Shape-Dependent Radiosensitization Effect of ... - ACS Publications

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Shape-Dependent Radiosensitization Effect of Gold Nanostructures in Cancer Radiotherapy: Comparison of Gold Nanoparticles, Nanospikes, and Nanorods Ningning Ma,† Fu-Gen Wu,*,† Xiaodong Zhang,† Yao-Wen Jiang,† Hao-Ran Jia,† Hong-Yin Wang,† Yan-Hong Li,† Peidang Liu,‡ Ning Gu,† and Zhan Chen*,§ †

State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, and ‡Institute of Neurobiology, School of Medicine, Southeast University, Nanjing 210096, People’s Republic of China § Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor, Michigan 48109, United States S Supporting Information *

ABSTRACT: The shape effect of gold (Au) nanomaterials on the efficiency of cancer radiotherapy has not been fully elucidated. To address this issue, Au nanomaterials with different shapes but similar average size (∼50 nm) including spherical gold nanoparticles (GNPs), gold nanospikes (GNSs), and gold nanorods (GNRs) were synthesized and functionalized with poly(ethylene glycol) (PEG) molecules. Although all of these Au nanostructures were coated with the same PEG molecules, their cellular uptake behavior differed significantly. The GNPs showed the highest cellular responses as compared to the GNSs and the GNRs (based on the same gold mass) after incubation with KB cancer cells for 24 h. The cellular uptake in cells increased in the order of GNPs > GNSs > GNRs. Our comparative studies indicated that all of these PEGylated Au nanostructures could induce enhanced cancer cellkilling rates more or less upon X-ray irradiation. The sensitization enhancement ratios (SERs) calculated by a multitarget singlehit model were 1.62, 1.37, and 1.21 corresponding to the treatments of GNPs, GNSs, and GNRs, respectively, demonstrating that the GNPs showed a higher anticancer efficiency than both GNSs and GNRs upon X-ray irradiation. Almost the same values were obtained by dividing the SERs of the three types of Au nanomaterials by their corresponding cellular uptake amounts, indicating that the higher SER of GNPs was due to their much higher cellular uptake efficiency. The above results indicated that the radiation enhancement effects were determined by the amount of the internalized gold atoms. Therefore, to achieve a strong radiosensitization effect in cancer radiotherapy, it is necessary to use Au-based nanomaterials with a high cellular internalization. Further studies on the radiosensitization mechanisms demonstrated that ROS generation and cell cycle redistribution induced by Au nanostructures played essential roles in enhancing radiosensitization. Taken together, our results indicated that the shape of Au-based nanomaterials had a significant influence on cancer radiotherapy. The present work may provide important guidance for the design and use of Au nanostructures in cancer radiotherapy. KEYWORDS: shape-dependent, gold nanostructures, X-ray radiotherapy, radiosensitizing effect, anticancer

1. INTRODUCTION Important progress has been made in conventional cancer therapeutic strategies such as surgical resection, chemotherapy, and radiotherapy. Among these therapeutic strategies, ionizing radiation therapy (i.e., radiotherapy) with high-energy γ rays or X-rays was mainly used for primary therapy of localized tumors and for adjuvant and palliative therapy. It is estimated that radiotherapy has contributed to tumor radical cure for approximately 50% of cancer patients.1 Currently, how to deal with the radiation resistance of hypoxic tumor cells is still a major challenge in radiation oncology. It is impossible to continuously raise the radiation dose to kill hypoxic tumor cells because then the radiation would cause damage to normal proximal tissues. It was found that the use of radiosensitizers is an effective way to enhance the radiotherapy efficacy against the © 2017 American Chemical Society

malignant tumor while drastically decreasing the needed radiation dose and reducing irreversible damage to normal tissues. Clinically, the most widely used radiosensitizers mainly include radiosensitive drugs and oxygen.2 With the advanced progress of nanoscience and nanotechnology, a large number of metal-based nanomaterials have been widely explored to enhance the therapeutic efficiency and specificity of radiotherapy, such as bismuth,3−5 gold,6−16 platinum,17−19 tungsten,20,21 tantalum,22,23 hafnium,24 silver,25−29 molybdenum,30 iron,31,32 and rare earth elements (e.g., gadolinium and cerium).33−37 Among these metal-based Received: January 22, 2017 Accepted: March 24, 2017 Published: March 24, 2017 13037

DOI: 10.1021/acsami.7b01112 ACS Appl. Mater. Interfaces 2017, 9, 13037−13048

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ACS Applied Materials & Interfaces

glioblastoma radiation therapy.14 To improve the local concentration of Au nanoparticles in the vicinity of cancer cells, the Au nanoparticle-loaded multilayer polyelectrolyte microdisks were designed to enhance cancer radiation therapy.15 Very recently, we reported that gold nanospikes can achieve an enhanced radiosensitization effect if they are used in the combined cancer radiation and photothermal therapy.16 These previous investigations have described the applications of spherical gold nanoparticles, gold nanorods, and gold nanospikes in cancer radiotherapy. However, few comparative studies have evaluated the shape effect of Aubased nanomaterials on their biological effects in cancer radiotherapy. In the present study, we carried out a detailed investigation to provide an in-depth understanding of the shape effect of Au nanomaterials on radiosensitization. We synthesized three types of Au-based nanomaterials, spherical gold nanoparticles (GNPs), gold nanospikes (GNSs), and gold nanorods (GNRs), with similar sizes but different shapes, and then compared their effects on cancer radiotherapy at clinically relevant megavoltage energies. To keep the same surface chemistry and increase the stability of these Au-based nanomaterials, they were all chemically modified with poly(ethylene glycol) (PEG) molecules. We have evaluated the cytotoxicity and cellular uptake efficiency of these Au nanostructures, and investigated the potential mechanisms of how these gold nanostructures induced enhanced radiosensitization.

