Nanosheets as Theranostic Agent for Synergistic - ACS Publications

May 21, 2018 - ABSTRACT: Phototherapy, including photothermal therapy (PTT)/photo- dynamic therapy (PDT), is usually considered as a promising strateg...
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Photosensitizer-Conjugated Bi2Te3 Nanosheets as Theranostic Agent for Synergistic Photothermal and Photodynamic Therapy Jing Bai,† Xiaodan Jia,† Yudi Ruan,†,‡ Chao Wang,†,‡ and Xiue Jiang*,†,‡ †

State Key Lab of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, Jilin, China ‡ University of Science and Technology of China, Hefei 230026, Anhui, China

Inorg. Chem. Downloaded from pubs.acs.org by ST FRANCIS XAVIER UNIV on 08/08/18. For personal use only.

S Supporting Information *

ABSTRACT: Phototherapy, including photothermal therapy (PTT)/photodynamic therapy (PDT), is usually considered as a promising strategy for cancer treatment due to its noninvasive and selective therapeutic effect by laser irradiation. A light-activatable nanoplatform based on bovine serum albumin (BSA)-coated Bi2Te3 nanosheets conjugated with methylene blue (MB) was successfully designed and constructed for bimodal PTT/PDT combination therapy. The resultant nanoconstruct (BSA-Bi2Te3/MB) exhibited high stability in various physiological solutions and excellent biocompatibility. Especially, the nanoconstruct not only possessed strong near-infrared absorption and high photothermal conversion as a photothermal agent for efficient tumor ablation but also could successfully load photosensitizer for PDT of tumor. When exposed to laser irradiation, tumors in mice with BSA-Bi2Te3/MB injection were completely eliminated without recurrence within 15 d, demonstrating the potential of the nanoconstruct as a bimodal PTT/PDT therapeutic platform for cancer treatment.

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INTRODUCTION Despite the great efforts made in cancer treatment over the past decade, alternative high-efficient cancer treatment approaches remain a challenge for both clinicians and patients. Phototherapy, including photothermal therapy (PTT) and photodynamic therapy (PDT), is a promising treatment strategy owing to a minimally invasive treatment, selective and localized therapeutic effect, and minimal side effects.1 In PTT, photoabsorbing agents could convert near-infrared light (NIR) into heat, resulting in cancer cells ablation.2,3 However, the laser in the light path inevitably travels some normal tissues, reducing heat effectiveness in the tumor cells and causing nonspecific damage to healthy tissues. For PDT, the apoptosis or necrosis of cells is caused by highly toxic singlet oxygen (1O2),4,5 which is generated from an activatable photosensitizer (PS). However, the limited penetration depth from visible or ultraviolet light and insufficient oxygen supply in cancer cells limited PDT therapeutic effects for solid tumors. So, the effect of single treatment on tumor growth is often unsatisfactory. Many efforts have been made in smart integration of PTT and PDT into one system, and improved therapeutic efficacy was obtained. Various nanoconstructs have been developed for the combination therapy of PTT and PDT, including gold nanostructures,6 graphene oxide,7 fullerene,8 and other inorganic or organic nanoagents,9 etc. Their practical applications in combination of PTT and PDT are often limited by poor structure stability upon NIR irradiation of gold nanorod, the complicated synthesis of carbon-based nanomaterials, and low loading efficiency for photosensitizers. In that © XXXX American Chemical Society

