Selenide Nanosheets for Highly Efficient Photonic Cancer Theranostics

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Biological and Medical Applications of Materials and Interfaces

Construction of 2D Antimony (III) Selenide Nanosheets for Highly Efficient Photonic Cancer Theranostics Yadan Zhou, Wei Feng, Xiaoqin Qian, Luodan Yu, Xiuguo Han, Gonglin Fan, Yu Chen, and Jiang Zhu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02104 • Publication Date (Web): 08 May 2019 Downloaded from http://pubs.acs.org on May 8, 2019

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ACS Applied Materials & Interfaces

Construction of 2D Antimony (III) Selenide Nanosheets for Highly Efficient Photonic Cancer Theranostics Yadan Zhou1, Wei Feng2*, Xiaoqin Qian3, Luodan Yu2, Xiuguo Han4 ,Gonglin Fan1,Yu Chen2*, Jiang Zhu1* 1Department

of Ultrasound, Sir Run Run Shaw Hospital, Zhejiang University School of

Medicine, Hangzhou 310016, P. R. China. 2State

Key Laboratory of High Performance Ceramics and Superﬁne Microstructure,

Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, P. R. China. 3Department

of Ultrasound, The Affiliated People's Hospital of Jiangsu University, Zhenjiang

212002, P. R. China 4Shanghai

Key Laboratory of Orthopedic Implants, Department of Orthopedic Surgery

Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, P. R. China

ABSTRACT: Photonic cancer hyperthermia has been considered to be one of the most representative non-invasive cancer treatments with high therapeutic efficiency and biosafety. However, it still remains a crucial challenge to develop efficient photothermal nanoagents with satisfactory photothermal performance and biocompatibility, among which twodimensional (2D) ultrathin nanosheets have recently been regarded as the promising multifunctional theranostic agents for photothermal tumor ablation. In this work, we report, for the first time, on the construction of a novel kind of PTAs based on the intriguing 2D antimony (III) selenide (Sb2Se3) nanosheets for highly efficient photoacoustic (PA) imaging1 ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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guided photonic cancer hyperthermia by near infrared (NIR) laser activation. These Sb2Se3 nanosheets were easily fabricated by a novel but efficiently combined liquid nitrogenpretreatment and freezing-thawing approach, which were featured with high photothermalconversion capability (extinction coefficient: 33.2 L g-1 cm-1; photothermal-conversion efficiency: 30.78%). The further surface engineering of these Sb2Se3 ultrathin nanosheets with polyvinyl pyrrolidone (PVP) substantially improved the biocompatibility of the nanosheets and their stability in physiological environments, guaranteeing the feasibility photonic antitumor applications. Importantly, 2D Sb2Se3-PVP nanosheets have been certificated to efficiently eradicate the tumors by NIR-triggered photonic tumor hyperthermia. Especially, the biosafety in vitro and in vivo of these Sb2Se3 ultrathin nanosheets have been evaluated and demonstrated. This work meaningfully expands the biomedical applications of 2D bionanoplatforms with planar topology through probing into new members (Sb2Se3 in this work) of 2D biomaterials with unique intrinsic physiochemical property and biological effect. KEYWORDS: Two-dimensional,

Antimony (III) selenide

therapy, Cancer, Nanomedicine

2 ACS Paragon Plus Environment

nanosheets, Photothermal

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INTRODUCTION As one of the most representative non-invasive cancer-therapeutic modalities, near infrared (NIR)-activated photothermal therapy (PTT) has drawn extensive research interests because of its intriguing noninvasiveness, high controllability and desirable therapeutic efficiency/biosafety, which typically requires photothermal agents (PTAs) to produce heat by converting light energy into thermal energy.1, 2 Therefore, the PTA plays a key role in PTT for cancer ablation. Especially, great endeavors have been recently dedicated to questing for original high-performance nanomaterials with strong NIR (λ = 750-1350 nm) absorbance and excellent photothermal-conversion efficiency.3 With the fast development of PTAs, such as precious metal nanoparticles (e.g., Au, Pt),4, 5 carbon material (e.g., graphene, carbon nanorods) ,6, 7 metal sulphides (e.g., CuS, FeS),8, 9 and some organic substances (e.g., indocyanine green, prussian blue),10,

