Atomically Precise Gold–Levonorgestrel Nanocluster as a

Jun 26, 2019 - 8) and Immunol Staining Fix Solution were purchased from Beyotime. Biotechnology Corporation. A LIVE/DEAD. TM. Cell Imaging Kit and ...
2 downloads 0 Views 10MB Size
Download PDF
www.acsnano.org

Atomically Precise Gold−Levonorgestrel Nanocluster as a Radiosensitizer for Enhanced Cancer Therapy Tong-Tong Jia,† Guang Yang,† Sai-Jun Mo,† Zhao-Yang Wang,† Bing-Jie Li,‡ Wang Ma,‡ Yue-Xin Guo,‡ Xiaoyuan Chen,§ Xueli Zhao,*,† Jun-Qi Liu,*,‡ and Shuang-Quan Zang*,† Downloaded via IDAHO STATE UNIV on July 17, 2019 at 16:13:20 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, China Department of Radiation Oncology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China § Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, Maryland 20892, United States ‡

S Supporting Information *

ABSTRACT: Gold nanoclusters have become promising radiosensitizers due to their ultrasmall size and robust ability to adsorb, scatter, and re-emit radiation. However, most of the previously reported gold nanocluster radiosensitizers do not have a precise atomic structure, causing difficulties in understanding the structure−activity relationship. In this study, a structurally defined gold−levonorgestrel nanocluster consisting of Au8(C21H27O2)8 (Au8NC) with bright luminescence (58.7% quantum yield) and satisfactory biocompatibility was demonstrated as a nanoradiosensitizer. When the Au8NCs were irradiated with X-rays, they produced reactive oxygen species (ROS), resulting in irreversible cell apoptosis. As indicated by in vivo tumor formation experiments, tumorigenicity was significantly suppressed after one radiotherapy treatment with the Au8NCs. In addition, compared with tumors treated with X-rays (4 Gy) alone, tumors treated with the nanosensitizer exhibited an inhibition rate of 74.2%. This study contributes to the development of atomically precise gold nanoclusters as efficient radiosensitizers. KEYWORDS: gold nanocluster, atom-precise, radiosensitizer, ROS burst, therapy

R

enhancing the radiotherapy effect. However, their large sizes (>6 nm) caused difficulties for efficient particle removal by the kidneys,18−20 causing liver damage. Fortunately, among Au-based materials,21−24 the size of gold nanoclusters (AuNCs) (1−3 nm) is smaller than the kidney filtration threshold,25 which could improve the renal clearance efficiency. For instance, Xie and colleagues reported a glutathione-protected Au25NC as an effective radiosensitizer that can escape reticuloendothelial system absorption for cancer therapy.19 Liang and colleagues investigated RGD peptide-templated AuNCs with active cancer cell targeting properties and employed these AuNCs as radiosensitizing agents for cancer radiotherapy.26 This previous study provided a proof of principle for the utilization of AuNCs as promising radiosensitizers. Although a series of AuNCs with radiotherapy

adiotherapy is one of the three major cancer therapeutic strategies in the clinic and treats 65−75% of local solid tumors at different stages.1,2 The main goal of radiotherapy is to shrink tumors and kill cancer cells by using high-energy radiation, such as X-rays. To maximize the radiation dose administered to cancer cells and weaken the fatal damage to normal cells, many studies have aimed to develop radiosensitizers with excellent performance. 3−5 Because of their high photostability and low cytotoxicity, a number of inorganic-based theranostic agents,6−8 such as semiconductor nanomaterials9,10 and nanostructures composed of high-Z elements,11−13 were developed and produced a marked effect. In 2015, Shi and colleagues demonstrated the integration of a scintillator and ZnO nanoparticles as an ionizing radiation-induced photodynamic therapeutic14,15 agent, achieving synchronous radiotherapy.16 In 2019, Choi and colleagues proposed porous platinum nanoparticles as a nanomedicine platform for effective radio-enhanced therapy.17 The high porosity and large surface area of the platinum nanoparticles convert endogenic H2O2 to O2, substantially © XXXX American Chemical Society

Received: May 15, 2019 Accepted: June 26, 2019 Published: June 26, 2019 A

DOI: 10.1021/acsnano.9b03767 ACS Nano XXXX, XXX, XXX−XXX

Article

Cite This: ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano

Scheme 1. (a) Schematic of the synthesis of Au8NCs; color codes: orange indicates Au, red indicates O, yellow indicates S, turquoise indicates Cl, and gray indicates C. Hydrogen atoms were omitted for clarity. (b) Au8NCs for cancer radiotherapy via the ROS burst.

Figure 1. Structure and characterization of the Au8NCs. Perspective views of the Au8NCs showing (a) the molecular structure and (b) the dihedral angle formed by the planes of two tetranuclear units. Color codes: orange indicates Au, red indicates O, and gray indicates C. Hydrogen atoms and some carbon atoms were omitted for clarity. (c) Positive mode ESI-TOF-MS spectrum of the Au8NCs. The inset shows an enlarged portion of the spectrum showing the measured (black line) and calculated (red line) isotopic distribution patterns. (d) TEM image of the Au8NCs. (e) DLS analysis of the Au8NCs. (f) Normalized excitation and emission spectra of the Au8NCs (inset: image of Au8NCs under 365 nm laser excitation in phosphate buffer, 10 μM) at room temperature.