nanomaterials, gold (Au)-based nanomaterials as emerging radiosensitizers have shown great radiosensitizing effect and also been widely employed for cancer radiotherapy due to their unique advantages such as good biocompatibility, excellent optical properties, facile synthesis, and ease of surface modification.6−16 The Au-based nanomaterials can enhance tumor radiosensitization because of the following potential mechanisms: (1) Gold is a high atomic number material (Z = 79) that can absorb high-energy γ rays or X-rays and emit photoelectrons, Auger electrons, Compton electrons, and fluorescence photons. Subsequently, these emissions cause the ionization of intracellular components or water molecules to generate reactive oxygen radicals.38,39 These generated free radicals diffuse via chain reactions in the cells and finally cause irreversible damage to intranuclear DNA double strands.40,41 (2) The effect of γ or X-rays on tumor cells also depends on the cell cycle distribution and DNA self-repair capability. Au-based nanomaterials induce a shorter G0/G1 phase while extending the radiosensitive G2/M phase in cancer cells, which will cause the cell cycle synchronization and kill cells more efficiently.42−44 The radiation enhancement effect induced by Au nanomaterials was influenced by many key parameters including the size, shape, surface chemistry, and concentration of Au nanomaterials, as well as the cell type and ionizing radiation. Furthermore, the cellular uptake efficiency of Au nanomaterials plays a critical role in inducing the radiation enhancement effect. It is commonly believed that more internalized nanoparticles will produce more reactive oxygen species (ROS) in cancer cells, thus leading to a larger extent of DNA damage. Some previous studies have investigated the cellular uptake of Au-based nanomaterials with different sizes and/or shapes. For instance, previous studies reported that the cellular uptake efficiency of spherical gold nanoparticles depends largely on their physical dimensions, and the cellular internalization of 50 nm nanoparticles was much higher than that of the 14, 30, 74, and 100 nm nanoparticles.45 Moreover, the spherical Au nanoparticles show a higher cellular internalization as compared to the rod-shaped ones with a similar size.45 Similarly, it has been reported that the spherical Au nanoparticles have higher cellular internalization than similar-sized rod-shaped or starshaped Au nanoparticles.46 Au-based nanomaterials with different sizes have been extensively used for cancer radiotherapy. It was found that the 50 nm-sized Au nanoparticles have a stronger radiosensitization effect than the 14 and 74 nm-sized ones because the 50 nm-sized gold nanoparticles have the highest cellular uptake.8 The result demonstrated that the radiosensitizing effect induced by Au nanomaterials is tightly related to the amount of internalized nanomaterials. In another study, it was shown through in vitro and in vivo experiments that the 12.1 and 27.3 nm PEG-coated gold nanoparticles have greater radiosensitization effects than the 4.8 and 46.6 nm counterparts.9 In addition to the size effect, the shape and surface coating effects of Au nanomaterials on the efficiency of radiotherapy have also been examined. For example, ultrasmall glutathione-protected gold nanoclusters with high tumor uptake were utilized as the next-generation sensitizers for cancer radiotherapeutic effect.10−12 The arginine-glycineaspartate (RGD) peptide-conjugated Au nanorods were found to enhance the efficiency of radiotherapy in A375 melanoma cancer cells.13 Bovine serum albumin (BSA)-capped Au nanoparticles were employed as radiation sensitizers for U87

2. MATERIALS AND METHODS 2.1. Materials. In this study, all chemicals of analytical grade were used. Silver nitrate (AgNO3), trisodium citrate dihydrate (Na3C6H5O7· 2H2O), HAuCl4·3H2O, L-ascorbic acid (LAA), hexadecyltrimethylammonium bromide (CTAB), sodium borohydride (NaBH4), dimethyl sulfoxide (DMSO), fluorescein isothiocyanate (FITC), and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO) and used as received unless noted otherwise. Thiol-terminated monomethoxy poly(ethylene glycol) (HS-PEG, MW = 2000 Da) was bought from JenKem Technology CO., Ltd. (Beijing, China). All aqueous solutions were prepared using ultrapure water with a resistivity of 18.2 MΩ cm obtained from a Milli-Q water purification system (Millipore Corp., Billerica, U.S.). Human oral epidermoid carcinoma (KB) cell lines were obtained from the Type Culture Collection of the Chinese Academy of Science, Shanghai, China. 2.2. Synthesis of PEG-Coated Au Nanostructures with Different Shapes. The spherical GNPs (also known as Au nanospheres) used here were prepared according to the previously reported protocol.47 Specifically, 50 mL of 0.01% HAuCl4 solution was heated to boiling. 0.25 mL of 1% sodium citrate solution was then added rapidly into the boiling solution of HAuCl4 with vigorous stirring, and the mixture was kept boiling for 15 min. When the reaction solution was cooled naturally, citrate-stabilized GNPs were obtained. To stabilize the as-synthesized GNPs, 1 mg of HS-PEG was added into the freshly prepared GNP solution, and the solution was stirred overnight at room temperature. Excessive HS-PEG was removed by centrifugation at 10 000 rpm and washed three times with deionized water to obtain PEGylated GNPs. GNSs were prepared via the galvanic replacement reaction between Ag nanoparticles (AgNPs) and HAuCl4 according to the reported method with a slight modification.48 AgNPs were synthesized by citrate reduction of AgNO3 according to the previous report.16 The GNS solution was prepared by dropping AgNP solution (5 mL) into 1% HAuCl4 aqueous solution (140 μL) with vigorous stirring followed by the addition of some LAA aqueous solution (10 mM, 1 mL). The color of the solution changed from light yellow to dark blue within a short period of time. To coat PEG on the GNS surfaces, HS-PEG (250 μg) was added to the GNS solution, and the mixture was kept stirring 13038