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photonic tumor

hyperthermia by PTT has been actively explored in the efficient treatment of tumors. Typically, the desirable PTAs should be featured with the following properties. Firstly, the most important performance of PTAs is the high photothermal-conversion efficiency, which is the crucial factor for their applications in PTT. Secondly, PTAs should be non-toxic with low or even tolerable biotoxicity. Thirdly, these PTAs should be easily functionalized or modified with desirable organic moieties such as PEGylation or targeting components for some specific purposes, or even with therapeutic molecules for achieving synergistically therapeutic outcome. Driven by the outstanding performance of graphene, two-dimensional (2D) nanomaterials with distinctive planar topology have received high attention to become extremely active research areas in the field of nanotechnology. The family of 2D nanomaterials have been substantially enriched in the past a few years, including the typical transition metal dichalcogenides (TMDCs),12,

13

transition metal carbides and nitrides

(MXenes) ,14 layered double hydroxides (LDHs) ,15, 16 hexagonal boron nitride (h-BN) ,17 the 3 ACS Paragon Plus Environment


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single element-composed metal or nonmetal materials (e.g., graphene, black phosphorus, antimonene and boron).18-21 Due to the unique planar structure and remarkable physicochemical properties, these 2D materials have found numerous potential applications in diverse areas including electronics,22 optoelectronic devices,23 catalysis,24 energy conversion and storage25 and biomedicine.26 2D nanosheets such as black phosphorus,

27

WS2,28 Ti3C2,29

antimonene nanosheets,30 have also been explored as PTAs for efficient photonic cancer hyperthermia.31 Antimony (III) selenide (Sb2Se3) has a unique crystal structure, which inherently tends to grow into 1D nanostructure, such as ribbon,32 nanotubes,33 and nanowire,34 making the difficulty in the fabrication of Sb2Se3 with the desirable 2D planar nanostructure. The typical bottom-up approaches including chemical vapor deposition and solvo- or hydro-thermal synthesis et al. are not suitable for the synthesis of Sb2Se3 nanosheets. The alternative topdown methodology including etching-assisted exfoliation,29 ion intercalation-assisted liquid exfoliation,35,

36

mechanical force-assisted liquid exfoliation,37,

38

and micromechanical

cleavage,39 request the presence of layered bulk crystals, which are further exfoliated into 0nanosheets by breaking the interlayer weak Van der Waal’s force. Zeng et al. reported that few-layer-thick Sb2Se3 nanosheets were prepared from Sb2Se3 layered bulk precursors through electrochemical lithium intercalation method.35 Recently, Song et al. also successfully fabricated Sb2Se3 nanosheets via the freezing-thawing approach.40 It was found that the liquid nitrogen pretreatment could cause small cracks in the layered powder.41 Herein, in this work, we proposed a novel and facile method by combined liquid nitrogen pretreatment and freezing-thawing approach to generate ultrathin Sb2Se3 nanosheets (Figure 1a). Initially, purity Sb2Se3 powers were soaked in liquid nitrogen for 1 h to form the cracks, which were then dispersed into deionized water by stirring vigorously for 12 h to make the water enter into the cracks. Hereafter, the mixture was placed into the refrigerator (set as 4 ACS Paragon Plus Environment

