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Sequential Treatment of Cell Cycle Regulator and Nanoradiosensitizer Achieves Enhanced Radiotherapeutic Outcome Xiaotong Cheng, Xiaodong Zhang, Peidang Liu, Liu-Yuan Xia, Yao-Wen Jiang, Ge Gao, Hong-Yin Wang, Yan-Hong Li, Ningning Ma, Huan-Huan Ran, and Fu-Gen Wu ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00085 • Publication Date (Web): 10 Apr 2019 Downloaded from http://pubs.acs.org on April 10, 2019

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ACS Applied Bio Materials

Sequential Treatment of Cell Cycle Regulator and Nanoradiosensitizer Achieves Enhanced Radiotherapeutic Outcome

Xiaotong Cheng,† Xiaodong Zhang,† Peidang Liu,‡ Liu-Yuan Xia,† Yao-Wen Jiang,† Ge Gao,† Hong-Yin Wang,† Yan-Hong Li,† Ningning Ma,† Huan-Huan Ran,† and FuGen Wu*,†

†State

Key Laboratory of Bioelectronics, School of Biological Science and Medical

Engineering, Southeast University, Nanjing 210096, China ‡Institute

of Neurobiology, School of Medicine, Southeast University, Nanjing 210009,

China

Corresponding Author *E-mail: [email protected] (F.G.W.)

KEYWORDS: gold nanomaterials, radiotherapy, reactive oxygen species, cellular internalization, radiosensitizers

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ABSTRACT Nanoradiosensitizers are promising agents for enhancing cancer radiotherapeutic efficiency. Although many attempts have been adopted to improve their radiation enhancement effect through regulation of their size, shape, and/or surface chemistry, few methods have achieved satisfactory radiotherapeutic outcomes. Herein, we propose a sequential drug treatment strategy through cell cycle regulation for achieving improved radiotherapeutic performance of the nanoradiosensitizers. Docetaxel (DTX), a clinically approved first-line drug in the breast cancer treatment, is first used to affect the cell cycle distribution and arrest cells in the G2/M phase, which has been proven to be the most effective phase for endocytosis and the most radiosensitive phase for radiotherapy. Then the cells are exposed to a commonly used nanoradiosensitizer, gold nanoparticles (GNPs), followed by X-ray irradiation. It is found that by arresting the cancer cells in G2/M phase via the DTX pretreatment, the cellular internalization of GNPs is significantly promoted, therefore enhancing the radiosensitivity of cancer cells. The sensitization enhancement ratio (SER) of this sequential DTX/GNP treatment reaches 1.91, which is significantly higher than that (1.29) of GNP treatment. Considering its low cost, simple design, and high feasibility, this sequential drug delivery strategy may hold great potential in radiotherapy.

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INTRODUCTION Clinically, surgery, chemotherapy, and radiotherapy are the three main therapeutic methods used for cancer treatment. Radiotherapy, which is applied in about half of cancer treatments, can kill cancer cells by depositing the energy into tumor tissues.1 In spite of its effectiveness, radiotherapy may not yet eradicate tumors completely because some tumor cells are radioresistant or the irradiation is outside the targeted area. Although a sufficient irradiation dose may eradicate tumors, the deposited energy may damage the healthy tissues in the mean time.2 To improve the radiotherapeutic performance, increasing the radiation dose specifically to the tumor cells would be an effective approach. Novel radiosensitizers based on nanoparticles that specifically target tumors have shown the tumor-specific damaging effect of the radiation (including γ or X-rays).3‒10 A variety of metal-based nanoparticles have been demonstrated as potential nanoradiosensitizers, such as gold11‒13 platinum,14,15 bismuth,6,8,16‒18 tungsten,19,20 tantalum,21,22 silver,23,24 and iron.25 It has been well known that the anticancer effect of radiotherapy is associated with many factors, including the size,26,27 shape,28 and surface chemistry29,30 of nanoradiosensitizers, the energy and dose of radiation,31 the types of tumor cells,32 etc. To improve the radiotherapeutic effect, one possible approach is the combined use of a nanoradiosensitizer and a chemotherapeutic drug which can synergistically improve the efficacy of each agent by attacking the tumor from two distinct aspects.33‒35 Such codelivery nanoplatforms have several inherent advantages including better drug4