B

DOI: 10.1021/acsnano.9b03767 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano

Figure 2. Relative cell viability of EC1 cells treated with different concentrations of (a) Au8NCs and (b) levonorgestrel. (c) Confocal imaging of EC1 cells after incubation with Au8NCs (2 μM) for different amounts of time (0, 2, 4, and 8 h). Scale bar: 50 μm.

enhancement have been developed,27−29 it remains a major challenge to precisely correlate radiosensitizer properties with the inner core structure and ligands. To overcome these longstanding challenges, colloidal metal nanoclusters (NCs) with an atomically precise structure30,31 are desirable for radiosensitizer32,33 investigations. However, most of these nanomaterials contain aromatic ligands34,35 and have poor water solubility,36−38 which significantly hampers their biological applications.39,40 To satisfy the above-mentioned multifaceted criteria, we reported the single-crystal structure of the alkynyl-protected41,42 AuNC Au8(C21H27O2)8 (∼2 nm). The current design consists of the following components: (I) excellent surface modification by levonorgestrel (a typical drug molecule)43,44 for good biocompatibility; (II) ultrasmall AuNCs that can be easily enriched in tumor areas and concentrate passing radiation; and (III) an atomically distinct structure that provides an opportunity to regulate the gold radiosensitizer by optimizing functional ligands at the atomic level. The major radio-enhancement mechanism was determined to be an increase in reactive oxygen species (ROS), causing irreversible cell apoptosis (Scheme 1b). Accordingly, a vital radiosensitizer that makes cancer cells more sensitive to radiation by increasing the local treatment efficiency using a relatively low and safe radiation dose was developed.

[Au4(C21H27O2)4] units were found to be connected by aurophilic interactions with a Au−Au bond length of 2.923 Å (Figure S1). Within the two tetranuclear [Au4(C21H27O2)4] units, Au2(I), Au4(I), Au5(I), and Au7(I) were linear and coordinated to two terminal C atoms from different ligands through σ bonds, and Au1(I), Au3(I), Au6(I), and Au8(I) were coordinated to two alkynyl units by π bonds in a η2/η2 bonding manner (Table S2). The Au−Au bond lengths in the molecule were in the range of 2.913−3.302 Å (Table S3), which was below the sum of two van der Waals radii.46 The chemical composition of the Au8NCs was further confirmed by electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) in positive mode. As shown in Figure 1c, the mass spectrum showed a predominant peak of a double-charged ion at an m/z of 2056.1679 (theoretical value: 2056.1620), corresponding to the [Au8(C21H27O2)8+2Na]2+ cation. Its isotopic distribution pattern exhibited perfect agreement with the simulated pattern. Additionally, the composition of the Au8NCs was also analyzed by elemental analysis (EA) and Fourier transform infrared (FT-IR) spectroscopy (Figure S2, Table S4). As shown in Figure S3, thermogravimetric analysis (TGA) showed a total weight loss of 60.7% as the temperature approached approximately 800 °C, which was similar to the theoretical content of alkynyl ligands in Au8NCs (61.26%). Transmission electron microscopy (TEM) images of the Au8NCs revealed a AuNC diameter of approximately 2 nm in phosphate buffer (Figure 1d), which was similar to the maximum C···C separation in the clusters (20.18 Å) indicated by X-ray analysis. Dynamic light scattering (DLS) measurements of the Au8NCs (Figure 1e) also confirmed a relatively narrow size distribution of approximately 2 nm with good dispersibility. Luminescence is one of the most appealing and crucial properties of AuNCs, with potential applications in biolabeling47 and biomedical targeting.48,49 Therefore, the normalized excitation and emission spectra of the Au8NCs were measured. As shown in Figure 1f, the Au8NCs were stable in phosphate buffer (10 μM) and showed intense yellow-green luminescence at room temperature with main broad emission bands at 518 (λem1) and 578 nm (λem2). The quantum yield reached 58.7%, which may be attributed to ligand-to-metal emission for

RESULTS AND DISCUSSION Au8(C21H27O2)8 nanoclusters (Au8NCs) were synthesized through a one-pot method45 at a high yield by reacting C21H28O2 (levonorgestrel) and Me2SAuCl in dichloromethane (DCM)/CH3CN (v/v = 1:1) in the presence of Et3N (Scheme 1a; the detailed synthetic procedure is described in the Experimental Section). Single-crystal X-ray diffraction (SCXRD) analysis revealed that Au8NCs are crystallized in the chiral space group P1 (Table S1, Supporting Information) and contain eight gold atoms and eight levonorgestrel ligands in each molecule, as shown in Figure 1a. The Au8NCs were shown to be divided into two parts, each containing an approximately planar tetranuclear structure and four levonorgestrel ligands [Au4(C21H27O2)4]. The dihedral angle of the two planes was 70.646° (Figure 1b). These two C

DOI: 10.1021/acsnano.9b03767 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano

Figure 3. (a) Live/dead imaging of EC1 cells after receiving different treatments (control group, 2 Gy X-ray, 4 Gy X-ray, Au8NC + 0 Gy Xray, Au8NC + 2 Gy X-ray, Au8NC + 4 Gy X-ray). Green: live; red: dead. Scale bar: 50 μm. (b) Representative images of the colony formation assay of EC1 cells with different treatments. (c) Statistical results of the surviving fraction of EC1 cells in the colony formation assay. (d) Wound healing assay and (e) statistical results of the wound healing assay. The magnification of microscopy pictures is 40×.

