Copper Oxide Nanoparticles Induce Enhanced Radiosensitizing Effect

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Copper Oxide Nanoparticles Induce Enhanced Radiosensitizing Effect via Destructive Autophagy Yao-Wen Jiang, Ge Gao, Hao-Ran Jia, Xiaodong Zhang, Jing Zhao, Ningning Ma, Jia-Bao Liu, Peidang Liu, and Fu-Gen Wu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01181 • Publication Date (Web): 05 Feb 2019 Downloaded from http://pubs.acs.org on February 9, 2019

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ACS Biomaterials Science & Engineering

Copper Oxide Nanoparticles Induce Enhanced Radiosensitizing Effect via Destructive Autophagy

Yao-Wen Jiang,† Ge Gao,† Hao-Ran Jia,† Xiaodong Zhang,† Jing Zhao,‡ Ningning Ma,† Jia-Bao Liu,† Peidang Liu,‡ and Fu-Gen Wu*,†

†State

Key Laboratory of Bioelectronics, School of Biological Science and Medical

Engineering, Southeast University, Nanjing 210096, P. R. China. ‡Institute

of Neurobiology, School of Medicine, Southeast University, Nanjing 210096, P. R.

China

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ABSTRACT: Emerging nanotechnologies for radiotherapy are attracting increasing interest from researchers in recent years. To improve the radiotherapeutic performance, developing nanoparticles that can efficiently generate toxic reactive oxygen species (ROS) under X-ray irradiation are highly desirable. Here, we investigated the potential of copper oxide nanoparticles (CuO NPs) as nanoradiosensitizers. Increased cancer cell inhibition was observed in colony formation assay and real-time cellular analysis after the combined treatment with CuO NPs and X-ray irradiation, whereas the CuO NPs alone did not have any negative influence on cell viability, indicating the radiosensitization effect of CuO NPs. Importantly, significantly increased ROS level in cells contributed to the enhanced damage to cancer cells under the combined treatment. Besides, the cell cycle was regulated to the X-ray-sensitive phase (G2/M phase) by CuO NPs, which may also account for the inhibited proliferation of cancer cells. Furthermore, results from Western blot analysis and colony formation assay revealed that the increased cell death might be mainly attributed to the excessive autophagy induced by both CuO NPs and X-ray irradiation. Moreover, in vivo experiments verified the radiosensitization of CuO NPs and their favorable biosecurity. The current study suggests that CuO NPs can be utilized as nanoradiosensitizers for increasing the efficiency of cancer radiotherapy.

Keywords: metal-based nanoparticles, radiosensitizer, autophagic cell death, reactive oxygen species, cell cycle regulation

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INTRODUCTION Radiotherapy (RT), one of the most successful ways in eliminating localized tumors, acts alone or in combination with other methods in treating various cancers clinically. Nevertheless, due to the soft tissue components of tumors, they can absorb only a small proportion of radiation energy. Therefore, for effectively killing tumors, high doses of radiation are often required, which leads to energy waste. More importantly, radiation with high doses would penetrate and damage adjacent healthy tissues due to the lack of spatial control of the energy deposition;1–3 this may affect the efficient delivery of energy to the tumor tissues and thus limit the application of RT.4 In addition, there are still many types of cancers that are radioresistant, and therefore, it is highly desirable to develop new means to enhance radiation responses of tumor cells and achieve enhanced RT cancer treatment with low doses. Meanwhile, nanoparticles (NPs) have found numerous applications in various fields, including RT.1,4 Metal-based NPs (especially those containing high-Z elements) that can enter the cells and act within the tumors possess unique advantages in RT. First, the strong radiation attenuation ability of high-Z elements makes these metal-based NPs potential radiosensitizers, which can absorb more radiation energy than soft tissues and deposit it within tumors during irradiation process, realizing greater radiation damage in tumor cells alone.5–7 Additionally, the secondary rays, especially the Auger electrons released from irradiated high-Z elements can produce a large number of free radicals and highly charged target atoms, which further increase the damage of DNA or other target molecules, producing excellent ionization effects in adjacent tumor regions and therefore enhancing the