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80 ℃) for 12 h. This step that transformed intercalated water into ice would increase the crack spacing. The frozen mixture would melt in bath sonication or sonicated by probe sonication for 12 h. 2D nanosheets reported for PTA application were synthesized by using organic solvents such as N-Methyl pyrrolidone, which were also needed to improve their production efficiency.42 However, Sb2Se3 nanosheets were fabricated with high productivity by using liquid nitrogen and water without introducing any impurity elements. Moreover, Sb2Se3 nanosheets are composed of non-toxic and earth-abundant constituents, such as selenium contained in Sb2Se3-PVP nanosheets that is an essential trace element for human body and antimonial drugs for the pharmaceutical industry,43 which further strengthens Sb2Se3-PVP nanosheets as a new powerful PTA with high therapeutic biosafety. In order to enhance the biological compatibility and physiological stability, after further surface modification with polyvinyl pyrrolidone (PVP), Sb2Se3-PVP nanosheets could efficiently accumulate into tumor tissue by the typical enhanced permeability and retention (EPR) effect (Figure 1b). Upon external NIR laser irradiation, these ultrathin Sb2Se3-PVP nanosheets exert the specific functionality of transforming light energy to heat energy, causing the non-invasive photonic tumor hyperthermia. RESULTS AND DISCUSSION Scanning electron microscopy (SEM) image displays the unique laminated structure of Sb2Se3 bulk crystals at different magnifications (Figure 2a, b). Before exfoliation, the morphology and structure of Sb2Se3 bulk crystal were further identified via transmission electron microscopy (TEM, Figure 2c). After liquid nitrogen pretreatment and freezingthawing approach, the Sb2Se3 ultrathin nanosheets were successfully composited, as demonstrated in TEM image (Figure 2d). The high-resolution TEM (HRTEM) image reveals that the lattice fringes is around 0.279 nm corresponding to the (301) lattice plane of Sb2Se3 nanosheets (Figure 2e). The atomic force microscopy (AFM) image confirms the typical 5 ACS Paragon Plus Environment


sheet microstructure of the Sb2Se3 nanosheets and the thickness as determined via the crosssectional analysis is less than 4 nm, indicating the successful fabrication of the few-layer Sb2Se3 nanosheets (Figure 2f, g). The distribution of Se atoms (red) and Sb atoms (green) in the Sb2Se3 nanosheets can be easily distinguished by atomic-resolution HAADF images (Figure 2h-k). X-ray photoelectron spectroscopy (XPS) was used to confirm the chemical composition of Sb2Se3 nanosheets. The O, Sb and Se elements are present in the full scan XPS survey spectrum of Sb2Se3 nanosheets (Figure S1). The Raman spectra of exfoliated severallayer Sb2Se3 nanosheets show the representative peaks similar to Sb2Se3 bulk crystals (Figure S2), The most intense peak at about 189 cm-1 corresponds to Ag mode of Sb-Se stretching, which is orthogonal to the ribbon direction.40,

44

A slight blueshift could observed, which

could be attributable to the weakened long-range coulombic interaction in the thinner material.40 The X-ray diffraction (XRD) patterns of the few-layer Sb2Se3 nanosheets and Sb2Se3 bulk crystal confirm their orthogonal structure (JCPDS:15-0861) (Figure S3). These results demonstrate the unchanged crystal structure of Sb2Se3 nanosheets during the exfoliation process. To further improve the dispersion and physiological stability of Sb2Se3, PVP as one of the mostly explored biocompatible surface stabilizers was applied to surface modification of 2D Sb2Se3 nanosheets. After PVP modification, the fabricated Sb2Se3-PVP nanosheets showed remarkably enhanced dispersibility and stability in H2O, phosphate buffer saline (PBS), Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) as compared to pristine 2D Sb2Se3 nanosheets (Figures 3a and S4 ). Fourier transform infrared (FT-IR) spectrometer and zeta potential were used to confirm the successful surface modification of Sb2Se3 nanosheets with PVP molecules. As shown in the FTIR spectra, the absorption bands at approximately 1652 cm−1 was ascribed to the vibration of C=O bond, and the C-N group stretch was found near 1286 cm-1 in PVP and Sb2Se3-PVP nanosheets. These bands further 6 ACS Paragon Plus Environment