gold(I)-alkynyl components in the Au8NCs.50 Furthermore, at 293 K, the photoluminescence lifetime of the Au8NCs at a wavelength of 518 nm was 4.11 μs, while the photoluminescence lifetime at a wavelength of 578 nm was 3.93 μs (Figures S4 and S5). Moreover, obvious optical adsorption of the Au8NCs was observed in the wavelength range of 200− 450 nm, displaying main bands at 236 and 393 nm, respectively (Figure S6). To examine the toxicity of the Au8NCs, human esophageal squamous cancer cells (EC1) were used as a model to assess cellular responses using a CCK-8 assay kit. EC1 cells were cultured with the Au8NCs and levonorgestrel ligand at different concentrations for 24 h. The data shown in Figure 2a,b demonstrated that the IC50 (dose required to inhibit 50% cell proliferation) of the Au8NCs (7.8 μM, equal to 62.4 μM levonorgestrel ligand) was lower than that of the free levonorgestrel ligand (176.4 μM). Additionally, the IC10 of the Au8NCs was 2.1 μM, which was the concentration at which cells maintained good viability. Considering the intense luminescence and high stability of the Au8NCs in phosphate buffer (Figures 1f and S3), we investigated their practical application in live cell imaging. According to the IC10, 2 μM Au8NCs were added for the cell imaging and subsequent experiments, because cells could maintain good viability at this concentration, avoiding toxicity to normal tissues in the following in vivo therapy. The data shown in Figure 2c indicated that the intracellular luminescence increased as the co-incubation time between the Au8NCs and EC1 cells increased from 0 to 8 h, and cell contours were clear at 8 h. Then, cell medium containing

Au8NCs was removed and replaced with fresh medium. The intracellular luminescence gradually decreased and nearly disappeared at 24 h (Figure S7). These phenomena demonstrated that the Au8NCs were endocytosed, emitted luminescence in the EC1 cells, and were subsequently eliminated, making Au8NCs a promising candidate for biological and biomedical imaging. Due to the promising biocompatibility of Au materials and their capability of concentrating local radiation,51 as-fabricated AuNCs were investigated as a potential radiotherapy sensitizer. Six groups of cells that received different treatments (control, 2 Gy X-ray, 4 Gy X-ray, Au8NC, Au8NC + 2 Gy X-ray, Au8NC + 4 Gy X-ray) were analyzed, followed by live/dead imaging to assess the therapeutic effect. As shown in Figure 3a, cells in the control group and Au8NC group had extremely high survival rates, whereas the viability of cells in the Au8NC combined with irradiation groups decreased in a dose-dependent manner. We also conducted additional control groups that were pretreated with levonorgestrel (a equal quantity of Au8NC) for radiotherapy. The irradiated cells (Figure S8) displayed extremely high survival rates, which indicated good biocompatibility of the protecting ligands of Au8NC. A cell colony formation assay52 was conducted to further assess the enhanced radiotherapy of the Au8NCs over a longer period of time. The corresponding results revealed similar dose-dependent radiosensitization effects (Figures 3b,c and S9). In the control and Au8NC groups, the colonies were densely packed, indicating that the Au8NCs exhibited outstanding biocompatibility at the experimental concentration and had no observed effect on cell proliferation. Compared to D

DOI: 10.1021/acsnano.9b03767 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano

Figure 4. Intracellular ROS imaging of EC1 cells at 6 h with different treatments. Scale bar: 50 μm.

the body weights were observed every other day (Figures 5a−e and S17). The tumor volumes in the control group increased approximately 5 times, while the tumor volumes in the Au8NC + 4 Gy group were markedly reduced. Moreover, the relative body weights of the mice under different conditions remained nearly unchanged over 14 days (Figure 5d), indicating low toxicity in vivo. Additionally, hematoxylin and eosin (H&E) staining of the tumors and organs was conducted to evaluate the treatments. Widespread damage was observed in the tumor tissue from the Au8NC + 4 Gy group compared to the other two groups, and no histopathological abnormalities in the organs were observed (Figure 5f). These results demonstrated the significantly enhanced tumor-suppressing efficacy of the asfabricated nanosensitizer. To further evaluate the in vivo behaviors, we investigated the biodistribution of the Au8NCs. The tumors and major organs collected from the BALB/c-nude mice following the intraperitoneal injection of the Au8NCs were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) to determine the concentration of Au. At 24 h, a high quantity of Au8NCs accumulated at the tumor site, and the Au8NCs were rarely observed in most of the organs except for the bladder (Figure S18), indicating that the NCs had a sufficient transit time in the systemic circulation for accumulation at the tumor site. These results could be attributed to the ultrasmall hydrodynamic size and excellent levonorgestrel surface modification. Therefore, EC1 tumor-bearing mice were intraperitoneally injected with Au 8NCs or Dulbecco’s phosphate-buffered saline (DPBS), and after 24 h, the tumors were irradiated with 4 Gy X-ray radiation (Figure S19). According to the tumor growth curves, Au8NC + 4 Gy X-ray therapy inhibited tumor growth by 74.2% (compared with the 4 Gy X-ray group), indicating the efficient radiosensitization induced by the Au8NCs in vivo (Figures S20 and S21). Furthermore, the H&E staining results of the mice in the combined therapy group showed widespread damage of the tumor tissue, and histopathological abnormalities in the organs were not observed (Figure S22). The cancer treatment results

