Action of Gold Nanospikes-Based Nanoradiosensitizers: Cellular

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Action of Gold Nanospikes-Based Nanoradiosensitizers: Cellular Internalization, Radiotherapy, and Autophagy Ningning Ma, Peidang Liu, Nongyue He, Ning Gu, Fu-Gen Wu, and Zhan Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b09599 • Publication Date (Web): 17 Aug 2017 Downloaded from http://pubs.acs.org on August 18, 2017

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ACS Applied Materials & Interfaces

Action of Gold Nanospikes-Based Nanoradiosensitizers: Cellular Internalization, Radiotherapy, and Autophagy

Ningning Ma,† Peidang Liu,‡ Nongyue He,† Ning Gu,† Fu-Gen Wu,*,† and Zhan Chen*,§

†

State Key Laboratory of Bioelectronics, School of Biological Science and Medical

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

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

China §

Department of Chemistry, University of Michigan, 930 North University Avenue, Ann Arbor,

Michigan 48109, United States

KEYWORDS: gold nanostructures, ionizing radiation, cell penetrating peptide, autophagy inhibitor, sensitization enhancement ratio (SER)

1

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ABSTRACT: A major challenge to achieve effective X-ray radiation therapy is to use a relatively low and safe radiation dose. Various radiosensitizers, which can significantly enhance the radiotherapeutic performance, have been developed. Gold-based nanomaterials, as a new type of nanoparticle-based radiosensitizers, have been extensively used in researches involving cancer radiotherapy. However, the cancer therapeutic effect using the gold nanoparticle-based radiotherapy is usually not significant because of the low cellular uptake efficiency and the autophagy-inducing ability of these gold nanomaterials. Herein, using gold nanospikes (GNSs) as an example, we prepared a series of thiol-poly(ethylene glycol)-modified GNSs terminated with methoxyl (GNSs), amine (NH2-GNSs), folic acid (FA) (FA-GNSs), and the cell-penetrating peptide TAT (TAT-GNSs), and evaluated their effects on X-ray radiotherapy. For the in vitro study, it was found that the ionizing radiation effects of these GNSs were well correlated to their cellular uptake amounts, with the same order of GNSs < NH2-GNSs < FA-GNSs < TAT-GNSs. The sensitization enhancement ratio (SER), which is commonly used to evaluate how effectively radiosensitizers decrease cell proliferation, reaches 2.30 for TAT-GNSs. The extremely high SER value for TAT-GNSs indicates the superior radiosensitization effect of this nanomaterial. The radiation enhancement mechanisms of these GNSs involved the increased reactive oxygen species (ROS), mitochondrial depolarization, and cell cycle redistribution. Western blotting assays confirmed

that

the

surface-modified

GNSs

could

induce

the

up-regulation

of

autophagy-related protein (LC3-II) and apoptosis-related protein (active caspase-3) in cancer cells. By monitoring the degradation of the autophagy substrate p62 protein, GNSs caused impairment of autolysosome degradation capacity and autophagosome accumulation. Our data demonstrated that autophagy played a protective role against caner radiotherapy, and the inhibition of protective autophagy with inhibitors would result in the increase of cell apoptosis. Besides the above in vitro experiments, the in vivo tumor growth study also indicated that X-ray + TAT-GNSs treatment had the best tumor growth inhibitory effect, confirming the highest radiation sensitizing effect of TAT-GNSs. This work furthered our understanding on the interaction mechanism between gold nanomaterials and cancer cells and should be able to promote the development of nanoradiosensitizers for clinical applications.

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1. INTRODUCTION Clinically, conventional therapeutic approaches for cancer mainly include surgical resection, radiotherapy, chemotherapy, and their combinations, etc.1 Particularly, the ionizing radiation therapy (radiotherapy) with high-energy X- or γ-rays plays an important role in primary or adjuvant therapeutic approaches. However, the radiation resistance of hypoxic tumor cells to γ or X-rays still remains a challenge. Meanwhile, a number of side effects to the proximal normal tissues emerged at elevated radiation doses used for radiotherapy. Therefore, a variety of radiosensitizers have been developed to enhance the anticancer efficiency in radiation therapy, which can drastically reduce the radiation dosage and the irreversible damages to surrounding normal tissues. The widely applied radiosensitizers mainly include oxygen and radiosensitive drugs such as misonidazole, 5-fluorouracil (5-FU), and tirapazamine.2 Recently, metal nanoparticles as emerging radiosensitizers have been widely explored for achieving enhanced therapeutic efficiency in cancer radiotherapy, such as nanoparticles prepared using gold,3-11 silver,12-16 platinum,17-19 bismuth,20-24 tungsten,25,26 tantalum,27,28 hafnium,29 molybdenum,30 and rare earth elements (e.g., gadolinium and cerium).31-33 Among these metal-based nanomedicines, gold nanostructures have been applied as promising radiation sensitizing agents for cancer radiotherapy due to their facile synthesis, good biocompatibility, easy surface modification, and tunable optical properties. In general, the potential mechanisms of radiosensitization induced by gold-based nanostructures are proposed: (1) Upon X-ray irradiation, the internalized gold (a high-Z material with Z = 79) nanoparticles produce enhanced localized radiation dosage due to their strong absorption of X-rays as well as the subsequent emission of secondary electrons (e.g., Auger electrons, photoelectrons, Compton electrons) and fluorescence photons, which might cause the ionization of water molecules and/or intracellular components.34,35 (2) Gold-based nanostructures induce a series of biological effects, including oxidative stress caused by secondary electrons,36,37 cell cycle arrest,38 and DNA damage.39,40 To achieve excellent radiosensitization performance, gold nanostructures should be sufficiently taken up by cancer cells. With plenty cellular uptake of gold nanoparticles, the radiation dosage could be reduced while achieving the desired therapeutic effect, which could decrease the side effect of high radiation doses towards normal tissues. Therefore it is 3



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urgently desired to design gold nanoparticles with high cellular uptake efficiency for cancer radiotherapy. Currently, the effect of the size and shape of gold nanoparticles on the cellular uptake and/or cancer radiotherapy have been investigated.3,41,42 For instance, Chan et al. performed a comparison study on the cellular internalization of different sized and shaped spherical gold nanoparticles (GNPs).41 They found that the uptake of GNPs into mammalian cells is highest at the nanoparticle size of 50 nm as compared to those of 14, 30, 74, and 100 nm, and also the cellular uptake of rod-shaped GNPs is lower than their spherical counterparts. A similar study also found that the 50 nm-sized GNPs showed stronger radiosensitization effect than the 74 and 14 nm-sized GNPs because of the maximum cellular uptake.3 Besides, it was also demonstrated that the rod-shaped or star-shaped gold nanomaterials have lower cellular internalization than similar-sized spherical ones.42 Very recently, we reported that spherical gold nanoparticles could achieve higher cellular internalization and radiosensitization effect than gold nanospikes and gold nanorods.43 We believe that the radiosensitization effect induced by gold nanomaterials should be closely related to the amount of internalized gold nanomaterials. Research has been performed to enhance the cellular uptake efficiency and/or cancer radiotherapy via chemical modification of nanoparticles with organic molecules. For instance, the cellular uptake of citrate-stabilized GNPs was reported to be higher than transferrin-coated GNPs since the surface of citrate-stabilized GNPs probably contains a variety of serum proteins (e.g., α- and β-globulin proteins) which can facilitate the endocytosis of these GNPs via multiple receptors.41 Some previous studies reported that the cationic 16-mercaptohexadecyl trimethyl ammonium bromide (MTAB)-modified GNPs or polyethylenimine-modified gold nanorods exhibit high cellular uptake,44,45 which can be explained by the strong electrostatic interaction between cationic nanoparticles and negatively

charged

cell

surfaces.46

Moreover,

arginine-glycine-aspartate

(RGD)

peptide-modified gold nanorods were designed to enhance the efficiency of cancer radiation therapy because the conjugation of RGD peptides enables gold nanorods to recognize ανβ3 integrin abundantly expressed on tumor blood vessels.8 Besides, other surface ligand-modified Au-based nanostructures such as BSA-capped gold nanoparticles and ultrasmall glutathione (GSH)-protected gold nanoclusters were utilized as efficient 4