case, the new integrated nanoconstructs with strong NIR absorbance, large surface area for loading photosensitizers, as well as excellent biocompatibility are greatly demanded. Recently, two-dimensional (2D) transition-metal dichalcogenides (TMDCs) consisting of transition metal and chalcogen have drawn considerable attention because of their extraordinary layered structure features and fascinating application in electronics, energy storage, biosensing, disease diagnosis, and so on. Especially, bismuth-based chalcogenides, such as Bi nanocrystals, Bi2S3 and Bi2Se3, have been developed for PTT of cancer due to their strong NIR absorption ability. For example, the PVP-encapsulated Bi2Se3 nanosheets with high photothermal conversion efficiency and excellent photoacoustic performance were demonstrated as efficient hyperthermia agents for cancer therapy.10 Similarly, Yu group designed Bi2Se3 spherical sponge into a drug delivery vehicle with high photothermal conversion efficiency and triple-modal imaging ability.11 Recently, biocompatible bismuth nanocrystals (Bi-PEG NCs) were developed as theranostic agent for multimodal imaging and photothermal ablation of tumors.12 In another report, Bi2S3 nanourchins were developed as a new theranostic nanoplatform for triple-modal imaging-guided chemo- and photothermal combination therapy, which exhibited an excellent anticancer efficacy both in vitro and in vivo.13 Importantly, Bi2Se3@PDA nanocomplex was fabricated and applied simultaneously for PTT, chemo-therapy, and dualReceived: May 21, 2018

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DOI: 10.1021/acs.inorgchem.8b01385 Inorg. Chem. XXXX, XXX, XXX−XXX

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modal imaging.14 The Bi-based compounds may, therefore, be promising for integrated different therapeutic functions in one system. As a typical member of Bi-based chalcogenides, Bi2Te3 shows enormous application potentials in biomedical applications given the high X-ray attenuation of Bi as computed tomography (CT) contrast agent. However, the applications of Bi2Te3 nanostructures in cancer therapy have not been reported, to the best of our knowledge, and remain to be explored. Recently, we observed that Bi2Te3 nanosheets (Bi2Te3 NSs) also showed a significant absorption in the NIR region, which have potential for PTT of cancer. Major challenges limiting the novel Bi2Te3 nanoagents toward biological applications include the synthesis of Bi2Te3 nanosheets with reliable stability and the high toxicity of the nanoagents in biological systems. Owing to weak van der Waals forces between each layer, several groups including ours have explored the use of atomically thin TMDC nanosheets as efficient photothermal therapeutic agents for cancer therapy.15,16 Many methods, including chemical vapor deposition, thermal ablation method, mechanical and chemical exfoliation, and direct solvothermal synthesis, have been used to prepare reliable and scale-up production of TMDC nanosheets. Among them, chemical exfoliation, such as Li intercalation17 and organic solvents exfoliation,18 is the most commonly used method for the largescale synthesis of TMDC nanosheets. However, the reaction of Li intercalation is aggressive during exfoliation, leading to structural and electronic destruction of TMDC nanosheets. Although organic solvent exfoliation retains the pristine property of TMDC bulk crystals, the resulting TMDC nanosheets suffer poor water dispersity and biocompatibility from organic solvents, which limit their further clinical application. So some functionalization strategies are usually used to afford suitable TMDC biocompatibility and stable water dispersion, which might result in the increase of experimental complexity and impede the wide use of TMDC nanosheets. Recently, bovine serum albumin (BSA) as an effective exfoliating agent was successfully used to produce 2D materials19 as well as a strong stabilizing agent against reaggregation of single-layer nanosheets, improving biocompatibility. This protein exfoliation as a reproducible and biocompatible approach provides a viable method for producing other single-layer TMDC nanosheets. Herein, for the first time, we report a simple approach for preparing a new type of 2D Bi2Te3 nanosheets through employing BSA as an exfoliating agent and stabilizer. On the one hand, the as-prepared BSA-coated Bi2Te3 nanosheets (BSA-Bi2Te3 NSs) showed a broad absorption band in NIR region with photothermal conversion efficiency as high as 45.3% under 808 nm laser irradiation and suitable photostability. On the other hand, methylene blue (MB), as photosensitizer (PS), was loaded on the surface of BSABi2Te3 NSs via physical adsorption or electrostatic interaction as formed BSA-Bi2Te3/MB nanocomposites. In this design, Bi2Te3 nanosheets could absorb NIR light and generate heat as an efficient photothermal agent and also act as a carrier for photosensitizer. The resulting BSA-Bi2Te3/MB nanocomposites showed enhanced anticancer effects in vitro and in vivo by the combination of PTT and PDT. The results suggested a great potential of BSA-Bi2Te3/MB nanocomposites in cancer therapy and shed light on a new way to improve anticancer efficacy. Our work expands the application of 2D TMDCs in biological systems.