the irradiation groups in the absence of Au8NCs, the surviving fractions of the groups with Au8NCs were obviously lower. Specifically, the X-ray irradiation treatment (4 Gy) combined with Au8NCs decreased the cell survival fraction to 2.7%, indicating that the Au8NCs are excellent for radiosensitization. The observed survival fraction was smaller than that for other AuNC radiosensitizers (at a safe concentration) capped with proteins or peptides under the same irradiation dose.18,19,26−29 The radiation sensitization effect of Au8NCs was further investigated by a wound healing assay (Figure 3d).53 At 36 h, the rate of wound healing in the Au8NC + 4 Gy group was 4.9% (Figure 3e), which was lower than that in the other two groups, indicating the ability of the nanosensitizer to weaken tumor cell migration under X-ray irradiation. We further explored the underlying mechanisms of Au8NCmediated enhanced radiosensitization. The production of ROS upon X-ray irradiation is believed to play a crucial role during radiotherapy. The ROS burst likely attacks the covalent bonds of DNA, causing cell apoptosis or programmed cell death. Therefore, we analyzed intracellular ROS levels by confocal imaging using CellROX Deep Red Reagent. The cell-permeant dye is nonfluorescent in a reduced state and exhibits bright fluorescence upon oxidation by ROS. As shown in Figures 4 and S10−S16, the average intensity in the experimental group (Au8NC + 4 Gy X-ray) was 3.7 times greater than that in the group without the radiosensitizer, and the control group exhibited nearly no red fluorescence. These results confirmed that the electroactive surface of the Au8NCs catalyzed the formation of ROS. Motivated by the above-mentioned results, a tumor formation assay54 was conducted to evaluate the radiosensitization effect in vivo. First, EC1 cells were divided into three different treatment groups as follows: control group, phosphate-buffered saline (PBS)-treated group, and Au8NCtreated group. Next, EC1 cells (2 × 106 cells per mouse) were injected subcutaneously into the flanks of female BALB/c-nude specific-pathogen-free (SPF) mice and treated with different doses of X-ray irradiation (control group: 0 Gy; PBS-treated group: 4 Gy; Au8NC group: 4 Gy). Then, the tumor sizes and E

DOI: 10.1021/acsnano.9b03767 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano

Figure 5. In vivo tumorigenicity assay of Au8NCs under different conditions. (a) Representative images of mice under various conditions at days 0 and 14. (b) Images of dissected tumors. (c) Relative tumor volume curves of the mice. (d) Relative mouse body growth curves. (e) Statistical results of the tumor weights. (f) H&E histological staining of excised organs and tumor slices. Scale bar: 100 μm.

reduced the X-ray dose but also decreased the side effects of radiation in normal tissues. As an atomically precise radiosensitizer, the success of the Au8NCs might provide opportunities to analyze the structure−activity relationship and perspectives for designing radiosensitizers at the atomic level.

further confirmed the positive radiosensitization by the Au8NCs in vivo.

CONCLUSION In summary, an atomically precise gold nanocluster (Au8NC) with a diameter about 2 nm and satisfactory biocompatibility was synthesized as a superior radiosensitizer for cancer therapy. The cell colony formation assay indicated that the surviving fraction of cells treated with radiotherapy and Au8NCs was obviously smaller than that in groups that were only treated with radiation. The ratio of wound healing in the Au8NC + 4 Gy group was as low as 4.9%. The radioenhancement mechanism was identified to be the ROS burst upon irradiation, which caused irreversible cell apoptosis. As demonstrated by the in vivo tumor formation experiments, tumors were significantly suppressed after one treatment with radiotherapy and the Au8NCs. Notably, compared with the tumors that were treated with X-ray irradiation alone, tumors treated with an X-ray dose of 4 Gy and the nanosensitizer demonstrated an inhibition ratio of 74.2% in the cancer therapy experiments. The established nanosensitizer not only

EXPERIMENTAL SECTION Synthesis of Au8NCs. Two milliliters of 25 μM levonorgestrel solution (DCM/CH3CN = 1:1) was added in one portion followed by neat NEt3 (8 μL). Me2SAuCl (7.4 mg, 25 μM) was then added under stirring to yield a yellow-green transparent solution. The resultant solution was stirred for 5 min and allowed to evaporate slowly in the dark at room temperature for 1 day to yield yellowgreenish block crystals. The yield was 78.7% (based on Au). FT-IR (KBr, cm−1): 1652 (−CO); CCDC number 1908486. Cytotoxicity Analysis. EC1 cells were seeded into 96-well plates at a density of approximately 2 × 104 cells per well and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum at 37 °C in a humidified incubator with 5% CO2. After overnight incubation to allow for cell attachment, the cell medium was then replaced with 100 μL of fresh medium containing different concentrations of Au8NCs (0, 1, 2, 5, 10, 15, 20, and 30 μM) or levonorgestrel ligand F

DOI: 10.1021/acsnano.9b03767 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano

∼250 mm3, the mice were used for biodistribution and cancer therapy studies. Biodistribution. EC1 tumor-bearing mice were randomly divided into two groups followed by an intraperitoneal injection of 100 μL of Au8NCs (50 μM). The mice were euthanized at 5 and 24 h, and the tumors and major organs were harvested. Then, the tumors and major organs were dispersed in 5 mL of HNO3 overnight and digested with 5 mL of 30% H2O2 under boiling at 200 °C. Finally, the remaining solution was cooled and diluted to 2 mL with 4% diluted nitric acid. After filtration, water was added for a final volume of 200 μL, which was diluted 50 times to a volume of 10 mL. A standard curve was prepared using Au standard solutions at concentrations of 2.5, 5, 10, 20, and 50 ppb. The amounts of Au in each sample were measured using ICP-MS. Tumor Formation Assay. Female BALB/c-nude SPF mice (6 weeks old) were randomly divided into three groups (five mice per group) and subcutaneously injected with approximately 2 × 106 EC1 cells per mouse (group 1: control; group 2: PBS treatment; and group 3: 2 μM Au8NCs for 24 h incubation). Then, the subcutaneously injected EC1 cells were treated with different irradiation conditions (group 1: 0 Gy; group 2: 4 Gy; and group 3: 4 Gy). The tumor sizes and body weights of the mice were analyzed, and the mice were photographed every other day for 14 days. After approximately 2 weeks, the cancer cells proliferated to produce large tumors, and the length (L) and width (W) of the tumors were measured to calculate the tumor volume (V) according to the following equation: V = L × W2/2. Cancer Therapy. EC1 tumor-bearing mice were weighed and randomly divided into two groups (group 1 and group 2). Then, 100 μL of DPBS was intraperitoneally injected into the mice in group 1, while the mice in group 2 were intraperitoneally injected with 100 μL of Au8NCs (50 μM). After 24 h, all mice were treated with 4 Gy X-ray irradiation. All treatments were only administered once. The tumor sizes and body weights of the mice were measured, and the mice were photographed similar to the tumor formation assay. Histopathological Examination. The hearts, livers, spleens, lungs, kidneys, and tumors of the mice were harvested and fixed in 4% neutral buffered formalin solution overnight. Then, the samples were embedded in paraffin blocks, sectioned into 5 μm slices, and mounted onto glass slides. After H&E staining, images were acquired with a Nikon DIGITAL SIGHT DS-Fi2 digital light microscope. All the histopathological studies were blindly evaluated by a board-certified pathologist.