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radiosensitizers for tumor radiation therapy through increasing the accumulation of gold nanomaterials in tumors. Naturally-occurring protein or peptide (e.g., BSA or GSH) as the protecting shell can enhance the permeability and retention effect through prolonged systemic circulation time or avoiding the absorption by the reticuloendothelial system, respectively.7,9 In addition, it was reported that one of the most efficient ways for enhanced cellular uptake of gold nanostars is the surface modification with cationic or amphipathic cell-penetrating peptide (e.g., TAT peptide), which can facilitate the translocation across the cellular membrane.47 Autophagy (i.e., self-digestion) as a highly conserved and lysosome-based degradative process plays a crucial role in maintaining cellular survival, differentiation, development, and homeostasis (especially a low-level basal autophagy), by which cytosolic components are delivered into the lysosome for degradation and recycling. This process can be regulated by several autophagy associated genes and signaling pathways including class I, class III PI3K (phosphatidylinositol 3-kinases), and mTOR (mammalian target of rapamycin). Generally, autophagy mainly includes two stages: (1) Intracellular components are engulfed by double or multilayer vacuoles and hence autophagosomes gradually formed. (2) Autophagosomes undergo fusion with lysosomes to form hybrid autolysosomes, in which engulfed intracellular components are degraded. The role autophagy plays in cells could be very complex because totally opposite effects might occur when the autophagy level is up-regulated: pro-survival and pro-death.48 Autophagy can be frequently activated for overcoming various stimuli such as starvation, microbial infection, organelle damage, protein folding errors, DNA damage, radiotherapy, and chemotherapy, etc. Recently, some previous studies have demonstrated that gold nanoparticles,49,50 silver nanoparticles,14,15,51,52 quantum dots,53-55 rare earth oxide nanocrystals,56 dendrimers,57 and neodymium oxides58 can enhance the autophagy level, and therefore these nanoparticles are also defined as a novel class of autophagy activators.59 Especially, the dendrimers (e.g., PAMAM nanoparticles) and neodymium oxide have been reported to be able to induce autophagic cell death.57,58 It is also likely that oxidative environment (e.g., ROS production) as well as mitochondrial damage can trigger the autophagic process.60,61 However, the function of autophagy in gold nanostructures-induced

5



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cancer radiotherapy is still unclarified, although a previous study reported that the induction of autophagy by γ-radiation contributed to the radioresistance of glioma stem cells.62 In this study, gold nanospikes (GNSs) modified or conjugated with poly (ethylene glycol) (PEG) and three kinds of typical functional ligands including positively charged amine (NH2), folic acid (FA, could specifically target to the folate receptor), and cell-penetrating peptide (TAT) were prepared to systematically investigate the effects of these different modifications on the cellular uptake efficiency and radiosensitization effect of GNSs in cancer radiotherapy. Such a comprehensive and comparative study has not been performed before although surface functionalized gold nanoparticles in cancer radiotherapy have been already reported. As compared with most clinical radiosensitizers which have the disadvantages of rapid excretion and low uptake efficiency in tumor tissues, we believe that the surface-modified GNSs might have the following advantages: (1) easy functionalization with targeting ligands to maximize their accumulation at the tumor site, (2) low systemic clearance rate, and (3) easy tissue specific pharmacokinetic study due to the easy quantification of Au content in tissues. Herein, KB cells overexpressing folate receptors were chosen as the model cancer cells. In addition, the role of autophagy induced by different surface-modified GNSs in mediating radiosensitization was evaluated.

2. EXPERIMENTAL SECTION 2.1. Materials. Silver nitrate (AgNO3), trisodium citrate dihydrate, HAuCl4·3H2O, L-ascorbic acid (LAA), folic acid (FA), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), N-Hydroxysuccinimide (NHS), dimethyl sulfoxide (DMSO), fluorescein

isothiocyanate

(FITC), 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium

bromide (MTT), 4% glutaraldehyde, 3-methyladenine (3-MA), chloroquine diphosphate (CQ), and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO) and used as received. Thiol-terminated monomethoxy poly (ethylene glycol) (HS-PEG, M.W. ~ 5000 Da) and thiol-PEG-amine (HS-PEG-NH2, M.W. ~5000 Da) were ordered from JenKem Technology Co., Ltd. (Beijing, China). Cysteine-terminated TAT (CGGYGRKKRRQRRR) peptide was purchased from GL Biochem Ltd. (Shanghai, China). All primary antibodies 6


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(e.g.,

anti-LC3A/B

(N-term),

anti-p62

(Sequestosome-1),

anti-caspase-3

(active),

anti-GAPDH), secondary antibodies (e.g., goat anti-rabbit IgG (H + L) HRP, goat anti-mouse IgG(H + L) HRP), and enhanced chemiluminescence (ECL) reagent were purchased from EMD Millipore Corporation (Temecula, CA, USA). RFP-LC3-lentivirus, polybrene (hexadimethrine bromide), puromycin were purchased from Hanheng Biomedical Technology Co., Ltd. (Shanghai, China). All aqueous solutions were prepared using ultrapure water with a resistivity of 18.2 MΩ·cm obtained from a Milli-Q water system (Millipore Corp., Billerica, USA). Human oral epidermoid carcinoma (KB) cell lines which overexpress folate receptors were obtained from the Type Culture Collection of the Chinese Academy of Science, Shanghai, China. 2.2. Synthesis of Gold Nanospikes and Surface Functionalization. Gold nanospikes (GNSs) were prepared via the galvanic replacement reaction between Ag nanoparticles (AgNPs) and HAuCl4 according to the reported method with slight modification.11,63 Briefly, Ag nanoparticles (AgNPs) were synthesized by the reduction of AgNO3 with trisodium citrate.11 The GNSs solution was prepared by dropping AgNPs solution (5 mL) into 1% HAuCl4 aqueous solution (140 µL) with vigorous stirring followed by the addition of LAA aqueous solution (10 mM, 1 mL). The solution was stirred until its color turned from light yellow to dark blue within a short period of time, indicating the formation of GNSs. To increase the stability and dispersibility of the above-synthesized GNSs, HS-PEG (500 µg) was added to the above solution to coat the surface of GNSs through Au–S coupling, leading to the formation of methoxyl-PEG modified GNSs (denoted as GNSs). The Au concentration of GNSs solutions was determined by atomic absorption spectroscopy (AAS, Supporting Information). To prepare NH2-GNSs, FA-GNSs, and TAT-GNSs, the mixture (0.1 µmol) of HS-PEG and one of the following ligands HS-PEG-NH2, HS-PEG-FA (detailed synthetic process of HS-PEG-FA was described in Supporting Information) or cysteine-terminated TAT peptide was prepared in a molar ratio of about 4 : 1. The mixture was added into the freshly prepared GNSs solution (i.e., the above-mentioned dark blue solution before PEG addition) and stirred at room temperature for 24 h. Herein, to ensure the surface ligand distribution on GNSs as same as possible, the molar ratio of each functional ligand and HS-PEG should be carefully 7



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controlled and also all the PEG segments used have the same molecular weight of 5 kDa. Excess ligands were removed by several rounds of centrifugation and dialysis to obtain the respective NH2-GNSs, FA-GNSs, and TAT- GNSs. 2.3. Characterization of Surface-Modified Gold Nanospikes. The morphologies of the prepared GNSs were characterized by a JEOL-2100 transmission electron microscope (TEM) operating at an accelerating voltage of 200 kV and a field emission scanning electron microscope (FESEM, Zeiss Ultra Plus, Germany) at an accelerating voltage of 20 kV. The average sizes of GNSs were obtained from statistical analysis of the representative TEM images by Nano Measurer software (Version 1.2). The hydrodynamic diameters and zeta potentials of different surface-modified GNSs were measured by a Zetasizer Nano ZS (Malvern, UK). UV–vis absorption spectra were collected using a UV–vis spectrophotometer (UV-2600, Shimadzu, Japan) with a wavelength range of 300–900 nm. The successful surface modification of these GNSs was verified by a Fourier transform infrared (FTIR) spectrometer (Nicolet iS50, Thermo Scientific, USA). FTIR spectra of samples in KBr were collected at room temperature in the spectral region of 4000–400 cm–1 with a spectral resolution of 4 cm–1. 2.4. Cell Culture and Cytotoxicity Evaluation. KB cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), streptomycin (100 µg/mL), and penicillin (100 U/mL) at 37 °C in a 5% CO2 incubator. The cells were cultured to 80% confluence and the culture medium was changed every 2 days. For each experiment, the cells were seeded in culture plates and allowed to adhere for 24 h, and then the culture medium was replaced with fresh medium containing gold nanospikes. Cytotoxicities of GNSs against KB cells were determined by MTT assay. In brief, KB cells (5 × 103 cells) were seeded on 96-well plates in 100 µL of culture medium and continuously cultured for 24 h until ~ 80% confluence in a 37 °C incubator. After exposure to GNSs, NH2-GNSs, FA-GNSs, or TAT-GNSs with various concentrations (50, 100, and 200 µg/mL) for 24 h, the medium in each well was replaced with MTT in PBS buffer (5 mg/mL, 10 µL) followed by incubation for an additional 4 h at 37 °C. The formed formazan crystals were dissolved in 150 µL of DMSO. Absorbance in each well was measured at 570 nm with a 8