Article

EXPERIMENTAL SECTION

Materials. Sodium hydroxide, sodium borohydride, and H2O2 (30%) were obtained from Beijing Chemical Reagents Company. Bismuth chloride, tellurium powder, and MB were obtained from Aladdin Reagents Company. 1,3-Diphenylisobenzofuran (DPBF), dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny-ltetrazolium bromide (MTT), calcein acetoxymethyl ester (Calcein AM), and propidium iodide (PI) were purchased from Sigma-Aldrich. BSA, fetal bovine serum (FBS), and streptomycin were purchased from Beijing Dingguo Biotechnology Co. Intracellular reactive oxygen species (ROS) assay kit and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Thermo Scientific. Phosphate-buffered saline (PBS) used in cell culture was purchased from Invitrogen (10010). All of the chemicals were of analytical grade and used as received without further purification. Deionized water was used for all experiment. Preparation of Bi2Te3 Nanosheets. Bismuth chloride (2.0 mmol) and 3.0 mmol of tellurium powder were dissolved in 35 mL of ethanol−water mixture (6:1 v/v), and then 2.5 mmol of sodium hydroxide and 8.0 mmol of sodium borohydride were added into mixture with further stirring for 15 min. Then the above-mentioned mixture was transferred to a Teflon autoclave and heated at 180 °C for 20 h. After it cooled to room temperature naturally, the obtained Bi2Te3 bulk was washed with ethanol and deionized water several times and then dried at 60 °C. To obtain Bi2Te3 nanosheets, the 10 mg of Bi2Te3 powder was dispersed into 10 mL of deionized water containing 10 mg of BSA and then further sonicated at the power of 200 W for 5 h. The resultant solution was then centrifuged at 3000 rpm to remove all unexfoliated powder in the precipitates. The final supernatant was dialyzed against deionized water to remove excess BSA and other residue ions for obtaining BSA-Bi2Te3 nanosheets. In Vitro Photothermal Performance. To assess the photothermal conversion performance of the BSA-Bi2Te3 nanosheets, 1.0 mL of test solution was suspended in a quartz cuvette and irradiated by the 808 nm laser with a power density of 2.0 W/cm2 for 10 min. The temperature changes were measured with a digital thermometer at 30 s intervals for a total of 10 min. To further demonstrate the photostability of BSA-Bi2Te3 nanosheets, the samples were irradiated for 10 min with an 808 nm NIR laser (laser on), followed by naturally cooling to room temperature without NIR laser irradiation for 30 min (laser off). This cycle was repeated five times, and then UV−vis spectra of the irradiated samples were obtained for characterizing the absorption property. Methylene Blue Loading on BSA-Bi2Te3 Nanosheets. MB was loaded on the surface of BSA-Bi2Te3 NSs via electrostatic attraction. In brief, BSA-Bi2Te3 NSs (1.0 mg) was mixed with a PBS solution of MB (5 mL, 40 μg mL−1) stirring overnight in the dark at 25 °C. Then the mixture was dialyzed against buffer solution to remove unloaded MB, obtaining BSA-Bi2Te3/MB NSs. The MB-loading content (MLC) onto BSA-Bi2Te3 NSs was determined using UV−vis spectroscopy at 665 nm. The loading content can be calculated according to the following equation: MLC = (OMB − RMB)/ OBSA‑Bi2Te3, in which OMB, RMB, and OBSA‑Bi2Te3 is the original MB content, residual MB content, and original BSA-Bi2Te3 content, respectively. Singlet Oxygen Detection. Singlet oxygen (1O2) production was assessed by a chemical oxidation method using DPBF as a detector, which could react irreversibly with 1O2 to cause a reduction of DPBF absorbance. In brief, 2.7 mg of DPBF was dissolved in 1.0 mL of ethanol to obtain the stock solution of DPBF (10 mM). Then, 10 μL of DPBF solution was added to different sample solutions ([BSABi2Te3] = 50 μg/mL, [MB] = 2 μg/mL) and irradiated with a 650 nm laser at the power density of 50 mW/cm2 for 20 min. The absorption spectra of DPBF at various irradiation times were measured at 410 nm by a UV−vis spectrophotometer. The effects of alone laser, BSABi2Te3, BSA-Bi2Te3/MB, and BSA-Bi2Te3/MB with laser irradiation were also detected by the same process. Cell Culture. Cervical cancer cell line (HeLa cells) were cultured in a DMEM medium supplemented with 10% (v/v) FBS, 100 U mL−1 B