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ACS Applied Materials & Interfaces at room temperature for 2 h. The final PEG-coated GNS solutions were washed twice with water and then redispersed in water for further use. GNRs were prepared using a seed-mediated growth method as described previously.49 To do so, CTAB-coated Au seeds were first obtained by the chemical reduction of HAuCl4 and NaBH4, and were used within 2 h. In more detail, 5 mL of 0.01% HAuCl4 aqueous solution was mixed with 5 mL of 0.2 M CTAB. After that, 0.6 mL of ice-cold 0.01 M NaBH4 was rapidly added into the mixture with vigorous stirring. The seed solution then was kept under vigorous stirring for 2 min and stored at room temperature for further use. Under gentle stirring, 12 μL of the Au seed solution was added to the growth solution mixed by 1% AgNO3 aqueous solution (17 μL), CTAB aqueous solution (0.2 M, 5 mL), 0.03% HAuCl4 aqueous solution (5 mL), and LAA aqueous solution (10 mM, 5 μL). The temperature of the growth medium was kept at 30 °C overnight. This procedure resulted in the formation of GNRs with the aspect ratio of up to 4. The as-synthesized CTAB-stabilized GNRs were washed three times with deionized water to remove excess CTAB and then redispersed in water. HS-PEG aqueous solution (400 μL, 1 mg/mL) then was added to the GNR solution, and the resultant solution was stirred at room temperature for 12 h. The PEG-coated GNRs were collected by centrifugation, washed with water, and then redispersed in water. 2.3. Characterization of GNPs, GNSs, and GNRs. The size and morphology of synthesized Au nanostructures were investigated using a field emission scanning electron microscope (SEM, Zeiss Ultra Plus, Germany) at an accelerating voltage of 20 kV. For each sample, the mean size of Au nanostructure was calculated from representative images, and the size histogram was generated simultaneously by Nano Measurer software (Version 1.2). The elemental analysis of the assynthesized GNSs was performed with a field emission scanning electron microscope (FESEM) equipped with an energy-dispersive spectrometer (EDS). The hydrodynamic diameter and zeta potential of Au nanostructures were measured by dynamic light scattering (DLS) using a Malvern Zetasizer (Nano ZS, Malvern, UK). UV−vis spectra were collected using a UV−vis spectrophotometer (UV-2600, Shimadzu, Japan) with a wavelength range of 300−900 nm. The fluorescence spectra of Au nanostructures before and after FITC labeling were recorded by a spectrofluorophotometer (RF-5301PC, Shimadzu, Japan). 2.4. Determination of Au Nanostructure Concentration by Atomic Absorption Spectroscopy (AAS). The final concentration of the Au nanostructure solution was measured with atomic absorption spectroscopy (AAS, AANALYST 400, U.S.). Typically, Au nanostructures were digested by freshly prepared aqua regia, transferred to a 10 mL volumetric flask, and then diluted with deionized water to the mark. The standard stock solution of Au element was purchased from the National Research Center for Certified Reference Materials (NRCCRM, Beijing, China). The stock solution was diluted to a series of solutions with different concentrations (e.g., 1, 2, 3, 4, and 5 μg/ mL), and the absorbance values of those solutions were measured by AAS to create the standard curve. According to the standard curve, the concentration of the prepared Au nanostructure solution was determined under the same conditions. 2.5. Cell Culture and Proliferation Assay. KB cells were cultured in high-glucose complete Dulbecco’s modified Eagle’s medium (DMEM) plus 10% fetal bovine serum (FBS), streptomycin (0.1 mg/mL), and penicillin (100 U/mL) at 37 °C in a humidified incubator with 5% CO2. The cytotoxicity of Au nanostructures against KB cells was determined by MTT assay. In brief, KB cells were seeded onto 96well plates at a density of 5 × 103 cells per 100 μL of culture medium and continuously cultured overnight until ∼80% confluence in a humidified 37 °C incubator. After exposure to Au nanostructures with various concentrations for 24 h, the medium in each well was replaced with 100 μL of fresh medium containing 10 μL of 5 mg/mL MTT solution, followed by incubation at 37 °C for 4 h. The formed formazan crystals were dissolved in 150 μL of DMSO. Absorbance at 570 nm in each well was measured with a Multiskan FC microplate