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radiotherapeutic effect.8–10 Therefore, a variety of high-Z elements-containing NPs, such as Au NPs,8,11–14 Ag NPs,15–19 Pt NPs,20–22 Bi2S3,23 Bi2Se3,24–27 WS2,28 and HfO2,29 have been used as nanoradiosensitizers. Specifically, nanomaterials based on Au (Z = 79) have attracted particular attention8,30 because of their well-developed synthetic procedures, high inertness, good biocompatibility, and ease of functionalization.8,11,31,32 Extensive efforts have been made to investigate the effect of size, morphology, and surface modification on the radiosensitization efficiency of Au nanomaterials.12,33–36 In addition, rare earth element-based nanomaterials (including upconversion nanoparticles), such as CeO2,37 Gd-based NPs,38–40 and NaYbF4:Er/Gd,41 and some other metal oxide NPs, such as TiO2,1,42,43 and Fe3O4 NPs,44–46 have also shown promising radiosensitizing effect. On the other hand, RT eradicates tumors through the effect of ionizing radiation, and X-rays as a kind of ionizing radiation can not only directly damage cancer cells but also decompose and ionize water molecules, inducing the generation of reactive oxygen species (ROS). The produced ROS may damage DNA/lipids and disrupt signal transduction, which will cause cell death if the damage to the biomolecules cannot be repaired.44,46,47 Meanwhile, owing to their faster cell proliferation and metabolism, cancer cells are more susceptible to oxidative damage than normal cells, and therefore the appropriate ROS stress can break the relatively low antioxidant capacity of cancer cells and disrupt their redox homeostasis, realizing selective tumor cytotoxicity.48–50 Therefore, NPs capable of generating ROS after X-ray exposure may increase the therapeutic efficiency of RT, making them promising in eliminating tumors with significantly decreased radiation doses and reduced side effects of X-ray irradiation. Previous studies have already utilized the increased ROS generation of

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TiO2 and Fe3O4 NPs in realizing enhanced RT. Different from those high-Z element-containing NPs, TiO2 and Fe3O4 NPs were found to be effective in radiosensitization mainly due to the enhanced generation of ROS after X-ray exposure, which can increase the DNA damage and retard the DNA repair directly.46,51,52 In addition, autophagy induced by NPs has also drawn much attention recently.53–56 Some researchers reported that when exposed to ionizing radiation, cells may protect themselves by skipping cell apoptosis and allowing continued survival of cells through autophagy, which can remove impaired macromolecules and organelles (e.g., mitochondria) from cells,57,58 thus decreasing the efficiency of RT. However, it is suggested that when the autophagy at basal level is replaced by prolonged activation of autophagy in cancer cells, cell death instead of therapy resistance would occur due to the excessive cellular selfdegradation.57,59 Although it is known that autophagy is generally protective, uncontrolled or excessive autophagy could be detrimental, and it is still controversial whether autophagy will truly lead to cell death or merely play an innocent bystander or even a protective role in dying cells. Recently, there is increasing evidence that the cell death may be attributed to autophagy in certain settings.60 Therefore, autophagy rather than apoptosis may be a potential mechanism for radiosensitization. It has been reported that the autophagy-dependent non-apoptotic form of cell death, termed as autosis, can be triggered by autophagy-inducing peptides, starvation, and neonatal cerebral hypoxia-ischemia.60,61 Previous studies have achieved enhanced radiotherapeutic performance through an increased autophagy level in breast cancer cell line, glioblastoma cell line, and even radioresistant hepatocarcinoma cell line,58,62,63 and hence the induction of autophagy may be an effective way to overcome the radioresistance of tumors.

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Recently, it has been demonstrated that copper oxide nanoparticles (CuO NPs) can induce autophagy in cancer cells;64,65 however, the application of CuO NPs as nanoradiosensitizers for cancer RT remains unexplored so far. In this work, we investigate whether CuO NPs can be adopted as efficient nanoradiosensitizers for cancer RT. The CuO NPs prepared through a quick precipitation procedure exhibit autophagy-inducing ability after being internalized by cancer cells. After X-ray irradiation, the induced excessive autophagy leads to the autosis of cells (Figure 1). Furthermore, the enhanced efficiency of RT in vivo also confirms the radiosensitization of CuO NPs. Therefore, CuO NPs possess the potential for use as effective radiosensitizers in RT.