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resistance management, synergistic therapeutic efficacies, and the ability to control drug release.36 Another way to enhance the radiotherapeutic outcomes is to increase the cellular uptake of nanoradiosensitizers by tumor cells. In previous studies, many methods have been used to maximize the cellular uptake efficiency of nanoradiosensitizers. Size control is a direct way to achieve this goal.3,27,37,38 Besides, a variety of ligands have already been utilized to improve the tumor-targeted delivery of nanodrugs, such as aptamers, proteins, peptide, and small molecular compounds.39‒42 Although many co-delivery nanoplatforms and modified nanoradiosensitizers have been reported and some of them are in the clinical research stage, they still encounter some unfavorable factors such as the complex modification methods, the poor stability and biocompatibility of ligands, the uncontrollable regulation of some key parameters including

entrapment

efficiency

and

release

kinetics,

and

unsatisfactory

radiotherapeutic performance. Therefore, it is vital for us to develop a new approach with higher effectiveness and better clinical application feasibility to foster the development of nanoradiosensitizer-based radiotherapy. On the other hand, the cell cycle includes a series of events that take place in a cell leading to its division and DNA duplication for producing two daughter cells. It has been proven that not only the morphology and surface properties of nanoparticles but also the phase of cell cycle may have influence on the cellular internalization of nanoparticles.43 Cells in different cell-cycle phases have different nanoparticle uptake abilities with the following sequence: G2/M > S > G0/G1.43 Besides, it has been reported that G2/M phase is the most radiosensitive phase.44‒46 The above findings 5



inspire us to develop a new sequential treatment strategy for enhancing the endocytosis of nanoradiosensitizers, in which a cell cycle regulator is first introduced to arrest the cancer cells in the G2/M phase, followed by the addition of the nanoradiosensitizers. The G2/M phase-arrested cells are more radiosensitive and can endocytose more nanoradiosensitizers, thereby increasing the radiotherapeutic efficiency. Many compounds can arrest cells into G2/M phase, such as geldanamycin,47 decitabine,48 vinblastine,49 and docetaxel (DTX).50 Among them, DTX has been widely used in chemotherapy and can effectively arrest cells in the G2/M phase.51‒53 It can interact with the microtubular network of cells, facilitating the tubulin assembly into stable microtubules and preventing their disassembly.54,55 On the other hand, gold nanoparticles (GNPs) have become an important type of nanoradiosensitizers because of their several merits,56 such as relative inertia,57 good biocompatibility,58,59 and the well-established synthetic methods60,61 that allow the fabrication of GNPs with ideal sizes for optimized tumor penetration and delivery.62,63 Taking these points into consideration, we herein develop a sequential treatment strategy in which DTX, a cell cycle regulator is first used to tune the cell cycle distribution, followed by subsequent GNP (a typical representative of nanoradiosensitizers) treatment and X-ray irradiation (Scheme 1). Pretreating the cells with DTX can arrest more cells in the G2/M phase, which will allow the cells to endocytose more GNPs. The enhanced GNP uptake can remarkably improve the radiotherapeutic outcome, as demonstrated by colony formation assay and the calculation of sensitization enhancement ratio (SER). To exclude the synergistic effect of the two drugs (DTX and GNPs), the effect of different 6


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combinations of the drug treatment sequences on the radiotherapeutic efficacy was also investigated. Further, the potential mechanism of enhanced radiosensitization effect of the sequential DTX/GNP treatment was also discussed.

Scheme 1. Schematic Illustrating the Improved Radiotherapeutic Performance of the Sequential DTX/GNP Treatment via Cell Cycle Regulation in Cancer Cells.

EXPERIMENTAL SECTION Cell Cycle Analysis. MCF-7 breast cancer cells were seeded in 6-well dishes and incubated in the incubator. When the cells reached ~80% confluence, they were treated 7