photometer (Thermo Scientific). Each group was measured in triplicate. 2.6. Shape-Dependent Cellular Uptake of Au Nanostructures. The shape-dependent cellular uptake of gold nanostructures was determined by confocal laser scanning microscopy (CLSM, TCS SP8, Leica, Germany) using the following methods. In method one, the cellular uptake was visualized by labeling the Au nanostructures with FITC, a green fluorescent probe. FITC-labeled Au nanostructures were synthesized via the covalent binding of HS-PEGFITC onto the nanostructure surface. Briefly, HS-PEG-FITC was prepared by the addition reaction between FITC and HS-PEG-NH2. The HS-PEG and HS-PEG-FITC were mixed in a molar ratio of about 99:1 and immediately added into the freshly prepared Au nanostructure solutions under vigorous stirring for 2 h at room temperature. Excessive PEG derivatives were removed by centrifugation and washed three times with water to obtain FITC-labeled Au nanostructures (i.e., FITC-labeled GNPs, FITC-labeled GNRs, and FITC-labeled GNSs). Meanwhile, KB cells were seeded to eight-well chambered coverglass (Lab-Tek) at a density of 3 × 104 cells/mL (7000 cells per well) in a 37 °C incubator overnight. Cell culture medium then was replaced with freshly prepared FITC-labeled gold nanostructure solution in DMEM (50 μg/mL) and incubated for another 24 h. After that, the cells were washed with cold phosphate buffered saline (PBS) solution and stained with Hoechst 33342 (Beyotime, China). The fluorescence signals of the cells were observed using CLSM. Afterward, the cells were detached from wells using trypsin-EDTA. The green fluorescence signal within cells from FITClabeled Au nanostructures was quantified via a flow cytometer (NovoCyte, ACEA). In method two, Au nanostructures internalized by the cells were visually identified by dark-field microscopy,50 which can make full use of the scattering optical properties of Au materials. Optical excitation of Au nanostructures produces a localized surface plasmon resonance effect that results in a strong optical scattering in the visible light frequency range. Therefore, Au nanostructures inside the cells can be visualized as bright spots. In addition, the amount of internalized Au nanostructures can be statically quantified according to cellular side scatter (SSC) using flow cytometry.51,52 Internalized Au nanostructures inside the cells leads to increased cellular granularity that is related to the signal intensity of SSC measured by flow cytometry. Thus, quantitative analysis of cellular uptake of Au nanostructures can be conducted via measuring the intracellular signal intensity of SSC. In a typical experiment, the cells were seeded to eight-well chambered coverglass in a 37 °C incubator overnight. The cell culture medium then was replaced with DMEM medium containing one of the abovementioned freshly prepared PEGylated Au nanostructures (50 μg/ mL) and incubated for an additional 24 h. Afterward, the cells were washed twice with cold PBS to remove excessive Au nanostructures in the cell culture medium. The cell nuclei were stained blue with Hoechst 33342 dye for cellular localization. The samples were mounted, and the scattering dark-field images of the cells were observed using confocal microscopy. For the quantitative analysis of cellular side scatter, the cells were harvested and washed twice with PBS. The mean side-scattered light intensity from Au nanostructures in the cells was measured via flow cytometry. 2.7. Colony Formation Assay. KB cells were cultured in six-well plates at a density of 5 × 104 per well for 24 h. When the cells grew to ∼80% confluence in plates, they were divided into eight groups: (a) control, (b) GNPs only, (c) GNSs only, (d) GNRs only, (e) X-ray only, (f) GNPs + X-ray, (g) GNSs + X-ray, and (h) GNRs + X-ray. Group (a) was the control cell group without any treatment. Groups (b) and (f) were treated with GNPs (50 μg/mL) in DMEM for 24 h. Similarly, groups (c) and (g) were treated with GNSs (50 μg/mL) in DMEM for 24 h. Groups (d) and (h) were treated with GNRs (50 μg/ mL) in DMEM for 24 h. After 24 h treatment, the Au nanostructurecontaining culture media were replaced with fresh culture media. Concurrently, groups (e), (f), (g), and (h) received the irradiation of X-rays (6 MV, 200 cGy/min) by a linear accelerator (Primus-M, Siemens, Germany) at various doses of 2, 4, 6, and 8 Gy, respectively. After different treatments, the cells were washed twice with PBS buffer 13039


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ACS Applied Materials & Interfaces and trypsinized. Finally, 1500, 1500, 3000, 3000, and 3000 cells at corresponding doses of 0, 2, 4, 6, and 8 Gy were counted and seeded in 35 mm dishes with 2 mL of fresh culture medium. Depending on the cell proliferation and colony formation rates of KB cells, the cells were incubated for an additional 10 days followed by Giemsa staining (KeyGen Biotech, Nanjing, China). The colonies formed with more than 50 cells were counted to evaluate the effects of respective treatments. Note that for each group there were three replicates. 2.8. Calculation of Sensitization Enhancement Ratio (SER). The cell survival fraction of each group was calculated by the ratio of the number of colonis formed by seeded cells after various treatments versus the number of colonies formed by untreated cells. The classical multitarget single-hit model was applied to perform nonlinear fitting for the cell survival fraction of each group using both SPSS and origin 7.0 software. Meanwhile, the sensitization enhancement ratio (SER) of each group was also determined by a classical multitarget single-hit model (details of the SER calculation can be found in the Supporting Information). 2.9. Detection of ROS. To measure the generation of intracellular ROS in each group, the cells were treated with a fluorescent marker, dichlorodihydrofluorescein diacetate (DCFH-DA, KeyGen Biotech, Nanjing, China) (10 μM), in DMEM without phenol red for 25 min in dark (37 °C, 5% CO2). The cells were then washed with PBS and resuspended in culture medium. Especially, the cells pretreated with the ROS positive control reagent (Rosup) for 30 min were set as the positive control group. All of the cells were immediately trypsinized and analyzed by flow cytometry. The fluorescence signals were detected in the FL-1 channel (Ex/Em = 488 nm/525 nm). For each sample, the mean fluorescence intensity of 10 000 cells was recorded to determine the intracellular content of ROS. 2.10. Cell Cycle Distribution. The change of cell cycle was evaluated by a cell cycle detection kit (KeyGen Biotech, Nanjing, China). After respective treatments in each cell group, the cells were washed with PBS and fixed with precooled 70% ethanol solution and stored at 4 °C. Prior to staining, the ethanol solution was removed, and the cells were incubated with 100 μL of RNase A at 37 °C for 30 min. Next, 400 μL of 50 μg/mL propidium iodide (PI) was added, and the mixture was incubated at 4 °C for 30 min. Finally, the cell cycle was analyzed with a flow cytometer in the FL-2 channel (Ex/Em = 488 nm/630 nm). 2.11. Cell Apoptosis/Necrosis Analysis. To compare the apoptosis-inducing capabilities of different shaped Au nanostructures before and after X-ray irradiation, the Annexin V-FITC/PI apoptosis detection kit (KeyGen Biotech, Nanjing, China) was used for evaluating cell apoptosis and necrosis ratios. Typically, after respective treatments, KB cells (∼1 × 106 cells) in each group were trypsinized and resuspended in 500 μL of 1× binding buffer. Afterward, 5 μL of Annexin V-FITC (20 μg/mL) and 5 μL of PI (50 μg/mL) were sequentially added into the above 1× binding buffer, and the cells were incubated for another 15 min at room temperature in dark. Finally, the quantitative analyses of cell apoptosis and necrosis were performed immediately by flow cytometry.