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indicate the successful PVP coating onto the surface of Sb2Se3 nanosheets (Figures 3b). After being surface modified, the value of zeta potential was changed from -27.27 mV to -8.10 mV (Figures 3c). All these results indicated the successful surface modification of 2D Sb2Se3 nanosheets, guaranteeing the further in vitro and in vivo therapeutic applications. To explore the photothermal-conversion potential and efficacy of 2D Sb2Se3-PVP nanosheets as efficient PTAs, we systematically evaluated their UV-vis-NIR absorbance, photothermal stability and photothermal-conversion efficiency. The UV-vis-NIR absorbance of Sb2Se3-PVP nanosheets showed high photo-absorption in the NIR window at varied concentrations (Figures 3d). The digital photographs of Sb2Se3-PVP nanosheets dispersed in aqueous solution exhibited the typical Tyndall effect, indicating their outstanding hydrophilicity and high dispersity (inset of Figure 3d). The extinction coefficient of Sb2Se3PVP nanosheets at 808nm was calculated to be 33.2 L g-1 cm-1 (Figure S5), which is remarkably higher than that of traditional 2D graphene oxide (3.6 L g−1 cm−1, at 808 nm) .45 The photothermal performance of Sb2Se3-PVP aqueous solution was further evaluated at varied concentrations from 0 to 400 ppm under the NIR irradiation (808 nm, 1.0 W cm−2) . At a relatively low concentration (100 ppm), the temperature of the Sb2Se3-PVP aqueous solution could reach 51.8 °C within 10 min NIR irradiation (Figure 3e). In contrast, the temperature of water exhibited no significant temperature increase, indicating that Sb2Se3PVP nanosheets could effectively convert light energy into thermal energy. Moreover, the temperature of the Sb2Se3-PVP aqueous solution (100 ppm) could be raised to 60 °C with NIR laser (1.5 W cm−2, 808 nm), which was expected to effectively kill cancer cells by hyperthermia (Figure 3f). Furthermore, the photothermal-conversion effciency (η) of Sb2Se3PVP was calculated to be 30.78% according to the previously reported method26 (Figure 3g,h), which is much higher than those of traditional PTAs such as Au nanorods (21%)46 and Cu2−x-Se NCs (22%).47 In addition, the photostability of 2D Sb2Se3-PVP nanosheets was then 7 ACS Paragon Plus Environment


investigated by exposing the Sb2Se3-PVP aqueous solution to the irradiation of 808 nm laser for five irradiation cycles. There was no significant change of the photothermal performance during five laser on/off cycles with laser irradiation (1.5 W cm−2, 808 nm), indicating the high photostability of Sb2Se3-PVP nanosheets (Figure 3i). Before and after laser irradiation, UVvis-NIR spectra, TEM images and atomic-resolution HAADF images of the Sb2Se3-PVP aqueous solution showed almost no obvious change, further suggesting the high photothermal stability (Figure S6 and S7). The in vitro cytotoxicity of 2D Sb2Se3-PVP nanosheets as PTAs was initially evaluated. Typically, these Sb2Se3-PVP nanosheets could efficiently enter into the cancer cells by endocytosis and then kill the cancer cells under NIR irradiation by hyperpyrexia (Figure 4a). The standard CCK-8 experiment was applied to test the potential cytotoxicity of Sb2Se3-PVP nanosheets on both cancer cells and normal cells in vitro. Murine breast cancer 4T1 cells, MBA-MD-231 cells and mouse skin fibroblast L929 cells in this work were incubated with Sb2Se3-PVP nanosheets at various concentrations from 0 to 400 ppm for 24 and 48 h, respectively. After examination by CCK-8 assay, no evident cytotoxicity was observed in different cell lines even at the concentrations up to 400 ppm (Figure 4b, c and S8a, b), indicating the low cytotoxicity of these Sb2Se3-PVP nanosheets. To survey the cytophagy of Sb2Se3-PVP nanosheets, confocal laser scanning microscopy (CLSM) and flow cytometry were applied to detect the fluorescence signal of FITC-labeled Sb2Se3-PVP nanosheets within cancer cells, respectively. The CLSM images of 4T1 cells intuitively displayed the green fluorescence stemming from FITC-labeled Sb2Se3-PVP nanosheets after 4 h coincubation, indicating the efficient intracellular uptake of Sb2Se3-PVP nanosheets. After 8 h incubation with 4T1 cells, a significant increase in fluorescence intensity was measured from 0% (0 h) to 99.3% (8 h) by using flow cytometry (Figure S9a-c). This result indicates that as the coincubation duration increases, the more nanosheets are uptaken by the cancer cells. 8 ACS Paragon Plus Environment