with different solutions (cell culture medium, cell culture medium containing 5 nM DTX, or cell culture medium containing 50 μg/mL GNPs) for 6 h. After that, the cells were treated with a cell cycle detection kit (KeyGen Biotech, Nanjing, China) according to the protocol. The cell cycle distribution was examined using a flow cytometer (NovoCyte 2060, ACEA, USA). Microtubule Polymerization Analysis. Microtubules were visualized by indirect immunofluorescence microscopy with Tubulin-Trakcer Red (Beyotime Institute of Biotechnology, China). MCF-7 cells were seeded onto coverslips in 96-well plates and incubated in the incubator. After that, cell culture medium and cell culture medium containing 5 nM DTX were used to treat the cells for 6 h, respectively. Immunofluorescence staining with Tubulin-Trakcer Red was carried out according to the protocol after the above treatment. After being washed by phosphate-buffered saline (PBS), the cells were stained by Hoechst 33342 (Beyotime Institute of Biotechnology, China) for 10 min. The cells were then observed under a confocal laser scanning microscope (TCS SP8, Leica, Germany). Cellular Uptake Analysis. First, the dark-field imaging was used to observe the cellular endocytosis of GNPs. MCF-7 cells were seeded onto coverslips in 96-well plates and incubated at 37 oC for 24 h. Cells were exposed to 5 nM DTX or cell culture medium for 6 h, washed by PBS twice, and then incubated with GNPs (50 μg/mL) for 6 h. Whereafter, the cells were washed with PBS and stained with Hoechst 33342 (to visualize nuclei) and Chol-PEG-FITC (to visualize plasma membranes). The dark-field images of the cells were then collected by the confocal microscope. To evaluate the 8


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cellular uptake of GNPs, an ImageJ software was used to analyze the cells and quantify the white dots in the corresponding confocal images. Besides, we also evaluated the Au contents in MCF-7 cells after treatment with GNPs using inductively coupled plasma optical emission spectrometry (ICP-OES). Briefly, the cells left untreated or pretreated with DTX were incubated with GNPs for various time periods (0.5, 1, 2, 4, 6, 8, and 12). Then the cells were collected by trypsinization, re-suspended in PBS, and counted by flow cytometry. Aqua regia was used to lyse the cells and dissolve GNPs. ICP-OES measurements were conducted to determine the Au content of each sample, which was then used to calculate the number of GNPs via a previous method.64 The number of GNPs per cell was obtained by dividing the number of GNPs by the number of cells in the lysis solution. Colony Formation Assay. MCF-7 cells were seeded in 6-well plates and incubated in the incubator for 24 h. After that, they were divided into 6 groups: (1) culture medium (12 h), (2) DTX (6 h)→culture medium (6 h), (3) GNPs (6 h)→culture medium (6 h), (4) “DTX + GNPs” (6 h)→culture medium (6 h), (5) DTX (6 h)→GNPs (6 h), and (6) GNPs (6 h)→DTX (6 h). Thereafter, each group was treated with or without X-ray irradiation. Specifically, cells in different groups were first treated with different media (cell culture medium containing 50 μg/mL GNPs, cell culture medium containing 5 nM DTX, or cell culture medium) in different sequences. Then the groups with X-ray treatment were irradiated with different doses of X-rays (0, 2, 4, and 6 Gy). After that, cells in different groups were washed and trypsinized, and 2000 cells in each group were counted and seeded in a 35 mm dish. There were three replicates for each group. 9



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After further incubation for 6 days, the cells were stained with Giemsa and counted to calculate the survival fractions to obtain the survival curves. Other Experimental Details. Materials, synthesis, and characterization of GNPs, other in vitro experiments (including cell culture and cytotoxicity assay, evaluation of reactive

oxygen

species

(ROS)

levels,

DNA

double-strand

breaks,

cell

apoptosis/necrosis assay), calculation of SER, and statistical analysis can be consulted in the Supporting Information.

RESULTS AND DISCUSSION Preparation and Characterization of GNPs. The spherical GNPs were prepared via the citrate reduction of chloroauric acid and further stabilized with the thiolterminated monomethoxy poly(ethylene glycol) (HS-PEG, M.W. ~5000 Da). To characterize the PEG-modified GNPs, we first used transmission electron microscopy (TEM) to detect the size and morphology. The PEG-modified GNPs were spherical (or close to spherical) in shape and displayed good water-dispersity with an average size of 28.0 ± 2.4 nm (Figure 1A). The PEG-modified GNPs shows a UV‒vis absorption peak at ~530 nm (Figure 1B), which is consistent with the previously reported GNPs with a similar size.65 To verify the successful PEGylation of the GNPs after the treatment of HS-PEG, dynamic light scattering (DLS) was used to examine the hydrodynamic diameters of GNPs before and after PEG modification (Table 1). For PEG-modified GNPs, the larger hydrodynamic diameter value (52.5 ± 23.5 nm) in 10


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comparison with the average size obtained from TEM image mainly results from the existence of the surface hydration layer. Meanwhile, the hydrodynamic diameter of the GNPs after PEG modification was larger than that of the GNPs before modification. In addition, the zeta potential value of the GNPs changed from –30.2 ± 10.8 mV to –5.7 ± 10.3 mV after PEG modification. Both of the above results verify the existence of the PEG chains after the surface modification. In the following parts, we will refer the PEGmodified GNPs as GNPs.