Figure 1. Shapes, aqueous dispersibility, and UV−vis spectra of the three Au nanostructures. (A) Schematics of the PEGylated GNPs, GNSs, and GNRs. (B) Photographs of the PEGylated GNPs, GNSs, and GNRs dispersed in phosphate buffered saline (PBS) solutions. (C) UV−vis absorption spectra of the PEGylated GNPs (1), GNSs (2), and GNRs (3). The maximum absorption peaks for GNPs, GNSs, and GNRs are at 528, 670, and 700 nm, respectively.

maximum absorption peaks for GNPs, GNSs, and GNRs centered at 528, 670, and 700 nm, respectively. Especially, GNSs exhibited a very broad absorption band in the wavelength range of 500−900 nm, which may originate from the high density of surface spikes. SEM experiments were carried out to investigate the size and morphology of the three Au nanomaterials. The SEM images presented in Figure 2 (left) clearly confirmed the unique morphologies of the three nanomaterials. The size distribution histograms (Figure 2, right) obtained by counting over 200 particles in the corresponding SEM images revealed that the average diameters of GNPs and GNSs were 53.2 ± 13.4 and 54.0 ± 9.3 nm, respectively, while the average length and width of GNRs were 50.2 ± 6.1 and 12.4 ± 2.0 nm (with an aspect ratio of around 4:1), respectively. Furthermore, elemental analysis by EDS revealed the presence of a very small quantity of silver atoms inside the resultant GNSs (Figure S1) due to the incomplete dissolution of silver atoms on the GNS surface. We have also measured the hydrodynamic diameters of these Aubased nanostructures by DLS, and the obtained values were all larger than the average sizes obtained from representative SEM images, which were mainly due to the presence of the PEG chains and the surface hydration layer (Table 1). In addition, the zeta potential measurement demonstrated that the PEGylated gold-based nanostructures were all negatively charged (Table 1). The negative surface charge and the surface PEG layer ensured that these Au nanomaterials could be stable for up to one month in PBS solutions. Here, we would also like to mention that all of the Au nanomaterials used below for tumor treatment refer to the PEG-coated nanomaterials unless noted otherwise. 3.2. Shape-Dependent Cellular Uptake of Au Nanostructures. The amount of Au nanostructures internalized by

3. RESULTS AND DISCUSSION 3.1. Characterization of Au-Based Nanostructures. Gold-based nanomaterials with different geometries (including GNPs, GNSs, and GNRs) were schematically displayed in Figure 1A. In general, GNPs and GNRs were prepared by citrate reduction and seed-induced growth methods, respectively. GNSs were synthesized via the galvanic replacement reaction between AgNPs and HAuCl4. Finally, PEG, a commonly used stabilizer and protecting agent,53,54 was conjugated on the surface of all three Au-based nanostructures through the coupling between Au atom and thiol-terminated PEG.55 Figure 1B showed that these PEGylated gold-based nanomaterials were well dispersed in PBS solution (pH 7.4, 10 mM). The UV−vis spectra of the three Au nanomaterials showed different absorption patterns (Figure 1C): The 13040


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Figure 2. Representative SEM images (left) and size distribution histograms (right) of GNPs (A), GNSs (B), and GNRs (C). The size distribution histograms were obtained by counting over 200 particles in the corresponding SEM images for each sample. Scale bars: 200 nm. The scale bars in the insets correspond to 20 nm.

with the similar sizes (∼50 nm) for a comparative study in cancer radiotherapy. In this study, confocal fluorescence imaging, dark-field imaging, and flow cytometry were employed to study the cellular uptake efficiency of the three Au nanostructures. Although the three Au nanostructures have the same surface functionalization (i.e., PEG layer) as well as similar sizes (∼50 nm) and surface charges (−14 ∼ −17 mV), they displayed different uptake degrees by KB cells in the following order: GNPs > GNSs > GNRs. Before fluorescence imaging, we first measured the fluorescence intensity of Au nanostructure solutions (50 μg/mL) before and after FITC labeling using fluorescence spectroscopy. As depicted in Figure S2, the fluorescence signal of FITC conjugated on the surface of Au nanostructures could be clearly detected, confirming the feasibility of using fluorescence-based techniques for qualitative

Table 1. Hydrodynamic Diameters and Zeta Potentials of GNPs, GNSs, and GNRs sample

hydrodynamic diameter (d, nm)

polydispersity index (PDI)

zeta potential (mV)

GNPs GNSs GNRs

107.8 ± 32.4 118.1 ± 46.4 108.9 ± 50.6

0.228 0.450 0.351

−16.1 ± 5.4 −16.5 ± 8.2 −14.2 ± 7.1

cancer cells is crucial for inducing irreversible damage after Xray irradiation. Extensive investigations have revealed that either cellular adhesion or cellular uptake of nanomaterials was strongly affected by the main component element, size, shape, and surface chemistry, etc.56−61 Meanwhile, spherical Au nanoparticles with size of 50 nm were reported to be internalized by cells more than the smaller and larger ones. Therefore, we synthesized three types of Au nanostructures 13041