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Multiskan FC microplate photometer (Thermo Scientific). Each group was assayed in triplicate and the results were presented as mean ± SD of three independent experiments. 2.5. Cellular Uptake of Surface-Modified Gold Nanospikes. The surface ligand-dependent cellular uptake behaviors of gold nanospikes were determined by confocal laser scanning microscopy (CLSM) using three methods as follows: In method one, the cellular uptake was visualized by labeling the gold nanospikes with fluorescein isothiocyanate (FITC), a green fluorescence labeled probe. The FITC-labeled gold nanospikes were synthesized via the covalent binding of HS-PEG-FITC onto the nanostructure surface. Briefly, HS-PEG-FITC was prepared following the addition reaction between HS-PEG-NH2 and FITC. Surface coatings containing HS-PEG-FITC, HS-PEG, and one of the following three ligands HS-PEG-NH2, HS-PEG-FA, or cysteine-terminated TAT peptide were mixed in the molar ratio of 1 : 79 : 20 followed by the addition of freshly prepared GNSs under gentle stirring at room temperature for 24 h. Excess ligands were removed by centrifugation to obtain FITC-labeled gold nanospikes (i.e., FITC-labeled GNSs, FITC-labeled NH2-GNSs, FITC-labeled FA-GNSs, and FITC-labeled TAT-GNSs). Meanwhile, KB cells (7000 cells per well) were seeded onto 8-chambered coverglass in a 37 °C incubator. After 24 h of incubation, the culture medium was replaced with the above-prepared FITC-labeled gold nanospikes in DMEM (50 µg/mL of Au atoms) and incubated overnight. Finally, the nuclei were stained with Hoechst 33342 (Beyotime, China). The green fluorescence signals (coming from the various FITC-labeled gold nanospikes) of the cells in each 8-chambered coverglass were observed using CLSM. Flow cytometric assays were conducted by harvesting the cells, rinsed twice with cold PBS, and detached from wells using trpsin-EDTA. The green fluorescence signal from the various FITC-labeled gold nanospikes was measured via a flow cytometer (NovoCyte, ACEA). In method two, the amount of the internalized GNSs was visually identified by dark-field microscopy and quantified by the side scatter (SSC) signal using flow cytometry (details of dark-field imaging and SSC intensity can be found in the Supporting Information). In method three, we also employed the inductively coupled plasma atomic emission spectroscopy (ICP-AES) to measure the quantity of Au-based nanoparticles in KB cells (see details in the Supporting Information). 2.6. Colony Formation Assay and Real-Time Cell Analysis. KB cells were cultured in 9



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6-well plates (5 × 104 cells per well) for 24 h. When the cells grew to ~ 80% confluence in plates, they were divided into ten groups: (a) control, (b) GNSs, (c) NH2-GNSs, (d) FA-GNSs, (e) TAT-GNSs, (f) X-ray, (g) X-ray + GNSs, (h) X-ray + NH2-GNSs, (i) X-ray + FA-GNSs, and (j) X-ray + TAT-GNSs. The groups (b–e) were respectively treated with GNSs, NH2-GNSs, FA-GNSs, or TAT-GNSs in DMEM (50 µg/mL of Au atoms) for 24 h. Concurrently, group (f) received the irradiation of X-rays by a linear accelerator at various doses of 2, 4, 6, and 8 Gy, respectively. Groups (g), (h), (i), and (j) were first incubated with GNSs, NH2-GNSs, FA-GNSs, and TAT-GNSs in DMEM (50 µg/mL of Au atoms) for 24 h, respectively, and then immediately irradiated with different doses (2, 4, 6, and 8 Gy) of X-ray. Finally, the KB cells were trypsinized, counted, and then 1500, 1500, 3000, 3000, and 3000 cells at the corresponding radiation doses of 0, 2, 4, 6, and 8 Gy were seeded in 35 mm dishes with 2 mL of fresh culture medium. Depending on the colony formation rates of KB cells, the cells were incubated for an additional 10 days followed by Giemsa staining (KeyGen Biotech., China). The colonies formed with more than 50 cells were counted to evaluate the effects of the above treatments. Each group was assayed in triplicate and the results were presented as mean ± SD of three independent experiments. For real-time cell analysis (RTCA) experiments, the trypsinized KB cells after the above-mentioned treatments (a–j) were counted and seeded on an electronic 8-well plate (with each well containing 1.5 × 103 cells in 500 µL of culture medium) of RTCA iCELLigence analyzer (ACEA Biosciences, Inc.) for cell proliferation and growth assays. The RTCA instrument was placed in a standard CO2 cell culture incubator where it transmitted data wirelessly to an iPad housed outside the incubator. The software in the iPad friendly allowed for real-time interfacing with the electronic eight-well plate and included real-time data display and analysis functions. 2.7. Calculation of Sensitization Enhancement Ratio (SER). Survival fraction of cells in each group was calculated by the ratio of the cell colonies formed by the cells receiving different treatments and the cell colonies formed by the untreated cells. Cell survival curves and SER values were determined by a classical multi-target single-hit model (Details for SER calculation are shown in Supporting Information). 10


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2.8. Detection of DNA Double-Stranded Breaks. Phosphorylated histone H2AX was widely regarded as molecular marker for DNA double-stranded break. Because there is a one-to-one correspondence between the amount of phosphorylated histone H2AX and the degree of DNA damage, so that specific immunofluorescence assay was performed to detect phosphorylated Ser 139 on histone H2AX (γ-H2AX). Briefly, after treatment with different surface-modified GNSs for 24 h, the cells were washed twice with PBS, fixed with 4% glutaraldehyde for 10 min, and permeated with 1% Triton X-100, blocked in 1% bovine serum albumin for 1 h, and then incubated with anti-human phospho-H2AX (S139) Alexa Fluor 488 mouse mAb (eBioscience, USA) overnight at 4 °C. Nuclei were stained blue with Hoechst 33342. 2.9. Reactive Oxygen Species Generation. To measure the generation of intracellular reactive oxygen species (ROS) in each group, KB cells after various treatments were loaded with fluorescent probes, 10 µM dichlorodihydrofluorescein diacetate (DCFH-DA, KeyGen Biotech., China) in DMEM without phenol red for 25 min in dark (37 °C, 5% CO2) and then washed with PBS, finally re-suspended in PBS. The cells in the positive control group were only pretreated with Rosup (ROS positive reagent) for 30 min. For each sample, the cells were trypsinized and immediately analyzed with flow cytometry, by which the mean fluorescence intensity (MFI) of 10000 cells was recorded to verify the intracellular generation of ROS. 2.10. Mitochondrial Membrane Potential Analysis. Mitochondrial membrane potential (MMP, ∆Ψ) was measured using JC-1 (Fanbo Biochemicals, Beijing, China). JC-1, a lipophilic cationic dye, easily enters and aggregates inside the healthy mitochondria, and then emits red fluorescence light. Once mitochondrial depolarization occurs, the dye will not accumulate within the mitochondria and emits green fluorescence signal. The MMP loss was determined by the percentage of green fluorescence intensity. Briefly, after treatment with different GNSs for 24 h, the cells were harvested and incubated with JC-1 at 37 °C for 20 min, and then washed twice with PBS solution. The cells pretreated with a mitochondrial electron transport chain inhibitor (CCCP, 10 µM) for 20 min was set as the positive control group. Finally, the green fluorescence intensity of the stained cells was assayed using flow cytometry (NovoCyte, ACEA, USA). 11



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2.11. Cell Cycle Distribution Assay. Cell cycle distribution was assayed using cell cycle detection kit (KeyGen Biotech., China). In brief, the cells after treatment were washed with PBS, fixed with chilled 70% ethanol, and then kept at 4 °C. Prior to staining, the 70% ethanol was removed followed by incubation with RNase A (100 µL) for 30 min at 37 °C. Then propidium iodide (PI) solution (400 µL, 50 µg/mL) was added and the mixture was co-incubated for 30 min at 4 °C. Finally, the cell cycle distribution was analyzed by flow cytometry. 2.12. Apoptosis

Assay

by

Annexin V-FITC/PI

Staining.