Figure 1. (A) TEM image and size distribution histogram (inset) of GNPs. (B) UV–vis spectrum of GNPs (10 μg/mL) dispersed in water.

Table 1. Hydrodynamic Diameters and Zeta Potentials of GNPs before and after PEG Modification

Sample

Hydrodynamic diameter (d, nm)

11


Zeta potential (mV)


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GNPs before PEG modification

25.9 ± 17.5

–30.2 ± 10.8

GNPs after PEG modification

52.5 ± 23.5

–5.7 ± 10.3

Cytocompatibility of GNPs and DTX. Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay was applied to evaluate the cytotoxicities of GNPs and DTX. The results in Figure 2A suggest that GNPs had low cytotoxicity to MCF-7 cells even when the Au concentration of GNPs reached 100 μg/mL. In particular, the cell viability was over 85% when the concentration was lower than 50 μg/mL. Therefore, the dose of GNPs was chosen at 50 μg/mL for the following experiments. For DTX, the results in Figure 2B indicate that it elicited low toxicity toward the cells at the concentrations of lower than 50 nM. Especially, when the concentration was lower than 5 nM, the cell viability was above 90% after incubation for 6, 12, and 24 h. In the following experiments, the concentration of DTX was selected at 5 nM and incubation time was chosen at 6 h.

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Figure 2. Effects of GNPs (A) and DTX (B) on the relative viabilities of MCF-7 cells. For (A), the cells were exposed to GNPs for 24 h, while for (B), the cells were exposed to DTX for 6, 12, and 24 h, respectively.

Cell Arrest in G2/M Phase Induced by DTX and GNPs. Flow cytometry was used to investigate whether the MCF-7 cells can be arrested in G2/M phase by DTX or GNPs. It was observed that the DTX treatment significantly increased the cell percentage in the G2/M phase from 23.3% to 34.5% (Figure 3A); in contrast, the GNP treatment only slightly elevated the percentage from 22.3% to 24.1% (Figure S1). The results indicate that the ability of GNPs to regulate the cell cycle is much weaker compared with that of DTX. To further demonstrate the G2/M phase regulation ability of DTX, confocal imaging experiments were carried out. As shown in Figure 3B, the cells showed intact, uniformly dispersed microtubule networks before DTX treatment. In contrast, bundle-like microtubule networks were formed after treatment with 5 nM of DTX, which demonstrated that DTX could promote the tubulin assembly into a stable microtubule. Besides, more cells in the nuclear division process (M phase) were observed after the treatment with DTX, indicating that the mitosis process was inhibited. Cellular Uptake Efficiency of GNPs. To investigate the time-dependent cellular uptake of GNPs, the dark field images of the MCF-7 cells after exposure to the GNPs for different time periods were obtained by confocal microscopy and analyzed with the ImageJ software. The results shown in Figure 4A reveal that the endocytosis of GNPs 13



rapidly increased during the first 2 h, followed by a slow rise, and plateaued after about 6 h. The above results were confirmed by the results of the time-dependent cellular uptake of GNPs measured by ICP-OES (Figure S2). Therefore 6 h was selected as the incubation time of GNPs in the following experiments. Then we tested whether the endocytosis of GNPs could be enhanced after the DTX pretreatment. The confocal imaging results indicate that the uptake was indeed higher after DTX pretreatment (Figure 4B). The corresponding statistical results (Figure 4C) and the ICP-OES results (Figure S3) of the number of GNPs per cell show an impressive enhancement of the cellular uptake of GNPs after DTX pretreament. This enhancement may be beneficial for the enhanced radiosensitization effect of the GNPs, which will be discussed below.

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Figure 3. (A) Cell cycle distribution results (measured by flow cytometry) of MCF-7 cells left untreated (control) and treated with 5 nM DTX for 6 h. (B) Representative confocal fluorescence images showing the microtubule structures in the MCF-7 cells left untreated (control) and treated with 5 nM DTX. Nuclei and α-tubulins were stained with Hoechst 33342 (blue) and Tubulin-Trakcer Red (red), respectively.