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Figure 1. Schematic showing the preparation route and radiosensetization effect of CuO NPs.

RESULTS AND DISCUSSION Characterization. CuO NPs were synthesized via a precipitation reaction between copper acetate and NaOH based on a reported approach.66 The size and morphology of the prepared CuO NPs were characterized by transmission electron microscopy (TEM). As shown in the TEM image and its corresponding size distribution histogram (Figure 2a), the average diameter of the spherical CuO NPs was 5.4 ± 1.2 nm, in good agreement with the results reported by Zhu et al.66 In addition, according to Figure 2b, the average hydrodynamic diameter of CuO NPs was 14.8 ± 4.8 nm, which is larger than the average size (5.4 ± 1.2 nm) obtained from TEM observation. This discrepancy can be explained by the presence of the hydration layer of the nanoparticles. Furthermore, the ultraviolet–visible (UV–vis) spectrum of the CuO NP dispersion (Figure 2c) shows a single broad peak at around 280 nm, which also agrees well with the result in the previous report,66 indicating the successful preparation of CuO NPs. Besides, the color of CuO NP aqueous dispersions changed with the CuO concentration: yellow for 0.1 mg/mL and dark brown for 3.5 mg/mL (inset of Figure 2c). In addition, the X-ray diffraction (XRD) data of CuO NPs were consistent with Joint Committee on Powder Diffraction Standards (JCPDS) card of CuO (JCPDS 80-1268), indicating that the single-phase CuO with a monoclinic structure and a high purity was obtained (Figure 2d). Besides, as shown in Fourier-transform infrared (FTIR) spectrum (Figure S1), the asymmetric stretching band (~1560 cm–1) and symmetric stretching band (~1400 cm–1) of COO– can be clearly observed, and the ∆v of ~160 cm–1 for the two stretching vibrations is

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associated with the interaction between bare CuO NPs and the COO– groups from acetic acid molecules. Furthermore, the size and morphology of the CuO NPs after storage in water or complete Dulbecco's modified Eagle's medium for 14 days (Figure S2) almost did not change as compared with those of the freshly prepared CuO NPs in water (Figure 2a), demonstrating the good stability of the NPs in these aqueous media.

Figure 2. (a) TEM image and size distribution histogram (inset) of CuO NPs (freshly prepared in water). (b) Dynamic light scattering result of CuO NPs dispersion. (c) UV−vis spectrum of CuO NPs and the photographs (inset) of 0.1 and 3.5 mg/mL CuO NP dispersions, respectively. (d) XRD spectrum of CuO NPs (in comparison with JCPDS: 80-1268 file shown).

Cellular Internalization and Cytotoxicity Evaluation of CuO NPs. To investigate the suitability of the CuO NPs for RT, we first evaluated the influence of CuO NPs on MCF-7 cells without X-ray irradiation. Considering the relationship between the amount of internalized particles and their functions, it is important to monitor the cellular uptake of the CuO NPs. Meanwhile, the internalized particles will lead to increased cellular granularity, 8


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which can be reflected by the change in the signal intensity of side scatter (SSC) in the flow cytometric data. Therefore, the cellular internalization of CuO NPs could be quantitatively analyzed by measuring the intracellular signal intensity of SSC. The SSC of CuO NP-treated MCF-7 cells increased with increasing NP concentration (Figure 3a). In addition, the dark-field optical images of MCF-7 cells after NP treatment verified the internalization of CuO NPs, and the plasma membrane and nucleus staining results further indicated that the CuO NPs were located in the cytoplasm (Figure S3). Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay was conducted to test the influence of CuO NPs on the viability of MCF-7 cells. Results in Figure 3b indicate that CuO NPs showed no cytotoxicity towards MCF-7 cells at 10 μg/mL or below; therefore 10 μg/mL of CuO NPs was suitable for further RT studies. Moreover, the hemocompatibility of CuO NPs was studied by hemolysis test on red blood cells (RBCs). Photographs of RBCs after treatment with phosphate-buffered saline (PBS), 30 μg/mL CuO NPs, or 1% Triton X-100 demonstrated the excellent hemocompatibility of CuO NPs (Figure 3c). Besides, hemoglobin released from lysed RBCs can be detected in the supernatant with a photometer. Compared with the negative control (PBS-treated RBCs) and positive control (1% Triton X-100-treated RBCs), negligible hemolysis was detected in the RBCs even after treatment with 30 μg/mL CuO NPs (Figure 3d), further confirming the excellent biocompatibility of CuO NPs. Moreover, the apoptosis assay was conducted via flow cytometry using annexin V-isothiocyanate (FITC)/propidium iodide (PI) dyes to evaluate the capability of CuO NPs to induce apoptosis. The FITC-labeled annexin V can identify the membrane-translocated phosphatidylserine, which is an indicator of apoptosis, and PI stains the nucleic acids in the