To compare the

apoptosis-inducing capabilities of different GNSs before and after X-ray irradiation, the Annexin V-FITC/PI apoptosis detection kit (KeyGen Biotech., China) was used for evaluating cell apoptosis rate. Briefly, KB cells (5 × 105 cells) were exposed to GNSs for 24 h, and then the cells were harvested with trypsin solution without EDTA and washed twice with PBS solution. Afterwards, the cells were incubated with 500 µL 1× binding buffer containing 5 µL of Annexin V-FITC and 5 µL of PI for 25 min at room temperature in dark. Cell apoptosis and necrosis rates were immediately analyzed by flow cytometry. 2.13. In Vivo Studies. All experimental procedures at the animal level were performed under protocols approved by the Animal Care and Use Committee of Southeast University. U14 cells (2 × 106, mouse uterine cervical cancer cells) in PBS solution (100 µL) were injected into the right flank of each BALB/c-nu mouse to generate the U14 xenograft tumor model. When the tumor volume had reached approximately 90 mm3, the mice were randomly divided into the following six groups at day 0 and then received different administrations: (1) Control, (2) GNSs only, (3) TAT-GNSs only, (4) X-ray only, (5) X-ray + GNSs, and (6) X-ray + TAT-GNSs. Among these, the mice in groups 2 and 3 were injected intravenously with GNSs and TAT-GNSs in PBS solutions (100 µL, 400 µg/mL), respectively. The mice in group 4 received X-ray radiation (6 MV, 6 Gy). Subsequently, at 24 h postinjection, the mice in groups 5 and 6 were exposed to X-ray radiation (6 MV, 6 Gy). For in vivo biodistribution analysis, the mice in goups 2 and 3 were sacrified at 24 postinjection and then the tissues were taken out and weighed. The tissues in each group were digested in aqua regia and the Au content was measured using ICP-AES. Finally, for in vivo study, the tumor volume and mouse weight were measured daily until day 8. The tumor volume was measured using a 12


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vernier caliper and calculated as the volume = (tumor length) × (tumor width)2/2. The relative tumor volume was calculated as V/V0, where V0 was the tumor volume when the treatment was initiated, and V was the volume of the tumor measured daily after the treatment. At the end of the experiments, all of the mice were sacrificed and the tumor tissues were separated and pictured. Then, the tumor tissues in different samples were fixed in 4% paraformaldehyde solution, processed routinely into paraffin, and stained with hematoxylin and eosin (H&E). Pathology was examined using an inverted microscope. 2.14. Cyto-ID Staining. Autophagosome precursors, autophagosome, and autolysosomes were stained and analyzed using Cyto-ID autophagy detection kit (Enzo Life Sciences, New York) according to the manufacturer’s instruction. Cyto-ID green autophagy dye only weakly stains lysosomes, while it serves as a marker of autolysosomes and earlier autophagic compartments. Typically, the cells were washed twice with PBS after treatment with GNSs for 24 h, and then stained with 1 mL of complete cell culture medium containing 1 µL of Cyto-ID green detection solution and 0.5 µL of Hoechst 33342 solution (to stain nuclei) for 25 min. Then the cells were observed under the confocal microscope and the green fluorescence from Cyto-ID dye indicated the formation of intracellular autophagosomes and autolysosomes. To quantify the fluorescence intensity of Cyto-ID dye in KB cells, the cells were trypsinized, stained, and analyzed with flow cytometry (NovoCyte, ACEA, USA). The mean fluorescence intensity (MFI) of green Cyto-ID dyes was recorded to quantify the autophagy level. 2.15. RFP-LC3 Stable Transfection for Autophagic Flux Measurement. KB cell lines which stably expressed RFP-LC3 (red fluorescent protein-tagged microtubule-associated protein light chain 3) were established through RFP-LC3-lentivirus (108 PFU/mL) transfection (see details in the Supporting Information). Cells were seeded on an 8-chambered coverglass slide (Lab-Tek, Nunc), and after treatments with different surface-modified GNSs for 24 h, the level of cell autophagy was monitored under a confocal microscope. Meanwhile, the RFP-LC3 puncta per cell were quantified by counting. Typically, the RFP-LC3 puncta in at least fifty representative images were counted to determine the average amount of RFP-LC3 puncta per cell. 13



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2.16. Western Blotting Analysis. KB cells were washed with cold PBS buffer and lysed in CytoBuster Protein Extraction Buffer (Novagen, San Diego) with 1% Protease Inhibitor Cocktail Set Ⅲ, EDTA-Free (Calbiochem., Germany) on ice. Cell lysates were centrifuged (12,000 rpm) for 10 min at 4 °C, and then the protein supernatant was transferred into cold tubes. The total protein concentration of each sample was determined with BCA protein assay kit (KeyGEN Biotech., China). An equal amount (40 µg) of protein under denaturing conditions was resolved and separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 100 V before transferring to 0.22 µm polyvinylidene difluoride (PVDF) membrane. Immediately, PVDF membranes were washed twice with freshly prepared Tris-buffered saline Tween-20 (1× TBST) and then the blotted PVDF membrane was blocked in TBST containing 5% nonfat milk for 1.5 h with constant agitation at room temperature. The PVDF membrane was then incubated with the primary antibody (1/2500) like anti-LC3A/B, anti-p62, or anti-caspase-3 (active) at 4 °C overnight with agitation,

and

then

incubated

with

the

corresponding

horse

radish

peroxidase

(HRP)-conjugated secondary antibody (1/5000) at room temperature for 2 h followed by washing three times with 1× TBST. Finally, target proteins were detected after incubation with ECL reagent (Tanon, China) for 5 min and then visualized by chemiluminescence imaging system (Tanon-5200, China). Western blotting film of each band was quantified by measuring integrated optical density (IOD). GAPDH was used as the loading control. 2.17. Biological TEM (BioTEM) Observation of Autophagosome in KB Cells. KB cells were grown in the six-well plates and then treated with TAT-GNSs for 24 h. After incubation for 24 h, the cells were harvested with trypsin. The cells were pelleted by centrifugation at 1500 rpm for 5 min and immediately fixed with 4% glutaraldehyde for 1 h. After postfixing in 1% OsO4 for 1 h at room temperature, the cells were then dehydrated with gradient ethanol solutions and embedded in epoxy resin. Ultrathin sections (70–90 nm) were prepared with an ultra-microtome (Leica Microanalysis) and then stained with uranyl acetate/lead citrate. The prepared sections were imaged under the BioTEM (H-600(4), Hitachi, Japan). 2.18. Statistical Analysis. All data presented are the mean ± standard deviation (Mean ± SD) of three independent experiments. The one-way ANOVA was performed to analyze the difference between the control and treated groups, in which a p value less than 0.05 was 14


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considered as significant difference. The data are indicated with (*) for p < 0.05 and (**) for p < 0.01.

3. RESULTS AND DISCUSSION 3.1. Characterization of Surface-Modified GNSs. GNSs with different surface coatings were synthesized as illustrated in Figure 1A. Generally, gold nanospikes (GNSs) were firstly obtained via the galvanic replacement reaction between AgNPs and HAuCl4. To fabricate the multifunctional GNSs with good monodispersity and stability, HS-PEG-NH2, HS-PEG-FA, or cysteine-terminated TAT (CGGYGRKKRRQRRR) was mixed with HS-PEG in a molar ratio of 1 : 4 and incubated with the freshly prepared GNSs to obtain the corresponding PEG-modified GNSs (denoted as NH2-GNSs, FA-GNSs, and TAT-GNSs, respectively). TEM and SEM images showed that the obtained GNSs had high density of branched structure on their surfaces and hollow interiors (Figure 1B, C). Size distribution histogram obtained from the representative TEM image verified that the average diameter of GNSs was 54 ± 9 nm (Figure 1B, inset). Besides, the UV–vis spectra of GNSs, NH2-GNSs, FA-GNSs, and TAT-GNSs all exhibited a similar spectral pattern with the presence of a strong surface plasmon absorption peak at around 650 nm (Figure 1D), which should originate from the spikes on the surfaces. Note that the two peaks at around 280 and 350 nm in the spectrum of GNSs-FA come from the absorption of folic acid. FTIR spectroscopy was used to confirm the successful conjugation of the PEG ligands on the surfaces of these GNSs (Figure 1E). The strong absorption peaks at 2924/2851, 1460/1375, and 1101/1016 cm–1 correspond to the CH2 stretching, CH2 bending, and C–O–C stretching vibrations in the PEG segments, respectively, confirming the presence of PEG molecules on GNSs. In addition, the two peaks at 1658 and 1631 cm–1 could be ascribed to the C=O stretching and N–H bending vibrations of the amide bonds in the TAT moiety, which further proved that the covalent binding between TAT and GNSs. These results confirmed the successful conjugation of different ligands on the GNSs surfaces. The hydrodynamic diameters of different surface-functionalized GNSs in PBS solutions (pH = 7.4) were measured using dynamic light scattering (DLS). The results summarized in 15