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Figure 4. (A) Time-dependent cellular uptake amount of GNPs. (B) Representative dark-field and confocal fluorescence images of the MCF-7 cells treated with GNPs (50 μg/mL) without (left) or with (right) the pretreatment of DTX. Nuclei and plasma membrane were stained with Hoechst 33342 (blue) and Chol-PEG-FITC (green), respectively. Scale bars: 10 μm. (C) Corresponding statistical results of the cellular GNPs. **p < 0.01.

Improved Radiotherapeutic Performance of Cancer Cells by Sequential DTX/GNP Treatment. It has been reported that cell-cycle phase will affect the radiosensitivity of cells, and the G2/M phase is the most radiosensitive phase.45 In 16


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chemotherapy involving the use of several chemotherapeutics, different treatment sequences of drugs sometimes lead to different therapeutic outcomes.66 Considering the radiosensitization effect of both DTX and GNPs, we compared the renewal efficiency of MCF-7 cells after exposure to one drug alone (DTX or GNPs), two drugs (added simultaneously), or two drugs with different treatment sequences, followed by X-ray irradiation at various doses (0, 2, 4, and 6 Gy). The results are presented in Figure 5 and S4. The SER calculation results (Figure 5C) indicate that DTX or GNPs alone exhibited only a weak radiosensitization effect on the MCF-7 cells, with an SER of 1.16 and 1.29, respectively. The group simultaneously treated with the two drugs showed a higher SER of 1.32. Interestingly, the sequential exposure of cells to DTX and GNPs significantly increased the radiosensitization effect—the SER of this sequential delivery reached as high as 1.91. The much higher SER value of the “DTX→GNPs” group compared with that of the “DTX + GNPs” group can be explained by the capability of DTX to arrest more cells in the most radiosensitive G2/M phase, which can notably promote the cellular internalization of GNPs.

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Figure 5. (A) Representative colony formation images of MCF-7 cells which experienced different treatments. Red dots represent the positions of cell colonies. For the enlarged images and corresponding quantitative results, please refer to Fig. S4. The concentration of DTX was 5 nM and the concentration of GNPs was 50 μg/mL. (B) Corresponding survival fraction curves of MCF-7 cells which experienced different treatments. (C) SER values of different drug treatments. *p < 0.05, **p < 0.01, ns: nonsignificant.

Potential Mechanisms of the Radiosensitization Effect of Sequential DTX/GNP Treatment: ROS Generation, DNA Damage, and Apoptosis. To investigate whether the sequential DTX/GNP treatment can cause DNA damage in cancer cells, 18


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phosphorylated histone H2AX (γ-H2AX) staining was used to evaluate the formation of DNA double-strand breaks (DSBs) in the cells. Confocal microscopy was applied to detect the γ-H2AX foci (stained by Alexa Fluor 488-labeled anti-phospho-H2AX (S139) and shown as red fluorescent dots) in the cell nuclei and the ImageJ software was used to analyze the foci in the nuclear region in the corresponding confocal images. The confocal images (Figure 6A) and the corresponding statistical results (Figure 6B) show that, for the groups without X-ray irradiation, there were few γ-H2AX foci in the “Control”, “GNPs”, and “DTX” groups. Because of the ability of DTX and GNPs to induce DNA DSBs,3,67 and the synergistic effect of the two drugs, the “GNPs + DTX”, “GNPs→DTX”, and “DTX→GNPs” groups showed slightly increased γ-H2AX foci inside the cells. For the groups after X-ray irradiation, they exhibited increased γ-H2AX foci within the cells and the “DTX→GNPs” group had the most γ-H2AX foci per cell, which is caused by the highest internalization level of GNPs in addition to the combined effect of DTX and GNPs.

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Figure 6. Evaluation of DNA damage level and ROS generation in MCF-7 cells which experienced different treatments. (A) Representative immunofluorescence images of γH2AX foci in the cells after different treatments. Scale bars: 10 μm. (B) Corresponding statistical results of the samples in (A). (C) Statistical results of ROS levels in the cells after different treatments. *p < 0.05, **p < 0.01, ***p < 0.001, ns: non-significant.