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ACS Applied Materials & Interfaces and quantitative analyses in this research. Next, confocal fluorescence imaging was used for the visual assessment of cells treated with 50 μg/mL FITC-labeled GNPs, FITC-labeled GNSs, or FITC-labeled GNRs for 24 h. As shown in Figure 3A, the cell nuclei were stained blue with Hoechst 33342, and the presence of green fluorescent dots around the blue nuclei indicated the internalization of the FITC-labeled Au nanomaterials. To quantify the cellular uptake of Au nanomaterials by KB cells, the intracellular fluorescence signals were detected by flow cytometry. The mean fluorescence intensity of the cells treated with Au nanomaterials was about 5.5−7.6-fold larger than that of the untreated control group (Figure 3B). Furthermore, it was found that the fluorescence signals from FITC-labeled GNPs-treated cells were 1.2-fold higher than that from FITC-labeled GNSs-treated ones, and 1.4-fold higher as compared to the FITC-labeled GNRs-treated cells. In dark-field imaging experiments, KB cells were treated with 50 μg/mL GNPs, GNSs, and GNRs, respectively. After incubation for 24 h, the cell nuclei were also stained blue with Hoechst 33342 and the presence of bright dots around the nuclei indicated the internalization of intracellular Au nanomaterials. Dark-field imaging makes full use of the unique optical scattering characteristic of metallic element so as to avoid possible fluorescence interference from surface modification of Au nanomaterials. Consistent with the quantitative fluorescence observations, dark-field imaging results revealed that the amount of bright dots displayed clearly a declining trend in the cells treated with GNPs, GNSs, and GNRs, respectively. As shown in Figure 3C, the bright dots in the cytoplasm mostly concentrated around the cell nucleus. Especially, the dark-field imaging indicated the amount of Au in the GNPs-treated cells is larger than that in the GNSs or GNRs-treated cells. Similarly, to quantify the bright dots in the cells, the cellular side scatter signal was also detected by flow cytometry. On the basis of the statistical data, the mean side scatter intensity of the cells treated with Au nanomaterials was about 1.1−1.6-fold higher than that of the untreated cells after incubation for 24 h (Figure 3D). Interestingly, the side scatter intensity in the GNPstreated cells was 1.2-fold higher than that of the GNSs-treated cells, and 1.5-fold higher than that of the GNRs-treated cells. We have quantified their cellular uptake efficiencies of GNPs, GNSs, and GNRs using two distinct methods, while each set of quantitative values was just relative. Thereby, the qualitative conclusion obtained from the above two methods was well correlated with each other, which confirmed that the shape of Au nanomaterials played a key role in the cellular uptake efficiency. The difference in cellular uptake efficiency will further affect their therapeutic effect in cancer radiotherapy, which will be reported below. 3.3. Biocompatibility Evaluation of Au Nanostructures and Shape Effect of Au Nanostructures on Cancer Radiotherapy. For biomedical applications, it is essential to evaluate the cytotoxicity of a potential nanomaterial. To determine the cytotoxicity of Au nanostructures, the cell proliferation ability was examined by MTT assay (Figure 4A). The GNPs, GNSs, and GNRs with different concentrations ranging from 3 to 200 μg/mL exhibited decreased cell viability in a dose-dependent manner after incubation for 24 h, implying that the cytotoxicity of Au nanostructures increased as a function of the nanomaterial concentration. Obviously, the cell viability of the cells treated with respective Au nanostructures at the concentration of 50 μg/mL (which will be used below) was still maintained at approximately 90%, confirming negligible

Figure 3. Cellular uptake of the PEGylated Au nanostructures. (A) Confocal fluorescence images of KB cells incubated with 50 μg/mL FITC-labeled GNPs, GNSs, or GNRs for 24 h, respectively. Nuclei were stained with Hoechst 33342 (blue). Scale bars: 25 μm. (B) Representative flow cytometric analysis of KB cells before and after treatment with FITC-labeled GNPs, FITC-labeled GNSs, or FITClabeled GNRs (50 μg/mL), respectively. The mean fluorescence intensity (MFI) values were also listed: 6644 for control, 50 766 for FITC-labeled GNPs, 40 087 for FITC-labeled GNSs, and 36 800 for FITC-labeled GNRs. (C) Dark-field images of KB cells treated with GNPs, GNSs, or GNRs (50 μg/mL) for 24 h, respectively. Nuclei were stained blue with Hoechst 33342. The white dots surrounding the nucleus were from the internalized Au nanostructures. Scale bars: 5 μm. (D) Quantitative analysis of the cellular SSC intensity in cells treated without (control) and with GNPs, GNSs, or GNRs. Data presented were the mean ± SD from n = 3 replicate experiments. * indicates p < 0.05, ** indicates p < 0.01 as compared to the control group.

toxicity toward the cells at this concentration. Even at a much higher concentration of 200 μg/mL, the cytotoxicity of Au 13042


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Figure 4. Cytotoxicity evaluation and X-ray irradiation-induced anticancer effects of GNPs, GNSs, or GNRs toward KB cells. (A) Viabilities of KB cells assessed by MTT assay after treatment with GNPs, GNSs, or GNRs at various concentrations ranging from 3 to 200 μg/mL. Data presented were the mean ± SD of three independent measurements. Error bars were based on the standard deviation of three parallel samples. (B) Colony formation images of KB cells treated with GNPs, GNSs, or GNRs at the concentration of 50 μg/mL combined with X-ray irradiation (4 Gy). (C) Corresponding survival fraction of KB cells after respective treatments obtained from the images shown in (B). Data presented were the mean ± SD of at least three independent experiments.