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Table 1 showed that the hydrodynamic diameters all exceeded 100 nm. The larger hydrodynamic diameter values as compared with the average diameter obtained from TEM images can be explained by the presence of the PEG chain and the hydration layer on the surfaces of these GNSs. Meanwhile, the zeta potential measurements indicated that GNSs, FA-GNSs, and TAT-GNSs were negatively charged (with the values of – 13 mV, – 18 mV, and – 8 mV, respectively), while NH2-GNSs were positively charged (+ 7 mV). The zeta potential results further confirmed the successful conjugation of the various functional groups (NH2, FA, or TAT) on GNSs surfaces. Up to now, we have used several characterization methods to validate the successful conjugation of different functional groups on GNSs surfaces. However, it was still one of most challenging tasks for us to quantitatively analyze the amount of funtional groups on the nanostructure surfaces due to the low quantity of funtional groups and the complexity of chemical constituents of the NP surfaces. However, to ensure the same funtional group amount on the NP surfaces, we should carefully control the molar ratio of funtional groups and HS-PEG during preparation procedure.

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Figure 1. Synthesis and characterization of GNSs. (A) Schematic illustrating the preparation of PEG-modified GNSs. (B) Representative TEM image and the corresponding size distribution histogram (inset) and (C) the representative SEM image of the freshly prepared GNSs without PEG modification. The size distribution histograms in TEM image were obtained by size analysis of over 200 particles. The mean diameter is 54 ± 9 nm. Scale bar: 50 nm. (D) UV–vis spectra and (E) FTIR spectra of GNSs, NH2-GNSs, FA-GNSs, and TAT-GNSs.

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Table 1 Hydrodynamic diameters and zeta potentials of different surface-functionalized GNSs measured by Zetasizer Nano ZS. Z-average

Polydispersity index

Zeta potential

(d. nm)

(PDI)

(mV)

GNSs

113 ± 39

0.365

– 13 ± 9

NH2-GNSs

117 ± 52

0.264

+7±5

FA-GNSs

128 ± 31

0.401

– 18 ± 6

TAT-GNSs

114 ± 40

0.316

–8±4

Sample

3.2. Cellular Uptake and Cytotoxicity Evaluation of GNSs. Internalization of nanostructures into cancer cells plays a crucial role in inducing changes of cellular behaviors that may dictate the efficiency of cancer radiotherapy. Therefore nanostructures need to be sufficiently delivered to the target cancer cells without distinct cytotoxicities. Herein, we employed the KB cells to investigate the cellular uptake efficiency of GNSs using fluorescence imaging, dark-field imaging, and ICP-AES. In fluorescence imaging, the cellular uptake of GNSs was visualized by treating the cells with FITC-labeled GNSs. The green fluorescence signals (from FITC) within the cells indicated the internalization of GNSs (Figure 2A). It can be seen from the fluorescence image and bright field image that all the four GNSs adhered to the cell plasma membranes while an evident cellular uptake was only observed for the TAT-GNSs- and FA-GNSs-treated cells. Note that the TAT-GNSs-treated group exhibited the highest cellular uptake amount of GNSs as can be seen from the large, black aggregates within the cells in the bright field image. The cellular internalization of GNSs into KB cells was also quantified by flow cytometry (Figure 2B). It was found that the fluorescence intensity of cells treated with NH2-GNSs, FA-GNSs, or TAT-GNSs was about 2.4–6.9-fold larger than that of untreated cells (control group) after 24 h of incubation. Besides, the fluorescence intensity detected in NH2-GNSs-, FA-GNSs-, or TAT-GNSs-treated cells was about 1.2, 2.0, or 2.8-fold higher than that detected in the GNSs-treated cells, respectively, indicating that the cellular uptake of gold nanospikes was significantly enhanced 18


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with the presence of functional groups (NH2, FA, or TAT). In addition, the dark-field imaging and flow cytometry (based on signal from the scatter light of GNSs) were both adopted to further evaluate the internalization of GNSs (Figure S1). The dark-field microscopy makes full use of the unique property of metallic element (Au) which can avoid possible fluorescence interference. Internalized GNSs are shown as bright spots in dark-field images (Figure S1A). Similarly, the mean side scatter light intensity from different modified GNSs was quantified using flow cytometry (Figure S1B). Consistent with the quantitative fluorescence observations, dark-field imaging revealed that the number of bright spots displayed a declining trend in cells treated with TAT-GNSs, FA-GNSs, NH2-GNSs, and GNSs, respectively. Based on the statistical data in Figure S1B, after treating the cells with Au nanomaterials for 24 h, the intensity of side-scattered light in the NH2-GNSs, FA-GNSs, and TAT-GNSs-treated cells were 1.1-, 1.6-, and 3.0-fold higher than that of the GNSs-treated cells. Finally, we also measured the quantity of Au-based nanoparticles in KB cells by ICP-AES. The ICP-AES data shown in Figure S2 (Supporting Information) clearly indicated that the order of the Au content in cells after various treatments was consistent with that revealed by fluorescence imaging and dark-field imaging. Collectively, the above three methods confirmed the cellular uptake efficiency of GNSs by KB cells had the following order: TAT-GNSs > FA-GNSs > NH2-GNSs > GNSs. The maximal cellular internalization of TAT-GNSs might be attributed to the ability of the TAT peptides to either directly penetrate the plasma membrane or facilitate endocytosis. Furthermore, it was found that the TAT peptides operated by anchoring on the plasma membrane via the multidentate hydrogen bonding of cationic arginines with the anionic biomacromolecules on the membrane, causing cytoskeleton reorganization to enhance membrane translocation.47,64 Meanwhile, the results also revealed that FA modification outperformed the amine modification in promoting the cellular uptake of GNSs, possibly due to the stronger receptor–ligand binding interaction between folate ligands on GNSs surfaces and overexpressed folate receptors on KB cell surfaces as compared with the electrostatic interaction between NH2-GNSs and cell surfaces. On the other hand, it is essential to evaluate the cytotoxicity of different GNSs with varied cellular internalization degrees if they are to be used for biomedical applications. MTT assays were carried out for KB cells after treatment with GNSs, NH2-GNSs, FA-GNSs, and 19



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TAT-GNSs solutions with different concentrations (50, 100, or 200 µg/mL) (Figure 2C). The results showed that the cell viability decreased in a dose-dependent manner: a higher GNS uptake efficiency leads to a higher cytotoxicity. However, even at a high concentration of 200 µg/mL, the differently modified GNSs exhibited negligible cytotoxicity towards KB cells. The results clearly showed that GNSs with different surface coatings had low cytotoxicity, enabling the potential applications of GNSs for cancer therapy.

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Figure 2. Confocal images for cellular uptake of surface-modified GNSs and cell cytotoxicity assay results. (A) Representative confocal fluorescence (FL) images and bright field (BF) images of KB cells incubated for 24 h with FITC-labeled GNSs, NH2-GNSs, FA-GNSs, or TAT-GNSs at same concentration (50 µg/mL of Au atoms). Nuclei were stained blue with Hoechst 33342. The green fluorescence signals in KB cells indicated internalized FITC-labeled surface-modified GNSs. Scale bar: 25 µm. (B) Cellular uptake of FITC-labeled surface-modified GNSs was quantified by flow cytometry (n = 10000 cells) and expressed as mean fluorescence intensity (MFI). (C) Cell viabilities of KB cells treated with different surface-functionalized GNSs at various concentrations (50, 100, or 200 µg/mL) for 24 h. n = 3; *p < 0.05 versus the control group.