(0, 50, 100, 150, 200, 500, and 1000 μM). After 24 h, CCK-8 reagent (10 μL per well) was added to the wells, and the cells were incubated for 1 h. The absorbance was measured at a wavelength of 450 nm using a SpectraMax absorbance reader. The relative cell viability (%) was calculated as follows: (Atest/Acontrol) × 100. Au8NCs for Cell Imaging. EC1 cells were cultured on glassbottomed Petri dishes at an initial density of 2 × 104 cells/dish. The medium was then replaced with cell medium containing Au8NCs (2 μM). Before the confocal imaging procedure, the cells on the Petri dish were washed three times with DPBS. Confocal images of the cells were acquired at different incubation time points with a Zeiss LSM 880 microscope (at an excitation wavelength of 405 nm). Colony Formation Assay. EC1 cells were seeded in cell culture dishes (35 mm × 12 mm) and incubated over 4 h for cell attachment. Then, the cells were divided into a control group (without the addition of nanomaterials) and a treatment group (2 μM Au8NCs). After 24 h, the cell medium was removed from the wells, which were then washed with DPBS three times. After treating the cells with different doses of X-ray (6 MV) irradiation (0, 2, and 4 Gy), macroscopic cell colonies formed after cultivation for 2 weeks. The cells were fixed with 4% paraformaldehyde for 10 min and stained with 0.2% crystal violet for 3 min. Then, the cells were washed three times with DPBS and counted to evaluate the effects of the treatments. Each group was analyzed in triplicate. The cell survival fraction was calculated as the ratio of the number of colonies formed in the treated wells to the number of colonies formed by the untreated cells (control group). Live/Dead Assay. EC1 cells were cultured on glass-bottomed Petri dishes with an initial density of 2 × 104 cells/dish. The same setup of cloning assay was applied with the live/dead assay until washing with DPBS three times and then X-ray treatment. After X-ray irradiation, cells were cultured for another 24 h before the viability assay. EC1 cells were stained with a LIVE/DEAD cell imaging kit according to the manufacturer’s instructions. Confocal images were taken with a Zeiss LSM 880 confocal fluorescence microscope (excitation: 488 and 552 nm). Wound Healing Assay. EC1 cells were cultured in six-well culture plates until the bottom was fully covered with a cell monolayer. The same setup of the colony formation assay was applied with the wound-healing assay until washing with DPBS buffer three times and then the X-ray treatment. Wounds were made by scraping the confluent cell monolayer using 200 μL pipet tips, and the displaced cells were removed. Then, the remaining cells were incubated with fresh culture medium at 37 °C with 5% CO2, and images were acquired at 0, 12, 24, and 36 h after scraping. Intracellular ROS Detection. EC1 cells were seeded in glassbottomed Petri dishes at an initial density of 2 × 104 cells/dish. Then, the cells were divided into a control group (without the addition of nanomaterials) and a treatment group (2 μM Au8NCs). After 24 h, the cells were treated with X-ray irradiation and incubated for different amounts of time (0, 0.5, 1, 2, 4, 6, and 8 h). To measure the generation of intracellular ROS in each group, the medium was replaced with cell medium containing CellROX Deep Red Reagent (a ROS indicator) at a final concentration of 5 μM, and the cells were treated according to the manufacturer’s instructions. Finally, the cells were fixed with 4% paraformaldehyde for 10 min and stained with 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining according to the quick protocol. Confocal images were acquired with a Zeiss LSM 880 confocal fluorescence microscope at excitation wavelengths of 405 and 647 nm. Animals and Tumor Model. All animal procedures were conducted in accordance with the Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Zhengzhou University. BALB/c-nude SPF mice (5-week-old females) were housed in a controlled environment with a 12 h/12 h light/dark cycle. A maximum of five animals were housed together and provided food and water ad libitum. EC1 tumors were established by subcutaneously injecting approximately 2 × 106 EC1 cells into the left lower flank of each mouse. When the tumor reached a volume of

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.9b03767. Experimental section, figures, and tables (PDF) Crystallographic information files (CIF)

AUTHOR INFORMATION Corresponding Authors

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

Guang Yang: 0000-0002-1379-0684 Shuang-Quan Zang: 0000-0002-6728-0559 Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Science Fund for Distinguished Young Scholars (No. 21825106), the National Natural Science Foundation of China (Nos. 21671175, G