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cell, indicating the cell death (disrupted membrane). Hence, the apoptosis rates can be measured through the fluorescence distinguishment (FITC: green fluorescence; PI: red fluorescence) using a flow cytometer. As shown in Figure 3e, only a very small portion (< 1%) of apoptotic cells was detected even in the presence of 30 μg/mL CuO NPs, further demonstrating the negligible influence of CuO NPs on the viability of MCF-7 cells.

Figure 3. (a) Endocytosis analysis results of CuO NP-treated MCF-7 cells using flow cytometry. (b) Viabilities of MCF-7 cells treated with CuO NPs at different doses. (c) Photographs of RBCs after different treatments as indicated. Triton X-100-treated sample serves as the positive control. (d) Hemolysis rates of CuO NPs at different doses. (e) Apoptosis rates of MCF-7 cells treated with CuO NPs at different doses.

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Radiosensitization Evaluation of CuO NPs. Based on the above results, CuO NPs at a concentration of 10 μg/mL were chosen for the subsequent X-ray irradiation assays. The colony formation assay was carried out first to study the radiosensitization effect of CuO NPs. Results in Figure 4a and b showed that the cell surviving fraction decreased with increasing radiation dose, and a significant decrease of survival fraction could be seen with the introduction of CuO NPs, indicating the enhanced radiosensitization. Besides, real-time cell analysis (RTCA) was utilized since it can quantify the cell proliferation in a label-free, real-time manner using noninvasive electrical impedance monitoring. Results of RTCA showed that after X-ray irradiation, the proliferation of cells was inhibited significantly, especially when combined with CuO NPs; whereas no inhibition effect of CuO NPs was observed without X-ray irradiation (Figure 4c), in consistence with the conclusion obtained by colony formation assay.

Figure 4. In vitro radiosensitization evaluation for CuO NPs. (a) Photographs of colonies formed by 11



MCF-7 cells treated without (control) and with CuO NPs in the presence of X-rays at different doses (0, 2, 4, 6, and 8 Gy). The positions of colonies after Giemsa staining are marked by red dots. (b) Corresponding cell surviving fractions of the samples in (a). (c) Real-time growth results of MCF-7 cells (monitored via an real-time cell analyzer) after different treatments as indicated.

Mechanisms of the Enhanced Radiotherapeutic Effect. To further explore the mechanism of the enhanced radiotherapeutic effect, the changes of ROS generation, cell cycle, and apoptosis/necrosis level were investigated using a flow cytometer. First, a remarkable elevation in ROS level was detected in MCF-7 cells in the groups of “X-ray” and “CuO NPs + X-ray” (Figure 5a and b). Cells receiving both CuO NPs and X-ray irradiation treatments had the most significant increase of the ROS level, which is similar to the radiosensitization mechanism of other non-high Z-element NPs (e.g., TiO2 and Fe3O4 NPs43,46). The determination of ROS using dichlorodihydrofluorescein diacetate (DCFH-DA) is non-discriminatory, which can detect various kinds of ROS, such as superoxide anions (O2•–), hydroxyl radicals (•OH), hydrogen peroxide (H2O2), etc.. It is well known that the CuO NPs have the ability to generate ROS such as O2•– and •OH,67 and thus O2•– and •OH may be the main ROS generated by CuO NPs in this work. In addition, it is proposed that Cu can react with endogenous H2O2 to generate ROS in a Fenton-type reaction to produce O2•– or •OH.67,68 Besides, it has been reported that the surface defect sites of nanocrystalline CuO could directly produce a large number of ROS (e.g., O2•–).69 Therefore, the electron transfer, rather than energy transfer, contributed to the ROS generation here, which was induced by the CuO NP-involved Fenton-type reaction. Second, the cell cycle distribution demonstrated that the CuO NP treatment increased the proportion of G2/M phase (Figure 5c and d), which

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is more sensitive to X-ray irradiation, leading to a more pronounced damage to the cells after subsequent X-ray irradiation treatment. Third, through the apoptosis analysis, it can be found that the CuO NP treatment alone would not alter the apoptosis level of MCF-7 cells; after X-ray treatment, the apoptosis level was slightly increased (Figure 5e and f). However, the degree of apoptosis was still very low, indicating that there must be other mechanisms for cell death.