3.3. Radiosensitization Effects of Surface-modified GNSs. To investigate the radiosensitization effects of GNSs, NH2-GNSs, FA-GNSs, and TAT-GNSs, colony formation assays were conducted to evaluate the self-renewal efficiency of KB cells after GNSs and X-ray treatments. To this end, KB cells were divided into ten groups: (a) Control, (b) GNSs, (c) NH2-GNSs, (d) FA-GNSs, (e) TAT-GNSs, (f) X-ray, (g) X-ray + GNSs, (h) X-ray + NH2-GNSs, (i) X-ray + FA-GNSs, and (j) X-ray + TAT-GNSs. Colonies were counted to evaluate the self-renewal efficiency of KB cells post-irradiation. The dose-dependent radiation enhancement effects of modified GNSs in the KB cells were shown in Figure 3A and B. It was found that all these GNSs exhibited enhanced radiosensitization and the survival fraction of cells decreased significantly at increasing radiation doses without and with the treatment of these modified GNSs. For example, based on the normalized survival fraction curves after nonlinear fitting calculation by using multitarget single-hit model (Figure 3B), we found that X-ray irradiation alone only decreased the cell viability to 48% at the radiation dose of 4 Gy. However, the cell viabilities were drastically reduced to 42%, 34%, 30%, and 24% in the presence of GNSs, NH2-GNSs, FA-GNSs, and TAT-GNSs, respectively. This result clearly indicated that the decreased cell viabilities were attributed to the sensitization of modified GNSs to X-ray radiotherapy but not the additive action of the nanostructures and X-ray radiation since all these survival fraction curves had already excluded the impact of surface-modified GNSs themselves. Besides, the sensitization 21



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enhancement ratio (SER), an evaluation standard of how effectively radiosensitizers decrease cell viability, was calculated based on the survival fraction of cells by multitarget single-hit model (Figure 3C). The calculated SERs were 1.34, 1.57, 1.84, and 2.30 for the GNSs-, NH2-GNSs-, FA-GNSs-, and TAT-GNSs-treated groups at the same concentration (50 µg/mL of Au atoms), respectively. The calculated SER value of GNSs was only about 1.34, which was a little higher than tungsten sulfide quantum dots (WS2, SER = 1.22) or gold nanorods (GNRs, SER = 1.21) and lower than spherical gold nanoparticles (GNPs, SER = 1.62).26,43 Particularly, the extremely high SER (2.30) of TAT-GNSs exceeds most of the previous radiosensitizers whose SER values are usually below 1.6.65 To further evaluate the effect of surface modification of GNSs on radiosensitization performance, we studied how much more X-ray radiation dose needs to be used for GNSs, NH2-GNSs, and FA-GNSs to reach the same therapeutic effect as TAT-GNSs. To achieve the cell survival fraction of 24% in TAT-GNSs-treated cells, the radiation dose should be increased by 33%, 29%, 20%, and 13% when the cells were treated with PBS solution only, GNSs, NH2-GNSs, and FA-GNSs, respectively. Importantly, the radiosensitization performance order (TAT-GNSs > FA-GNSs > NH2-GNSs > GNSs) of these GNSs was in good consistence with their corresponding cellular uptake efficiencies. To further confirm the above results, real-time cell analysis (RTCA) was performed to monitor the cell growth and proliferation of KB cells after various treatments (GNSs and/or X-ray). As shown in Figure 3D, after 50–60 h, the cell survival viabilities (reflected as cell index values) of the X-ray-treated groups became significantly lower than those of the respective groups without X-ray treatment. The results were in accordance with the conclusion obtained from the above colony formation assays.

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Figure 3. Radiosensitization effects of different GNSs in KB cells. (A) Colony formation images of KB cells treated with GNSs, NH2-GNSs, FA-GNSs, or TAT-GNSs at the same Au atom concentration (50 µg/mL) without and with X-ray irradiation (6 MV, 4 Gy). Colonies formed with more than 50 cells in each group were counted and each group was assayed in triplicate. (B) Radiation dose-dependent survival fractions of KB cells before and after treatments of various GNSs. (C) Sensitization enhancement ratios of GNSs, NH2-GNSs, FA-GNSs, and TAT-GNSs calculated by the multitarget single-hit model. (D) Cell proliferation curves of KB cells treated with various GNSs without and with X-ray irradiation (6 MV, 4 Gy).

3.4. Underlying Mechanisms of Radiosensitizing Effects Induced by Different Surface-Modified GNSs: ROS Generation, DNA Damage, Mitochondrial Membrane Potential Depolarization, Cell Cycle Redistribution, and Apoptosis. To further explore the reasons why GNSs could induce radiosensitization upon X-ray irradiation, we performed various assays to study ROS level, DNA damage, mitochondrial membrane potential, cell cycle distribution, and apoptosis. Among these, ROS generation has been widely reported to 23



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be induced by noble metal nanostructures, leading to oxidative stress.49 Generally, ROS refers to active oxygen atoms or groups of atoms like the super oxygen anion (•O2-), hydrogen peroxide (H2O2), hydroxyl radical (•OH), etc. Moreover, high energy X-rays can greatly increase the •OH production within cells, and therefore •OH is regarded as one of the most toxic reactive oxygen radicals that may lead to the ionizing radiation-mediated cytotoxicity. In this work, the ROS levels within the KB cells before and after treatments with GNSs and/or X-ray were quantified by flow cytometry using the ROS detection kit (where the relative ROS levels were expressed as the mean fluorescence intensity, MFI). The results showed that the MFI values in the X-ray (4 Gy)-treated groups were significantly up-regulated as compared to those in groups without X-ray irradiation (Figure 4A). There was a 1.18, 1.15, 1.39, or 1.74-fold increase of ROS level in KB cells treated with X-ray + GNSs, X-ray + NH2-GNSs, X-ray + FA-GNSs, or X-ray + TAT-GNSs as compared to those treated only with X-ray irradiation. The result confirmed that GNSs with different surface coatings caused increased intracellular ROS generation after exposure to X-ray irradiation. Excessive ROS in the cells will perturb the equilibrium of the oxidation–reduction potential and lead to intracellular peroxide production followed by a series of adverse biological effects such as the oxidation of unsaturated fatty acids in lipids and amino acids in proteins. Most often, the harmful effect mediated by ROS generation was manifested via mitochondrial depolarization, DNA double-strand break, cell cycle redistribution, and apoptosis. It is known that mitochondria are the active sites for intracellular ROS production, and a high ROS level can lead to mitochondrial dysfunction. Mitochondrial depolarization was considered to be one of the earliest omens of cell apoptosis. Herein, mitochondrial membrane potential (MMP) was determined using JC-1 dye. Once mitochondrial depolarization occurred, the dye will not accumulate within the mitochondria and emit green fluorescence signal. MMP loss was quantified by the ratio of green fluorescence intensity (Figure 4B). The results showed that TAT-GNSs combined with X-ray irradiation led to the highest loss of MMP, which agreed well with the highest cellular uptake efficiency of TAT-GNSs.

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Subsequently, the DNA double-strand breaks caused by excess ROS were investigated by immumohistochemical staining (e.g., γ-H2AX staining) using confocal microscopy. The bright green fluorescence spots in the nuclei came from the expression of γ-H2AX, indicating the presence of DNA damage. The average number of green fluorescence spots in the cell nuclei was manually counted for the assessment of irreversible DNA damage. After X-ray irradiation, the ROS levels in the cells increased. It was found that the number of γ-H2AX per cell in the X-ray + GNSs, X-ray + NH2-GNSs, X-ray + FA-GNSs, and X-ray + TAT-GNSs groups increased to 1.02, 1.13, 1.36, and 1.57-fold as compared to those treated with X-ray radiation alone, respectively. This result verified the main reason for inducing irreversible DNA damage was the increased ROS levels. To further evaluate the underlying mechanisms of radiosensitization induced by modified GNSs, we carried out the cell cycle distribution assays by flow cytometry using cell cycle detection kit (Figure 4D, the corresponding statistical data are shown in Table S1 in Supporting Information). After 24 h treatments, more cells in modified GNSs-treated groups were arrested at G2/M phase before exposure to X-ray radiation. While the number of G1 phase cells treated with modified GNSs was obviously less than that in the untreated group. That is to say, different modified GNSs played a crucial role in altering the cell cycle redistribution. It is well-known that the cells at G2/M phase were the most sensitive to X-ray radiation. The radiosensitization mediated by modified GNSs could be greatly enhanced once more cells were arrested at G2/M phase. Finally, the amount of cells at G2/M phase gradually decreased but those at G1 or S phase relatively increased. In brief, cell cycle distribution assay confirmed that one of the main mechanisms of radiosensitization induced by modified GNSs was attributed to cell cycle redistribution. Moreover, the radiosensitization effects of different modified GNSs on cell apoptosis or necrosis were also evaluated before and after X-ray irradiation using Annexin V-FITC/PI staining kit (Figure 4E). These data indicated that the apoptotic ratios of cells treated with modified GNSs combined with X-ray irradiation were higher than those treated with X-ray irradiation only. Compared with the GNSs, NH2-GNSs, and FA-GNSs, TAT-GNSs combined with X-ray irradiation led to the highest cell apoptotic ratio of 15.5% (a sum of the early apoptotic ratio of 8.8% and the late apoptotic ratio of 6.7%) and the lowest viability of 81.4%. 25



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In summary, the radiation effects of modified GNSs were enhanced through the up-regulation of cell apoptosis.