It seemed that the radiation sensitization effects of the three Au nanostructures were related to the respective cellular uptake efficiency. To clarify the relationship between SER and cellular uptake of these Au nanostructures, we evaluated the relative ratio of SER to the cellular uptake amount for the three Au nanomaterials (Figure 5). The cellular uptake efficiency of these

nanomaterials toward the cells was still not substantial, suggesting that the three Au nanostructures were suitable for potential biomedical applications. To evaluate the radiosensitization effects of the GNPs, GNSs, and GNRs, colony formation assays were conducted to evaluate the self-renewal efficiency of KB cells following the respective treatments. As discussed above, KB cells were divided into the following eight groups: (a) control, (b) GNPs only, (c) GNSs only, (d) GNRs only, (e) X-ray only, (f) GNPs + X-ray, (g) GNSs + X-ray, and (h) GNRs + X-ray. In each sample group, cell colonies were counted to evaluate the self-renewal efficiency of KB cells 10 days postirradiation. The dosedependent radiation sensitizing effects of the three Au nanostructures are shown in Figure 4B,C. The survival rate of the tumor cells after X-ray irradiation was found to depend on the type of Au nanostructures used. The survival fraction of the cells treated with the three Au nanostructures decreased significantly as the X-ray irradiation dose increased, indicating that all of these Au nanostructures are excellent radiosensitizers. Figure 4C presented the survival fraction of the cells in each group following X-ray irradiation at different doses. At the Xray irradiation dose of 4 Gy, the X-ray irradiation treatment alone decreased the cell survival fraction to 55%. When combining X-ray irradiation with GNPs, GNSs, and GNRs, the cell viability drastically reduced to 38%, 42%, and 47%, respectively. The sensitization enhancement ratio (SER) of each group was also calculated by the multitarget single-hit model. Generally, the SER was used to evaluate how effectively the radiosensitizer decreased cell proliferation. The calculated SER values were 1.62, 1.37, and 1.21 corresponding to the treatments of GNPs, GNSs, and GNRs at the same concentration, respectively. The results show that GNPs have a higher therapeutic effect than GNSs and GNRs at the same concentration, which may be attributed to the higher cellular uptake efficiency of GNPs.

Figure 5. SER/cellular uptake ratios of Au nanostructures in KB cells. The relative values of SER/SSC (sensitization enhancement ratio/side scatter) were 1.00 for GNPs, 1.12 for GNSs, and 1.10 for GNRs; the relative values of SER/MFI (sensitization enhancement ratio/mean fluorescence intensity) were 1.00 for GNPs, 1.07 for GNSs, and 1.02 for GNRs.

gold nanostructures was quantified by flow cytometry according to the intracellular side scatter (SSC) and mean fluorescence intensity (MFI) following gold nanostructure treatments. It was found that the relative ratio of SER to cellular uptake for GNPs, GNSs, and GNRs was nearly the same using either SSC or MFI data. The results indicated that the radiation enhancement effects of gold nanostructures must be dictated by the shapedependent cellular internalization of the Au nanostructures. In 13043


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ACS Applied Materials & Interfaces other words, the radiation enhancement effect in cancer radiotherapy likely is determined by the amount of the internalized Au elements. The Au nanomaterial that can achieve the highest cellular uptake is the most effective material for cancer radiotherapy. 3.4. Potential Mechanisms of Shape-Dependent Radiosensitization Effects Based on Au Nanostructures: Generation of ROS, Cell Cycle Distribution, and Apoptosis Assays. To further investigate how the Au nanostructures induced enhanced radiosensitization upon Xray irradiation, the intracellular ROS levels stimulated by Au nanostructures were detected. ROS usually refers to superoxide anion (•O2−), hydrogen peroxide (H2O2), hydroxyl radical (•OH), and singlet oxygen (1O2). Excessive ROS in the cells could perturb the equilibrium of the oxidation−reduction potential and lead to intracellular peroxide production followed by a series of adverse biological effects. After ionizing radiation, the generation of hydroxyl radical (•OH) in the cells significantly increased, and thus •OH as one of the most toxic reactive oxygen radicals plays an important role in X-rayinduced cytotoxicity. The intracellular ROS levels in each sample group after 24 h of incubation with three types of Au nanostructures were assessed with flow cytometry using the ROS assay kit (Figure 6). The mean fluorescence intensity (MFI) obtained by flow

Figure 7. Cell cycle distribution histograms of KB cells treated with Au nanostructures for 24 h without (A) or with (B) X-ray irradiation (4 Gy). The data were obtained by flow cytometry using cell cycle detection kit.

Table 2. Statistical Data of the Cell Cycle Distribution Ratios of KB Cells after Various Treatments (Au Nanostructures and/or X-ray Irradiation) Obtained from the Results Displayed in Figure 7a Figure 6. Generation of ROS within KB cells without (control) and with the treatment of GNPs, GNSs, or GNRs under the X-ray irradiation of 0 or 4 Gy. The cells were incubated with the fluorescent probe DCFH-DA after various treatments. The mean fluorescence intensity (MFI) in each group was quantified by flow cytometry, which can reflect the intracellular ROS level. Rosup was used as the positive control.

sample control GNPs GNSs GNRs X-ray X-ray + GNPs X-ray + GNSs X-ray + GNRs

cytometry can reflect the ROS levels in the cells. It was found that the MFI values in each group combined with X-ray irradiation (4 Gy) largely increased as compared to those without irradiation. In addition, there are 1.6-, 1.2-, and 1.1-fold increases of ROS levels in the cells treated with GNPs, GNSs, and GNRs as compared to the control group after X-ray irradiation. The above results demonstrated that the increased ROS generation should be one of the main reasons that led to the enhanced radiosensitization of Au nanostructures with different shapes. To further assess the potential mechanism of Au nanostructure-induced radiosensitizing effects, we carried out the cell cycle distribution assay by flow cytometry using the cell cycle detection kit (Figure 7 and Table 2). As compared to the control group, Au nanostructure-treated groups induced slightly more cells to distribute in the G2/M phase before X-ray irradiation. Meanwhile, the results indicated the above effect was more obvious with spherical GNPs than GNSs and GNRs