DOI: 10.1021/acsnano.9b03767 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano

therapy and Deep Photodynamic Therapy with Diminished Oxygen Dependence. Angew. Chem., Int. Ed. 2015, 54, 1770−1774. (17) Li, Y.; Yun, K. H.; Lee, H.; Goh, S. H.; Suh, Y. G.; Choi, Y. Porous Platinum Nanoparticles as a High-Z and Oxygen Generating Nanozyme for Enhanced Radiotherapy In Vivo. Biomaterials 2019, 197, 12−19. (18) Ghahremani, F.; Kefayat, A.; Shahbazi-Gahrouei, D.; Motaghi, H.; Mehrgardi, M. A.; Haghjooy-Javanmard, S. AS1411 AptamerTargeted Gold Nanoclusters Effect on the Enhancement of Radiation Therapy Efficacy in Breast Tumor-Bearing Mice. Nanomedicine 2018, 13, 2563−2578. (19) Zhang, X. D.; Chen, J.; Luo, Z.; Wu, D.; Shen, X.; Song, S. S.; Sun, Y. M.; Liu, P. X.; Zhao, J.; Huo, S.; Fan, S.; Fan, F.; Liang, X. J.; Xie, J. Enhanced Tumor Accumulation of Sub-2 nm Gold Nanoclusters for Cancer Radiation Therapy. Adv. Healthcare Mater. 2014, 3, 133−141. (20) Zhang, X. D.; Luo, Z.; Chen, J.; Shen, X.; Song, S.; Sun, Y.; Fan, S.; Fan, F.; Leong, D. T.; Xie, J. Ultrasmall Au10−12(SG)10−12 Nanomolecules for High Tumor Specificity and Cancer Radiotherapy. Adv. Mater. 2014, 26, 4565−4568. (21) Cheng, K.; Sano, M.; Jenkins, C. H.; Zhang, G.; Vernekohl, D.; Zhao, W.; Wei, C.; Zhang, Y.; Zhang, Z.; Liu, Y.; Cheng, Z.; Xing, L. Synergistically Enhancing the Therapeutic Effect of Radiation Therapy with Radiation Activatable and Reactive Oxygen SpeciesReleasing Nanostructures. ACS Nano 2018, 12, 4946−4958. (22) Wang, Z.; Shao, D.; Chang, Z.; Lu, M.; Wang, Y.; Yue, J.; Yang, D.; Li, M.; Xu, Q.; Dong, W. F. Janus Gold Nanoplatform for Synergetic Chemoradiotherapy and Computed Tomography Imaging of Hepatocellular Carcinoma. ACS Nano 2017, 11, 12732−12741. (23) Kaur, N.; Aditya, R. N.; Singh, A.; Kuo, T. R. Biomedical Applications for Gold Nanoclusters: Recent Developments and Future Perspectives. Nanoscale Res. Lett. 2018, 13, 302. (24) Huang, Q.; Zhang, S.; Zhang, H.; Han, Y.; Liu, H.; Ren, F.; Sun, Q.; Li, Z.; Gao, M. Boosting the Radiosensitizing and Photothermal Performance of Cu2−xSe Nanocrystals for Synergetic Radiophotothermal Therapy of Orthotopic Breast Cancer. ACS Nano 2019, 13, 1342−1353. (25) Zhao, T.; Zhou, T.; Yao, Q.; Hao, C.; Chen, X. Metal Nanoclusters: Applications in Environmental Monitoring and Cancer Therapy. J. Environ. Sci. Health, Part C: Environ. Carcinog. Ecotoxicol. Rev. 2015, 33, 168−187. (26) Liang, G.; Jin, X.; Zhang, S.; Xing, D. RGD Peptide-Modified Fluorescent Gold Nanoclusters as Highly Efficient Tumor-Targeted Radiotherapy Sensitizers. Biomaterials 2017, 144, 95−104. (27) Ghahremani, F.; Shahbazi-Gahrouei, D.; Kefayat, A.; Motaghi, H.; Mehrgardi, M. A.; Javanmard, S. H. AS1411 Aptamer Conjugated Gold Nanoclusters as a Targeted Radiosensitizer for Megavoltage Radiation Therapy of 4T1 Breast Cancer Cells. RSC Adv. 2018, 8, 4249−4258. (28) Kefayat, A.; Ghahremani, F.; Motaghi, H.; Amouheidari, A. Ultra-Small but Ultra-Effective: Folic Acid-Targeted Gold Nanoclusters for Enhancement of Intracranial Glioma Tumors’ Radiation Therapy Efficacy. Nanomedicine 2019, 16, 173−184. (29) Zhang, X.; Chen, X.; Jiang, Y. W.; Ma, N.; Xia, L. Y.; Cheng, X.; Jia, H. R.; Liu, P. D.; Gu, N.; Chen, Z.; Wu, F. G. GlutathioneDepleting Gold Nanoclusters for Enhanced Cancer Radiotherapy through Synergistic External and Internal Regulations. ACS Appl. Mater. Interfaces 2018, 10, 10601−10606. (30) Kang, X.; Chong, H.; Zhu, M. Au25(SR)18: The Captain of the Great Nanocluster Ship. Nanoscale 2018, 10, 10758−10834. (31) Liu, C. P.; Wu, T. H.; Liu, C. Y.; Chen, K. C.; Chen, Y. X.; Chen, G. S.; Lin, S. Y. Self-Supplying O2 through the Catalase-Like Activity of Gold Nanoclusters for Photodynamic Therapy against Hypoxic Cancer Cells. Small 2017, 13, 1700278. (32) Liu, J.; Chen, Q.; Zhu, W.; Yi, X.; Yang, Y.; Dong, Z.; Liu, Z. Nanoscale-Coordination-Polymer-Shelled Manganese Dioxide Composite Nanoparticles: A Multistage Redox/pH/H2O2-Responsive Cancer Theranostic Nanoplatform. Adv. Funct. Mater. 2017, 27, 1605926.

81703158), the Program for Science & Technology Innovation Talents in Universities of Henan Province (164100510005), the Program for Innovative Research Team (in Science and Technology) in Universities of Henan Province (19IRTSTHN022), and Zhengzhou University. We would like to thank the Academy of Medical Sciences of Zhengzhou University Translational Medicine Platform for their kind help and support of this work.