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Figure 5. Flow cytometric results of MCF-7 cells. (a) ROS generation and (b) corresponding histogram of the fluorescence intensity of DCFH-DA in MCF-7 cells that experienced various treatments. (c) Cell cycle distribution and (d) corresponding statistics of MCF-7 cells that received various treatments. (e) Apoptosis/necrosis analysis results and (f) corresponding statistics of apoptosis and necrosis rates of MCF-7 cells that experienced various treatments.

Moreover, DNA break, one manifestation of radiation damage, was also evaluated. As an 14


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indicator of the early phosphorylation event, which plays a critical role in recruiting proteins involved in DNA repair, phospho-H2AX (γ-H2AX) can be specifically bound by anti-γ-H2AX antibody. By using the Alexa Fluor 488-conjugated anti-human phospho-H2AX (S139) mouse mAb, we can see that compared to the small increase of γ-H2AX (shown as fluorescent green dots) in MCF-7 cells induced by CuO NPs or X-ray treatment alone, the significantly increased green dots in the nuclei indicated the severer DNA damage induced by the combined treatment with CuO NPs and X-ray irradiation (Figure 6), and such a significant difference in the amount of fluorescent dots can be confirmed by the quantitative statistics as shown in Figure S4, further demonstrating the radiosensitization effect of CuO NPs.

Figure 6. Confocal images of MCF-7 cells that experienced various treatments as indicated. Hoechst 33342 and Alexa Fluor 488-conjugated anti-human phospho-H2AX (S139) mouse mAb were used for visualizing the nuclei and γ-H2AX, respectively. Scale bars: 25 μm.

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Autophagy Analysis. Since CuO NPs have the ability to induce autophagy, which is crucial in regulating cellular behaviors (including cell death), the autophagy levels of cells after different treatments were also evaluated. During the autophagy process, protein LC3-II is generated from LC3-I through the conjugation with phosphatidylethanolamine moiety, and therefore the autophagy level can be reflected by the LC3-II/LC3-I ratio, which increases as the autophagy level increases. As shown in Figure 7a, the treatment with CuO NPs or X-ray irradiation alone could induce a moderate degree of autophagy, which may play a protective role via the removal of damaged biomacromolecules and organelles and hence help cells to survive. Furthermore, the co-treatment with CuO NPs and X-ray irradiation significantly elevated the autophagy level, and therefore the self-degradation caused by excessive autophagy may be one of the cell death pathways for the MCF-7 cells. Quantitative results of western blot assay based on the integrated optical densitometry showed an increased LC3-II/LC3-I level after the co-treatment with CuO NPs and X-ray as compared to that after the single treatment with CuO NPs or X-ray, indicating the enhanced autophagy level of MCF-7 cells after the combined treatment (Figure 7b). Moreover, 3-MA, an autophagy inhibitor that can decrease the activity of phosphatidylinositol 3-kinase, reduced the radiosensitization effect of CuO NPs (Figure 7c). Specifically, in consistence with the western blot results, CuO NPs and X-ray alone could induce autophagy. Notably, after the introduction of 3-MA, increased inhibition of colony formation was observed in X-ray irradiated group (Figure 7c and S5), which might be attributed to the inhibition of protective autophagy. In contrast, a remarkable decrease in the number of colonies was observed in the group after the co-treatment with CuO NPs and X-ray (Figure 7c and S5), which might be