G1 (%) 57.3 53.0 53.3 53.0 42.3 43.3 40.5 42.9

± ± ± ± ± ± ± ±

1.7 1.2 1.4 2.1 2.3 1.5 2.4 2.6

S (%) 21.4 23.5 24.0 24.8 20.8 23.7 24.7 21.5

± ± ± ± ± ± ± ±

2.0 2.2 1.0 1.9 0.2 1.6 1.1 2.8

G2/M (%) 21.3 23.5 22.7 22.2 36.9 33.0 34.8 35.7

± ± ± ± ± ± ± ±

1.2 1.6 1.2 1.4 1.0 0.6 2.0 0.8

Data presented were the mean ± SD of three independent experiments. a

(Figure 7A). Besides, the number of G1 phase cells treated with Au nanostructures was less than that of the control group. The increased amount of G2/M phase cells in Au nanostructuretreated groups might contribute to their anticancer efficiencies after X-ray irradiation because the cells in the G2/M phase are known to be most sensitive to X-ray irradiation. In addition, the increase of the active oxygen radicals markedly arrested cell cycle in the G2/M phase after irradiation and facilitated more cells to carry out DNA double-stranded repair as compared to the groups without irradiation (Figure 7B). As mentioned above, Au nanostructures arrested more cells in the radiosensitive G2/M phase, and therefore they could sensitize the cells for X-ray radiation. Accordingly, Au nanostructures treated 13044


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Figure 8. Apoptosis ratios of KB cells treated without (control) and with GNPs, GNSs, or GNRs for 24 h (A) before and (B) after X-ray irradiation (4 Gy) exposure determined by flow cytometry using the Annexin V-FITC/PI staining kit.

nanostructures to the G2/M phase cells might reduce the amount of G2/M phase cells and relatively increase the amount of the cells in the G1 and/or S phases. Taken together, the cell cycle assay demonstrated that the Au nanomaterial-induced anticancer effect with X-ray radiation

cells were likely to be killed upon X-ray irradiation. Besides, it was found that the number of the G2/M phase cells in Au nanostructures treated groups upon X-ray irradiation was slightly less than the treated groups with X-ray alone. We believe that the enhanced cell-killing efficiency of Au 13045


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ACS Applied Materials & Interfaces could be partly attributed to the redistributed cell cycle caused by Au-based nanostructures. To estimate the three Au nanostructure-induced cell apoptosis ratios before and after X-ray irradiation at the radiation dose of 4 Gy, flow cytometry was carried out using the Annexin V-FITC/PI apoptosis detection kit (Figure 8). The quantitative results showed that the X-ray irradiation significantly increased the apoptosis ratios of the cells treated with the three Au nanostructures. Specifically, as compared to the cells treated with GNSs and GNRs, the cells treated with GNPs exhibited the highest total apoptosis ratio of 13.51% (a sum of the early apoptosis ratio of 8.14% and the late apoptosis ratio of 5.37%) and the lowest viability of 83.83%. In summary, the cell apoptosis assay indicated that the radiation enhancement effect caused by Au nanostructures could be mainly attributed to cell apoptosis.

Ning Gu: 0000-0003-0047-337X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Key Basic Research Program of China (973 Program) (2013CB933904), the National Natural Science Foundation of China (21673037 and 81571805), Six Talents Peak Project in Jiangsu Province (2015-SWYY-003), Fundamental Research Funds for the Central Universities, Scientific Research Foundation of Graduate School of Southeast University (YBJJ1450), and Graduate Students’ Scientific Research Innovation Project of Jiangsu Province Ordinary University (CXLX12_0119). Z.C. acknowledges support from the University of Michigan.

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4. CONCLUSIONS In this work, we evaluated the influence of three types of different shaped Au nanostructures (GNPs, GNSs, and GNRs) on the radiosensitization effect in X-ray radiotherapy in vitro. We found that the cellular internalization efficiency of these Au nanomaterials had the following order: GNPs > GNSs > GNRs. We then revealed that the radiosensitization effect of these gold nanomaterials had the same order as that of the cellular uptake efficiency. By dividing the cellular uptake amount of Au materials, we found that the normalized SER values were the same for the three Au nanostructures, which indicated that the radiation sensitizing effect of Au nanostructures was dictated by the internalized Au nanomaterial amount. These results demonstrated that if we hope to use Au nanomaterials for radiosensitization in X-ray radiotherapy, the ultimate goal is to increase their cellular internalization efficiency as much as possible. This perspective may also be true for other metalbased nanoparticles that may have a possible radiosensitization effect, which should be tested in the future. More importantly, the studies on the mechanism indicated that the different shaped gold nanostructures affected the radiosensitization effect through regulating the ROS level and cell cycle distribution, and exerted their cytotoxic roles via apoptosis. Finally, for clinical applications, because the potential in vivo accumulation of metal (including Au) nanomaterials may cause long-term toxicity of the nanomaterials, it is imperative to screen appropriate metal-based nanomedicine with effective clearance or reduced accumulation for cancer therapy.62,63

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b01112. Calculation of SER; statistical analysis; energy-dispersive spectroscopy (EDS) result of GNSs; and fluorescence spectra of Au nanostructures before and after FITC labeling (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Fu-Gen Wu: 0000-0003-1773-2868 Xiaodong Zhang: 0000-0003-4137-3535 13046


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