REFERENCES (1) Chen, Q.; Chen, J.; Yang, Z.; Xu, J.; Xu, L.; Liang, C.; Han, X.; Liu, Z. Nanoparticle-Enhanced Radiotherapy to Trigger Robust Cancer Immunotherapy. Adv. Mater. 2019, 31, 1802228. (2) Malyarenko, O. S.; Usoltseva, R. V.; Zvyagintseva, T. N.; Ermakova, S. P. Laminaran from Brown Alga Dictyota Dichotoma and Its Sulfated Derivative as Radioprotectors and Radiosensitizers in Melanoma Therapy. Carbohydr. Polym. 2019, 206, 539−547. (3) Chan, L.; Gao, P.; Zhou, W.; Mei, C. M.; Huang, Y.; Yu, X. F.; Chu, P. K.; Chen, T. Sequentially Triggered Delivery System of Black Phosphorus Quantum Dots with Surface Charge-Switching Ability for Precise Tumor Radiosensitization. ACS Nano 2018, 12, 12401− 12415. (4) Jiang, W.; Li, Q.; Xiao, L.; Dou, J.; Liu, Y.; Yu, W.; Ma, Y.; Li, X.; You, Y. Z.; Tong, Z.; Liu, H.; Liang, H.; Lu, L.; Xu, X.; Yao, Y.; Zhang, G.; Wang, Y.; Wang, J. Hierarchical Multiplexing Nanodroplets for Imaging-Guided Cancer Radiotherapy via DNA Damage Enhancement and Concomitant DNA Repair Prevention. ACS Nano 2018, 12, 5684−5698. (5) Song, X.; Xu, J.; Liang, C.; Chao, Y.; Jin, Q.; Wang, C.; Chen, M.; Liu, Z. Self-Supplied Tumor Oxygenation through Separated Liposomal Delivery of H2O2 and Catalase for Enhanced RadioImmunotherapy of Cancer. Nano Lett. 2018, 18, 6360−6368. (6) Yi, X.; Chen, L.; Chen, J.; Maiti, D.; Chai, Z.; Liu, Z.; Yang, K. Biomimetic Copper Sulfide for Chemo-Radiotherapy: Enhanced Uptake and Reduced Efflux of Nanoparticles for Tumor Cells under Ionizing Radiation. Adv. Funct. Mater. 2018, 28, 1705161. (7) Goel, S.; Ni, D.; Cai, W. Harnessing the Power of Nanotechnology for Enhanced Radiation Therapy. ACS Nano 2017, 11, 5233−5237. (8) Goswami, N.; Luo, Z.; Yuan, X.; Leong, D. T.; Xie, J. Engineering Gold-Based Radiosensitizers for Cancer Radiotherapy. Mater. Horiz. 2017, 4, 817−831. (9) Juzenas, P.; Chen, W.; Sun, Y. P.; Neto Coelho, M. A. N.; Generalov, R.; Generalova, N.; Christensen, I. L. Quantum Dots and Nanoparticles for Photodynamic and Radiation Therapies of Cancer. Adv. Drug Delivery Rev. 2008, 60, 1600−1614. (10) Kleinauskas, A.; Rocha, S.; Sahu, S.; Sun, Y. P.; Juzenas, P. Carbon-Core Silver-Shell Nanodots as Sensitizers for Phototherapy and Radiotherapy. Nanotechnology 2013, 24, 325103. (11) Ma, N.; Wu, F. G.; Zhang, X.; Jiang, Y. W.; Jia, H. R.; Wang, H. Y.; Li, Y. H.; Liu, P.; Gu, N.; Chen, Z. Shape-Dependent Radiosensitization Effect of Gold Nanostructures in Cancer Radiotherapy: Comparison of Gold Nanoparticles, Nanospikes, and Nanorods. ACS Appl. Mater. Interfaces 2017, 9, 13037−13048. (12) Rezaee, Z.; Yadollahpour, A.; Bayati, V.; Dehbashi, F. Gold Nanoparticles and Electroporation Impose Both Separate and Synergistic Radiosensitizing Effects in HT-29 Tumor Cells: An In Vitro Study. Int. J. Nanomed. 2017, 12, 1431−1439. (13) Su, X. Y.; Liu, P. D.; Wu, H.; Gu, N. Enhancement of Radiosensitization by Metal-Based Nanoparticles in Cancer Radiation Therapy. Cancer Biol. Med. 2014, 11, 86−91. (14) Pratx, G.; Kapp, D. S. Is Cherenkov Luminescence Bright Enough for Photodynamic Therapy? Nat. Nanotechnol. 2018, 13, 354. (15) Li, X.; Lee, S.; Yoon, J. Supramolecular Photosensitizers Rejuvenate Photodynamic Therapy. Chem. Soc. Rev. 2018, 47, 1174− 1188. (16) Zhang, C.; Zhao, K.; Bu, W.; Ni, D.; Liu, Y.; Feng, J.; Shi, J. Marriage of Scintillator and Semiconductor for Synchronous RadioH

