPs in HeLa cells after different time incubation. 31
Fig. 8 (a) Illustration of the changes of PEG-SeNPs inside the cells, (b) Changes of PEG-SeNPs fluorescence (20 μM, green fluorescence) in HeLa cells after exposure to X-ray (8 Gy) and incubation for another 4 h.
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Fig. 9 Changes of PEGW-SeNPs (a) and PEGW-SeNPs (b) (20 μM, green fluorescence) in HeLa cells after exposure to X-ray (8 Gy) and incubation for another 4 h.
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