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Figure 4. Effects of GNSs (GNSs, NH2-GNSs, FA-GNSs, and TAT-GNSs; 50 µg/mL of Au atoms) and X-ray irradiation (4 Gy) on the ROS generation, MMP change, DNA damage, cell cycle distribution, and apoptosis of KB cells. (A) ROS generation of KB cells quantified by flow cytometry. The cells treated with Rosup were set as the positive control group. (B) The change of MMP (△Ψm) quantified by the ratio of green fluorescence intensity. CCCP was used as the positive control. (C) Representative fluorescence images (left) and quantitative analyses (right) of DNA double-stranded breaks measured via the expression of γ-H2AX. Nuclei were stained with Hoechst 33342 (blue) and the expression of γ-H2AX was visualized by Alexa Fluor 488-labeled anti-human phospho-H2AX mouse mAb (left). Scale bar: 10 µm. The number of green fluorescent spots in the cell nuclei (n = 50 cells) was manually counted and the histogram was expressed as the average number of γ-H2AX per cell (right). (D) Cell cycle distribution histograms of KB cells determined by flow cytometry using cell cycle detection kit. (E) Apoptosis analyses of KB cells by flow cytometry using Annexin V-FITC/PI staining kit. n = 3; *p < 0.05, **p < 0.01.

3.5. Tumor Growth Inhibitory Effect of Surface-Modified GNSs in Vivo. To study the in vivo tumor growth inhibitory effect of the surface-modified GNSs, we used in vivo U14 tumor model to compare the biodistributions of GNSs and TAT-GNSs in various normal tissues (including heart, liver, spleen, lung, and kidneys) and tumor tissues after intravenous injection. It is well known that PEG molecules conjugated on NP surfaces can minimize the adsorption of serum proteins in the blood to prolong the blood circulation time of the NPs. As shown in Figure 5, the two PEG-modified GNSs were distributed predominantly in the liver and spleen, and approximately 2.4% ID/g (expressed relative to the injected dose per gram tissue) for GNSs and 5.2% ID/g for TAT-GNSs were found in the tumor tissues at 24 h postinjection, suggesting the significant tumor accumulation of the two PEG-modified GNSs due to the enhanced permeability and retention effect in tumor vasculature.

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Figure 5. In vivo biodistributions of GNSs and TAT-GNSs analyzed for U14 tumor-bearing mice sacrificed 24 h postinjection. The amounts of Au in the tissues were quantified by ICP-AES.

Subsequently, we performed a series of in vivo studies based on U14 tumor model. As shown in Figure 6A, at day 8 after different treatments, tumors in GNSs- or TAT-GNSs-treated groups showed similar relative tumor volumes (V/V0) of 7.1 and 7.0 as compared with that (7.2) of the control group, which indicated their negligible tumor growth inhibitory effect. Compared to the “X-ray + GNSs”-treated group (V/V0 = 4.7), a relatively slow growth of the tumor volume (V/V0 = 3.8) was observed in “X-ray + TAT-GNSs”-treated mice over a period of 8 days, confirming the most efficient tumor growth delay after X-ray + TAT-GNSs treatment. The good tumor growth inhibition performance of the X-ray + TAT-GNSs treatment was also verified by the smallest tumor weight and size of the corresponding mice measured at the end of the experiments (Figure 6B and D). The mean body weights of mice throughout the experimental period were monitored every day (Figure 6C). It can be seen that the body weights slowly increased in GNSs- and TAT-GNSs-treated groups, indicating that the two GNSs might have negligible side effects. Finally, the pathological results of tumor tissues (Figure 6E) clearly revealed that the tumor tissue was markedly destroyed after X-ray + TAT-GNSs treatment, while X-ray + GNSs treatment only induced a small degree of damage, demonstrating the distinct sensitization effect of TAT-GNSs in cancer radiotherapy.

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Figure 6. In vivo radiotherapy of U14 tumor-bearing mice. (A) Relative tumor growth profiles of mice after various treatments as indicated. Mice injected with PBS solution (100 µL) were used as the negative control. (B) Tumor weights of mice in different groups at the end of the experiments. (C) Body weights of mice in different groups. (D) Representative photographs of tumors collected from mice in different groups at the end of the experiments. (E) H&E-stained images of tumors collected from mice in different groups at the end of the experiments. Scale bar: 100 µm.

3.6. GNSs-Induced Autophagy. To explore the effects of GNSs and/or X-ray irradiation on the autophagy level, the two autophagy-related structures (including autophagosomes and autolysosomes) in KB cells treated with TAT-GNSs and X-ray irradiation were characterized 29



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by biological TEM. Figure 7A–D showed that large clusters of TAT-GNSs and some of cellular contents were clearly engulfed within vesicles containing double layer or multilayer membrane structures (termed as autophagosome or initial autophagic vacuole, AVi). It was likely that the formed AVi tends to fuse with lysosomes (Figure 7D) or aggregated around the lysosomes (Figure 7B). Figure 7E–H showed the occurrence of late/degradative autophagic vacuole (AVd) as a result of fusion between AVi and multivesicular lysosomes. Moreover, when mitochondria were impaired under oxidative stress, they might be engulfed by initial or late autophagic vacuoles, which was the typical characteristic of autophagy flux. As shown in Figure 7F, the engulfed intracellular contents in these AVd were partially degraded but the remnants of mitochondrion could still be identified.

Figure 7. Biological TEM micrographs illustrate the morphology of autophagosomes and autophagic vacuoles in KB cells treated with TAT-GNSs solution (50 µg/mL of Au atoms) for 24 h. (A–D) Autophagosome or initial autophagic vacuole (AVi) with multilayer or double layer membrane structures. Scale bar: 100 nm. (E–H) Late/degradative autophagic vacuole (AVd). In (F), the morphology of mitochondria (M) could be identified in AVd. Scale bar: 200 nm.

To evaluate the autophagy level, we investigated the autophagosome formation as well as the autophagy-related protein expression. First, the Cyto-ID green autophagy dye was used to detect autophagosomes. The bright green spots in confocal images indicated the presence of 30


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autophagosomes or autolysosomes in the cytoplasm (Figure 8A, left). It was found that a low level of autophagy was activated in the untreated cells. After 24 h of treatment with different modified GNSs, more green dots emerged in the treated cells as compared to the untreated cells. And also the X-ray irradiation indeed facilitated the up-regulation of autophagy level induced by modified GNSs. In addition, the autophagy level was quantified by flow cytometry after Cyto-ID staining. As shown in Figure 8A (right), the fluorescence intensity in the cells treated with NH2-GNSs (without X-ray irradiation) did not appear to be much different from that of the cells treated with FA-GNSs even though FA-GNSs displayed higher cellular uptake efficiency than NH2-GNSs, which might be due to the effect of positive charge on autophagosome/lysosome fusion. Moreover, TAT-GNSs induced the up-regulation of autophagy level because of their highest cellular uptake amount in KB cells. In addition, using KB cells that stably expressed red fluorescent protein (RFP)-tagged LC3 (RFP-LC3), we found that GNSs actually induced the increase of RFP-LC3 dots (Figure 8B). Similarly, the autophagy level in each group was quantified by manually counting the average number of puncta per cell. The results further confirmed the increase of autophagy in GNSs-treated cells. Consistent with the statistical data of Cyto-ID staining, TAT-GNSs exhibited the largest number of RFP-LC3 dots. Besides, western blotting analysis was adopted to measure the expression of the most studied autophagy- and apoptosis-related proteins like LC3 and caspase-3 (active). Regarding LC3 proteins, the LC3-I refers to the cytosolic form of the microtubule-associated protein light chain 3 while the LC3-II form is found on membranes of autophagosomes and serves as an indicator of autophagosome formation. During autophagy process, LC3-I is conjugated by the phosphatidylethanolamine (PE) moiety for generating LC3-II. Conversion of LC3-I to LC3-II which correlates with the autophagosome number is considered as the best marker of autophagy because LC3-II is the only well-characterized protein that specifically localizes to autophagic structures throughout autophagy (from phagophore to lysosomal degradation). The ratio of LC3-II to LC3-I was quantified by measuring the integrated optical densitometry (IOD), which was significantly higher in the GNSs-treated cells than in the untreated cells in the 0 Gy and 4 Gy groups (Figure 8C). Interestingly, the GNSs-induced autophagosome increase is in good accordance with the cellular internalization of GNSs, and a higher 31