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Based on the excellent photothermal performance and biocompatibility, the near-infrared mediated photothermal cytotoxicity of Sb2Se3-PVP nanosheets was further systematically evaluated. After co-incubation with Sb2Se3-PVP nanosheets for 4 h followed by an 808 nm laser irradiation for 10 min, the 4T1 cells and MBA-MD-231 cells were efficiently killed in the presence of Sb2Se3-PVP nanosheets, which revealed that the photothermal cytotoxicity of Sb2Se3-PVP

was both concentration-dependent (Figure 4d and S8c) and laser power

density-dependent (Figure 4e and S8d). Additionally, the intracellular photothermal hyperthermia mechanism of Sb2Se3-PVP nanosheets was investigated by a flow-cytometry apoptosis assay after the co-staining of cancer cells with AnnexinV-FITC and Propidium Iodide (PI). Cell population is divided into four areas including necrotic cells (Q1), late apoptotic (Q2), early apoptotic (Q3), and live (Q4). During the experiment, 4T1 cells were incubated for 4 h with different treatments in four conditions, including the control group, Sb2Se3-PVP group, NIR group, and Sb2Se3-PVP combined laser. A large amount of apoptosis cells were observed in Sb2Se3-PVP combined laser group (Figure 4f and g). Furthermore, CLSM was used to directly observe the high photothermal-ablation efficacy of Sb2Se3-PVP nanosheets against cancer cells. After near-infrared irradiation (1.0 W cm-2, 10 min), 4T1 cells was co-stained with calcein-A (live cells, green fluorescence) and PI (dead cells, red fluorescence). Numerous dead cells were observed in Sb2Se3-PVP combined with laser group (Figure 4h), demonstrating the remarkable photothermal cytotoxicity of Sb2Se3-PVP nanosheets. Because the data of the flow-cytometry apoptosis assay and CLSM illustrated that photothermal hyperthermia of Sb2Se3-PVP nanosheets caused cancer-cell apoptosis, especially late apoptosis, this experiment further explored the effects of photothermal ablation on the nucleus and cytoskeleton of cancer cells. To observe the influence of PTT on cancercell nucleus, breast 4T1 cells in different groups were stained with 4’,6-diamidino-29 ACS Paragon Plus Environment


phenylindole ( DAPI ,blue fluorescent). Morphological shrinkage of cancer-cell nucleus was observed in the Sb2Se3-PVP combined with NIR group (Figure 4h), indicating that the photothermal ablation destroyed the nucleus. The cytoskeleton plays an important part in important life activities, such as maintaining cell morphology, withstanding external forces, and maintaining the order of the internal structure of cells.48 Herein, the cytoskeleton of 4T1 cells in different groups was stained with Alexa Fluor488-Phalloidin. These cells which were respectively dealed with Sb2Se3-PVP and NIR laser irradiation maintained intact cell cytoskeleton, which showed no difference as compared to the control group. In contrast, the cytoskeleton of cells lost its own integrity after the treatment of Sb2Se3-PVP nanosheets combined with laser irradiation (Figure 4h). These results solidly demonstrate that Sb2Se3PVP-enabled photothermal hyperthermia efficiently causes cancer-cell apoptosis by damaging the nucleus and cytoskeleton of cancer cells. Furthermore, hemolysis assay was applied to explore the blood-contact safety of Sb2Se3PVP nanosheets before in vivo evaluation. Neglected hemolytic activity (percent hemolysis

Selenide Nanosheets for Highly Efficient Photonic Cancer Theranostics

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