DOI: 10.1021/acsnano.9b03767 ACS Nano XXXX, XXX, XXX−XXX

Article

ACS Nano (33) Zhang, C.; Yan, L.; Wang, X.; Dong, X.; Zhou, R.; Gu, Z.; Zhao, Y. Tumor Microenvironment-Responsive Cu2(OH)PO4 Nanocrystals for Selective and Controllable Radiosentization via the X-RayTriggered Fenton-Like Reaction. Nano Lett. 2019, 19, 1749−1757. (34) Zhang, Q. Y.; Zhao, L. Chiral Metal Cluster and Nanocluster Complexes and Their Application in Asymmetric Catalysis. Tetrahedron Lett. 2018, 59, 310−316. (35) Chen, Y.; Liu, C.; Tang, Q.; Zeng, C.; Higaki, T.; Das, A.; Jiang, D. E.; Rosi, N. L.; Jin, R. Isomerism in Au28(SR)20 Nanocluster and Stable Structures. J. Am. Chem. Soc. 2016, 138, 1482−1485. (36) Lei, Z.; Wan, X. K.; Yuan, S. F.; Guan, Z. J.; Wang, Q. M. Alkynyl Approach toward the Protection of Metal Nanoclusters. Acc. Chem. Res. 2018, 51, 2465−2474. (37) Lei, Z.; Chang, S. S.; Wang, Q. M. Vapochromic Gold(I)Silver(I) Cluster Protected by Alkynyl and Phosphine Ligands. Eur. J. Inorg. Chem. 2017, 44, 5098−5102. (38) Jin, R.; Zeng, C.; Zhou, M.; Chen, Y. Atomically Precise Colloidal Metal Nanoclusters and Nanoparticles: Fundamentals and Opportunities. Chem. Rev. 2016, 116, 10346−10413. (39) Tao, Y.; Li, M.; Ren, J.; Qu, X. Metal Nanoclusters: Novel Probes for Diagnostic and Therapeutic Applications. Chem. Soc. Rev. 2015, 44, 8636−8663. (40) Zheng, Y.; Lai, L.; Liu, W.; Jiang, H.; Wang, X. Recent Advances in Biomedical Applications of Fluorescent Gold Nanoclusters. Adv. Colloid Interface Sci. 2017, 242, 1−16. (41) Xu, L. J.; Wang, J. Y.; Zhu, X. F.; Zeng, X. C.; Chen, Z. N. Phosphorescent Cationic Au4Ag2 Alkynyl Cluster Complexes for Efficient Solution-Processed Organic Light-Emitting Diodes. Adv. Funct. Mater. 2015, 25, 3033−3042. (42) Wang, Y.; Su, H.; Ren, L.; Malola, S.; Lin, S.; Teo, B. K.; Hakkinen, H.; Zheng, N. Site Preference in Multimetallic Nanoclusters: Incorporation of Alkali Metal Ions or Copper Atoms into the Alkynyl-Protected Body-Centered Cubic Cluster [Au7Ag8(C≡CtBu)12]+. Angew. Chem., Int. Ed. 2016, 55, 15152− 15156. (43) Mohamed, M. E. F.; Trueman, S.; Feng, T.; Friedman, A.; Othman, A. A. The JAK1 Inhibitor Upadacitinib Has No Effect on the Pharmacokinetics of Levonorgestrel and Ethinylestradiol: A Study in Healthy Female Subjects. J. Clin. Pharmacol. 2019, 59, 510−516. (44) McNicholas, C.; Swor, E.; Wan, L.; Peipert, J. F. Prolonged Use of the Etonogestrel Implant and Levonorgestrel Intrauterine Device: 2 Years Beyond Food and Drug Administration-Approved Duration. Am. J. Obstet. Gynecol. 2017, 216, 586.e1−586.e6. (45) Lin, X.; Liu, C.; Sun, K.; Wu, R.; Fu, X.; Huang, J. Structural Isomer and High-Yield of Pt1Ag28 Nanocluster via One-Pot Chemical Wet Method. Nano Res. 2019, 12, 309−314. (46) Manbeck, G. F.; Brennessel, W. W.; Stockland, R. A.; Eisenberg, R. Luminescent Au(I)/Cu(I) Alkynyl Clusters with an Ethynyl Steroid and Related Aliphatic Ligands: An Octanuclear Au4Cu4 Cluster and Luminescence Polymorphism in Au 3 Cu2 Clusters. J. Am. Chem. Soc. 2010, 132, 12307−12318. (47) Lei, Z.; Zhang, J. Y.; Guan, Z. J.; Wang, Q. M. Intensely Luminescent Gold(I) Phosphinopyridyl Clusters: Visualization of Unsupported Aurophilic Interactions in Solution. Chem. Commun. 2017, 53, 10902−10905. (48) Kang, B.; Opatz, T.; Landfester, K.; Wurm, F. R. Carbohydrate Nanocarriers in Biomedical Applications: Functionalization and Construction. Chem. Soc. Rev. 2015, 44, 8301−8325. (49) Wang, J.; Ma, Q.; Wang, Y.; Shen, H.; Yuan, Q. Recent Progress in Biomedical Applications of Persistent Luminescence Nanoparticles. Nanoscale 2017, 9, 6204−6218. (50) Shen, D. F.; Wu, S. S.; Wang, R. R.; Zhang, Q.; Ren, Z. J.; Liu, H.; Guo, H. D.; Gao, G. G. A Silver(I)-Estrogen Nanocluster: GSH Sensitivity and Targeting Suppression on HepG2 Cell. Small 2016, 12, 6153−6159. (51) Zhao, X.; Xu, L.; Sun, M.; Ma, W.; Wu, X.; Xu, C.; Kuang, H. Tuning the Interactions between Chiral Plasmonic Films and Living Cells. Nat. Commun. 2017, 8, 2007.

(52) Li, N.; Yu, L.; Wang, J.; Gao, X.; Chen, Y.; Pan, W.; Tang, B. A Mitochondria-Targeted Nanoradiosensitizer Activating Reactive Oxygen Species Burst for Enhanced Radiation Therapy. Chem. Sci. 2018, 9, 3159−3164. (53) Keese, C. R.; Wegener, J.; Walker, S. R.; Giaever, L. Electrical Wound-Healing Assay for Cells In Vitro. Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 1554−1559. (54) Lee, J.; Abdeen, A. A.; Wycislo, K. L.; Fan, T. M.; Kilian, K. A. Interfacial Geometry Dictates Cancer Cell Tumorigenicity. Nat. Mater. 2016, 15, 856−862.

I

DOI: 10.1021/acsnano.9b03767 ACS Nano XXXX, XXX, XXX−XXX