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due to the destructive effect of excessive autophagy. In particular, “autosis”, is adopted as the name for this potentially lethal role of autophagy.61,70 Recently, many discussions on the relationship

between

autophagy

and

cell

death

have

emerged,

and

excessive

autophagy-induced cell death has been frequently mentioned.61,70–73 For instance, Zhou and Yu et al. reported that Cd treatment can induce a remarkable degree of autophagy, resulting in cell death in normal liver cells (L02 cells) via excessive self-digestion and degradation of important cellular components.71,72 In addition, Xu et al. utilized excessive autophagic cell death triggered by a small molecule RL71 in treating triple-negative breast cancer.73 Therefore, excessive autophagy leading to cell death can be a potential therapeutic strategy. Furthermore, autophagy has also been taken into consideration in researches on the enhancement of radiotherapeutic performance recently. It is believed that autophagy is related to radiation-induced cell death although the basal level of autophagy is often considered protective for cells.74 Autophagy, a double-edged sword, plays a dual role in radiotherapy (i.e., cell survival or cell death). There are some studies reporting that the autophagy could achieve cellular radioresistance through gene mutation or microRNA regulation, and then decrease the radiotherapeutic effect, 75–77 which means that the inhibition of autophagy (e.g., with chloroquine) could improve radiotherapy.77 On the contrary, the induction of autophagy enhancing the effect of radiation on cell death, including overcoming radioresistance, was also proposed by many studies,74,78–80 and therefore, the combination of an autophagy inducer (e.g., rapamycin) and radiation would facilitate the development of autophagy in cancer cells accompanied with reduction of cell survival.80 We speculate that such distinct conclusions (inhibition and promotion of autophagy both have the ability to improve radiotherapy) may

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be due to the difference of autophagic response in different cells: pancreatic cancer cells and lung cancer cells for the former but glioblastoma and breast cancer cells for the latter. In the current work, MCF-7 cells (human breast cancer cells) were adopted, and the autophagy inducer CuO NPs realized enhanced radiosensitizing effect via elevating the level of autophagy in MCF-7 cells. Nonetheless, the underlying mechanisms remain obscure and need further investigation.

Figure 7. (a) Western blot results of the autophagy-related proteins LC3-I and LC3-II in MCF-7 cells that experienced various treatments. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as the loading control. (b) Corresponding quantitative analysis results of western blot assay. (c) Colony formation images of MCF-7 cells that received different treatments. CuO NPs: 10 μg/mL; 3-MA: 3 mM; X-ray: 6 Gy. The positions of colonies after Giemsa staining are marked by red dots.

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Animal Experiments. Encouraged by the above results in vitro, we expect that CuO NPs may also act as a radiosensitizer in vivo. Mice bearing U14 tumors were divided into four groups and experienced CuO NPs and/or X-ray irradiation treatments or left untreated. Results showed that CuO NPs alone could not inhibit the growth of tumors, and X-ray irradiation alone inhibited the tumor growth only for a short time period (~7 days). By contrast, the co-treatment with CuO NPs and X-ray significantly enhanced the tumor-inhibitory activity, verifying the radiosensitization of CuO NPs (Figure 8a). On the other hand, we monitored the changes in the body weight of mice in the four groups. As shown in Figure 8b, the mice receiving X-ray irradiation lost some weight during the first 4 day; however, their body weight began to increase at day 5 and became relatively stable thereafter, which is a normal phenomenon during RT, and the negligible overall changes in body weight indicate that the treatments (CuO NPs and X-ray irradiation) elicited few side effects toward mice (Figure 8b). To further confirm the effect of various treatments on tumor growth, apart from the in situ monitoring of the tumor volume, the weight of excised tumor tissues was also measured after the sacrifice of mice on the 14th day. The results shown in Figure 8c indicated that the combination between CuO NPs and X-ray irradiation exhibited the highest inhibitory activity of tumor growth, in good consistence with the results of tumor volume (Figure 8a). It should be noted that the therapeutic efficiency of single radiotherapy (even with radiosensitizers) is often not satisfactory due to the hypoxic tumor microenviroment, local recurrence or metastasis,1,14,23,27,81 and therefore the excellent therapeutic performance achieved in this work by using the single CuO NP-assisted radiotherapy may hold great promise for treating tumors.