Besides, it has been proposed that the ROS level increases when radiosensitizers take 20


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effect.68 Hence, the intracellular ROS level in each group was detected using 2,7dichlorodihydrofluorescein diacetate (DCFH-DA), whose fluorescence intensity can reflect the level of intracellular ROS and can be detected by flow cytometry. As shown in the flow cytometric data (Figure 6C), the intracellular ROS level increased notably after receiving X-ray irradiation (4 Gy). In addition, for the groups with X-ray irradiation, the GNP- and DTX-treatments could not remarkably increase the ROS level. Meanwhile, the ROS levels of the “DTX→GNPs”, “GNPs→DTX”, and “DTX + GNPs” groups were 1.4-, 1.2-, and 1.1-fold of that in the control group after X-ray irradiation since both drugs can induce the ROS generation and the effect may be enhanced through their combination.69,70 The above results show that the “DTX→GNPs” group after receiving X-ray irradiation had the largest increase in the ROS level, which can be explained by the significantly increased cellular uptake of GNPs with the DTX pretreatment, indicating that the increased ROS production should be one of the main reasons of the enhanced radiosensitization of the “DTX→GNPs” treatment. Finally, the cell apoptosis rates induced by different treatments were measured before and after the 4 Gy X-ray via flow cytometry. As shown in Figure 7, the X-ray irradiation notably increased the apoptosis rates of the cells treated with “GNPs + DTX”, “DTX→GNPs”, and “GNPs→DTX”. In particular, the cells receiving “DTX→GNPs” and “X-ray irradiation” treatments had the highest apoptosis ratio of 12.82%. The flow cytometric results indicate that the radiosensitization effects induced by these drug treatments are mainly attributed to cell apoptosis. 21



Figure 7. Flow cytometric data of MCF-7 cells which experienced different treatments. DTX: 5 nM, GNPs: 50 μg/mL. 22


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CONCLUSION In this work, we develop a sequential dual-strike radiotherapy approach which significantly improves the cellular uptake and radiosensitization effect of nanoradiosensitizers (using GNPs as a representative). The pretreatment of a very low concentration of DTX (5 nM) can significantly arrest cells in the most radiosensitive G2/M phase, and promote the cellular internalization of nanoradiosensitizers, thus enhancing the radiotherapeutic effect. We also demonstrate that increases in the ROS level and cell apoptosis rate can markedly enhance the radiosensitization of the sequential DTX/GNP treatment. In addition, this sequential dual-strike approach is also an effective combination therapy that meets the criteria for a unique but complementary mechanism of action in which two drug systems work synergistically. Considering the wide use of the two-step infusion method in the clinic for combination chemotherapy, this sequential DTX/GNP treatment may also find clinical applications. Finally, due to its low cost, simple design, and high feasibility, the dual-strike radiotherapy may augur well for clinical applications. It is hoped that the present work will promote the clinical translation of nanoradiosensitizers for cancer therapy, and may have implications for enhancing the therapeutic efficacies of various other radiosensitizers.

ASSOCIATED CONTENT Supporting Information 23



The Supporting Information is available free of charge on the ACS Publication website at DOI: Other experimental details, cell cycle distribution results, ICP-OES results, enlarged colony formation images of Figure 5A, quantitative analysis results of colony formation assay, and the corresponding references (PDF)

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

ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (21673037 and 81571805).

REFERENCES (1) Haume, K.; Rosa, S.; Grellet, S.; Śmiałek, M. A.; Butterworth, K. T.; Solov'yov, A. V.; Prise, K. M.; Golding, J.; Mason, N. J. Gold Nanoparticles for Cancer Radiotherapy: A Review. Cancer Nanotechnol. 2016, 7, 8. (2) Hainfeld, J. F.; Dilmanian, F. A.; Slatkin, D. N.; Smilowitz, H. M. Radiotherapy Enhancement with Gold Nanoparticles. J. Pharm. Pharmacol. 2008, 60, 977‒985. (3) Su, X. Y.; Liu, P. D.; Wu, H.; Gu, N. Enhancement of Radiosensitization by MetalBased Nanoparticles in Cancer Radiation Therapy. Cancer Biol. Med. 2014, 11, 86‒91. (4) Shi, W. Q.; Yuan, L. Y.; Wang, C. Z.; Wang, L.; Mei, L.; Xiao, C. L.; Zhang, L.; 24


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Sequential Treatment of Cell Cycle Regulator and ... - ACS Publications

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