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internalization increases the formation of autophagosomes. Caspase-3 is a frequently activated death protease and one of the crucial mediators of programmed cell death (apoptosis). After X-ray irradiation, the content of caspase-3 (active) in the various GNSs-treated groups was significantly higher than that in the untreated groups, which was in agreement with the previous Annexin V-FITC/PI staining results. Previous studies have shown that gold nanoparticles can block autophagic flux and degradation of autophagy substrate.51 To further confirm the effects of differently modified GNSs on autophagic flux, additional experiments for monitoring the degradation of autophagy substrate such as p62 (also known as SQSTM1/sequestome 1) were required. As shown in Figure 8D, we found that starvation (an autophagy inducer) could induce the autophagy-related rapid degradation of p62 protein, while CQ (an autolysosome degradation inhibitor) blocked p62 degradation. However, it was found that the differently modified GNSs mainly caused the accumulation rather than the degradation of p62. These results suggested that the differently modified GNSs might impair the autophagic degradation capacity, which was consistent with the previous result.51 The blockade of autophagic degradation eventually caused autophagosome accumulation in the cells treated with the differently modified GNSs. Therefore, the up-regulation of autophagy level might be closely related with the intracellular oxidative environment, mitochondrial depolarization, and impairment of autophagic degradation capacity induced by modified GNSs.

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Figure 8. Induction of autophagy in KB cells treated with different surface-modified GNSs (GNSs, NH2-GNSs, FA-GNSs, or TAT-GNSs; 50 µg/mL of Au atoms). (A) Confocal fluorescence images of KB cells treated without (control) or with different GNSs for 24 h, irradiated without or with X-ray (4 Gy), and then stained with Cyto-ID green autophagy dye (left). Nuclei were stained blue with Hoechst 33342 and autophagosome and autolysosome were stained green with the Cyto-ID dye. The mean fluorescence intensity (MFI) of the Cyto-ID dye within the KB cells was quantified by flow cytometry (right, n = 10000 cells). n = 3; *p < 0.05 versus untreated control group. Scale bar: 10 µm. (B) KB cells stably expressed RFP-LC3 were treated with different GNSs for 24 h. The red dots indicated the formation of LC3B puncta, which could reflect the induction of autophagy (left). The average puncta per cell (n = 50 cells) was 33



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quantified (right). Scale bar: 5 µm. n = 3; *p < 0.05. (C) Representative Western blotting results showing the effects of different GNSs (50 µg/mL of Au atoms) and X-ray irradiation (4 Gy) on the expression of autophagy-related proteins LC3-I (cytosolic form, 18 KD) and LC3-II (conjugated form, 16 KD), and apoptosis-related protein caspase-3 (active, 17 kD) in KB cells. The differences in LC3-I and LC3-II levels were compared by immunoblot analysis of cell lysates with anti-LC3 antibody. GAPDH served as a loading control. (D) The expression level of p62, a selective autophagy substrate, in KB cells cultured in culture medium for 24 h (lane 1), under starvation condition for 4 h (lane 2), in culture medium containing 20 µM CQ for 2 h (lane 3), and in culture medium containing 50 µg/mL GNSs, NH2-GNSs, FA-GNSs, or TAT-GNSs for 24 h (lane 4, 5, 6, and 7, respectively). GAPDH served as a loading control.

3.7. Protective Autophagy Associated with GNSs. As mentioned above, the modified GNSs could enhance the radiosensitization effect in cancer radiotherapy and cause the up-regulation of autophagy level. However, the function of autophagy in radiotherapy is still not clearly understood. It was reported in a recent study that the inhibition of autophagy enhanced the anticancer activity of silver nanoparticles.50 In this work, we would like to study the relationship between autophagy and X-ray radiotherapy enhanced by GNSs. Based on the statistical data of western blotting analysis (Figure 9A), we found that the expression of LC3-II significantly increased in the cells treated with GNSs, X-ray, and X-ray + GNSs, while the up-regulation of autophagy level could be slightly reversed with 3-methyladenine (3-MA), an autophagy inhibitor via the depression of phosphatidyl inositol 3-kinase (PI3K) activity and block the formation of autophagosomes. To clarify the relationship between autophagy and apoptosis in GNSs-induced radiosensitization, we further investigated the expression of caspase-3 (active), an apoptosis marker protein, and found that the caspase-3 (active) level in the cells treated with GNSs and/or X-ray irradiation combined with 3-MA increased significantly as compared with those treated with GNSs and/or X-ray irradiation alone. To further confirm the above result, the cell apoptosis assay was performed by flow cytometry. The data in Figure 9B showed that the apoptosis rate of X-ray + GNSs group is 11.05% (a sum of the early apoptotic ratio of 8.18% and the late apoptotic ratio of 2.87%), which increased to 24.21% in the presence of 3-MA. This result suggested that the autophagy inhibitor 3-MA increased the sensitivity/susceptibility of the cells to radiotherapy and more 34


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cells tended to experience apoptosis once the autophagy was inhibited. Thus the autophagy induced by GNSs and/or X-ray irradiation played a protective role in X-ray radiation therapy. Motivated by this finding, the appropriate combined use of autophagy inhibitor and gold nanostructures might be a useful strategy for increasing the sensitivity of cancer cells to ionizing radiation in cancer radiotherapy.

Figure 9. Effect of autophagy inhibitor (3-MA, 5 mM) on autophagy level and apoptosis in KB cells treated with GNSs (50 µg/mL of Au atoms) and/or X-ray irradiation (4 Gy). (A) Effect of 3-MA, X-ray, and GNSs on the expression level of LC3-I, LC3-II, and caspase-3 (active) in KB cells. The relative ratio of LC3-II/LC-I and caspase-3 (active) were quantified by integrated optical densitometry (IOD). n = 3; *p < 0.05; *p < 0.01. GAPDH served as a loading control. (B) Effect of 3-MA on the apoptosis ratios of KB cells treated with culture medium (control), X-ray, GNSs, or X-ray + GNSs using Annexin V-FITC/PI staining.

4. CONCLUSIONS 35



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In this work, different surface-functionalized GNSs (GNSs, NH2-GNSs, and FA-GNSs, TAT-GNSs) were successfully synthesized and their radiosensitization effects were evaluated. It was found that the radiation sensitizing effect mainly depended on the amount of internalized GNSs. With their extremely high SER value (2.30), TAT-GNSs exhibited the most significant radiosensitization effect mainly because of their highest cellular internalization efficiency. Besides, we have also investigated the cellular uptake mechanisms of these different GNSs and the changes of ROS, MMP, and cell cycle distribution induced by these different GNSs in radiotherapy. In addition, the up-regulation of autophagy was found to act as a cellular self-defense response to prevent the cells from ionizing radiation damage. Although an enhanced cellular uptake induced a more pronounced ionizing radiation effect, the increased autophagy level which played a protective role in cancer cells would decrease the anticancer efficiency. By combining with the autophagy inhibitor 3-MA, a significantly enhanced apoptosis ratio for KB cells was observed. Besides the above in vitro experiments, we have also carried out in vivo experiments and found that the most efficient tumor growth delay was achived for mice after X-ray + TAT-GNSs treatment, confirming the highest radiation sensitizing effect of TAT-GNSs. The present work provides an in-depth understanding on the metal nanoparticle-based cancer radiotherapy and clarifies the role autophagy plays in the anticancer efficiency, which will promote the development of metal nanoparticle-based therapeutics for combating cancer.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Preparation of HS-PEG-FA; determination of Au content by AAS; cellular uptake amounts of different GNSs evaluated by dark-field imaging, flow cytometry, and ICP-AES; calculation of sensitization enhancement ratio (SER); statistical data of cell cycle distribution; establishment of KB cell lines which stably express RFP-LC3 (PDF) 36


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AUTHOR INFORMATION Corresponding Authors *(F.G.W.) E-mail: [email protected]. *(Z.C.) E-mail: [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the grants from the National Key Basic Research Program of China (973 Program) (2013CB933904), 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 the Graduate Students’ Scientific Research Innovation Project of Jiangsu Province Ordinary University (CXLX12_0119). Z.C. acknowledges the support from the University of Michigan.

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Action of Gold Nanospikes-Based Nanoradiosensitizers: Cellular

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