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Furthermore, to assess the potential long-time toxicity, the biodistribution of Cu in mice at day 1 and day 14 was analyzed. As shown in Fig 8d, at 1 day postinjection, the Cu content of tumor tissue was much higher than that of the main organs (heart, liver, spleen, lung, and kidney), which is due to the excellent tumor retention of the CuO NPs. After 14 days postinjection, the Cu content of the tumor tissue became very low. Besides, the Cu element in the main organs was almost completely excreted out of the mouse, because the contents of Cu at day 14 in these organs were similar to the intrinsic concentrations of Cu in main organs of mice.82,83 Cu, a fundamental trace element in both animals and plants, is vital to the proper functioning of these living beings due to its participation in metabolic processes such as activation of enzymes and protein synthesis. Besides, previous studies using Cu-based nanoparticles for cancer treatment have proven the safety of Cu.84,85 The Cu biodistribution results indicate that the CuO NPs can be successfully cleared from the mouse body, ensuring the good biosafety of this nanomaterial. Notably, liver, as a main metabolic organ, contained a relatively higher Cu content than other organs at day 1; however, the Cu content of liver became significantly lower at day 14, indicating that the liver accumulation of the NPs was only temporary and the hepatic metabolism may contribute a lot to the elimination of the CuO NPs. Further, histological analysis on hematoxylin and eosin (H&E)-stained slices of the main organs was performed to evaluate the biosafety of CuO NPs. Results in Figure 8e showed that the organs treated with CuO NPs maintained undisturbed structures, except that CuO NPs had some damage to the liver. The slight liver damage may be due to the higher Cu content in this organ (as shown in Figure 8d). However, based on the above body weight and Cu

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biodistribution results, and considering the regeneration and repair abilities of liver, it is expected that the CuO NPs would not cause evident systemic toxicity to animals and have acceptable biocompatibility in vivo, thus holding promise for use as a relatively safe nanoradiosensitizer. It is worth mentioning that radiation therapy was the only treatment modality adopted in the present work, and previous studies have reported the combined use of radiotherapy and photodynamic therapy via luminescent NPs, such as LaF3:Ce3+ NPs;86,87 therefore if other treatment modalities like chemotherapy and/or phototherapy were combined with the present radiotherapy, the amount of Cu administrated can be further decreased, thereby avoiding the health risk caused by potential hepatotoxicity of the nanoagent.

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Figure 8. In vivo experiments on the radiosensitization of CuO NPs. Relative tumor volumes (a) and body weights (b) of U14 tumor-bearing nude mice that experienced various treatments. *** for P < 0.001. (c) Tumor weights of different groups and corresponding photographs of excised tumors at day 14. (d) Biodistribution of Cu in CuO NP-treated mice at day 1 and day 14 postinjection. (e) Photographs of histological slices (after H&E staining) of the main organs obtained from mice treated with PBS (control) or CuO NPs at 14 d postinjection. Scale bar is 0.3 mm.

CONCLUSION The present work demonstrates that CuO NPs can be utilized as a potential nanoradiosensitizer to enhance the therapeutic efficiency of RT. It is found that without X-ray 22


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irradiation, CuO NPs elicited negligible toxicity toward the MCF-7 cells. However, the co-treatment with CuO NPs and X-ray could greatly increase the ROS level, which subsequently induced the damage of cancer cells. Besides, CuO NPs could modulate the cell cycle distribution by increasing the radiosensitive G2/M phase in MCF-7 cells, thereby enhancing the radiosensitivity of the cancer cells. More interestingly, apart from the apoptosis/necrosis, the autophagy-related process also played a vital role in the death of MCF-7 cells. Under X-ray irradiation, CuO NPs could induce an excess level of autophagy, and such a high degree of autophagy did not show a protective role for the MCF-7 cells but rather a cancer cell killing effect through the self-degradation process. Such a destructive role of autophagy has rarely been reported. Moreover, the excellent radiosensitizing effect and good biosafety of CuO NPs were also verified by the in vivo experiments. As far as we know, the current work reports for the first time that CuO NPs can be used as a highly efficient nanoradiosensitizer, which may find potential applications in cancer therapy.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Materials and experimental details, FTIR spectrum, TEM images, dark field images, quantitative analysis of DNA double-stranded breaks, and colony formation results

AUTHOR INFORMATION 23



Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was granted by the NSFC (21673037 and 81571805) and Scientific Research Foundation of Graduate School of Southeast University (YBJJ1778).

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