DOI: 10.1021/acs.inorgchem.8b01385 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry penicillin, and 100 μg mL−1 streptomycin at 37 °C under 5% CO2. The cells were routinely harvested by the use of trypsin and resuspended in fresh complete medium before plating. Cell Toxicity Assay. To study cytotoxicity of BSA-Bi2Te3 NSs or BSA-Bi2Te3/MB NSs, HeLa cells and human umbilical vein endothelial cells (HUVEC) were seeded into 96-well plates (1 × 104 cells per well) overnight and then incubated with different concentrations of BSA-Bi2Te3 or BSA-Bi2Te3/MB ranging from 0 to 400 μg/mL for 24 h. After the incubation, 100 μL of MTT solution (0.5 mg/mL) in pH 7.4 PBS was added to each well after carefully removing the original culture medium, and then the mixture was incubated for another 4 h. Finally, the medium was replaced with 100 μL of DMSO to dissolve blue formazan. After the wells were shaken at room temperature for 10 min, the absorbance at 570 nm of each well was measured to determine their relative cell viability. The hemolysis assay was also performed to evaluate the in vitro biocompatibility of BSA-Bi2Te3 nanosheets. Human blood samples were freshly provided by the local hospital (Affiliated Hospital of Northeast Normal University). First, a fresh blood sample (2 mL) was added into PBS solution (4 mL), and then red blood cells (RBCs) were isolated from serum by centrifugation at 3000 rpm for 10 min. After it was washed five times with 10 mL of PBS solution, the purified blood was diluted with PBS (10 times) as stock solution. For hemolysis measurement, 0.2 mL of diluted RBCs suspension was then mixed with 0.8 mL of PBS as a negative control, 0.8 mL of deionized water as a positive control, and 0.8 mL of BSA-Bi2Te3 suspension at concentrations ranging from 20 to 400 μg/mL. Then, all the mixtures were standing incubated at 37 °C for 5 h and centrifuged at 12 000 rpm for 10 min. The absorbance of supernatants at 541 nm was measured using a Synergy HT Multimode Microplate Reader. The hemolysis percent of the RBCs was calculated by the following equation hemolysis percent (%) =

A sample − A negative A positive − A negative

stained with Calcein-AM and PI at 20 nM and 4 mM and visualized with CLSM. In Vivo Antitumor Studies. Kunming female mice (six weeks old) were purchased from Center for Experimental Animals of Jilin University. All animal experiments were conducted according to national guidelines and approval of the regional ethics committee for animal experiments. The tumor model was established by subcutaneously injecting U14 cells into the right hind limb of each mouse. When the tumor volume reached ∼50−60 mm3, mice were randomly divided into five groups (n = 10 per group) and received intravenous injections of different therapeutic agents: (1) 0.9% saline solution as control group, (2) BSA-Bi2Te3/MB NSs alone, (3) BSABi2Te3/MB NSs with 650 nm laser irradiation (50 mW/cm2, 15 min, PDT group), (4) BSA-Bi2Te3/MB NSs with 808 nm laser irradiation (2.0 W/cm2, 10 min, PTT group), and (5) BSA-Bi2Te3/MB NSs with 808 nm laser irradiation (2.0 W/cm2, 10 min) and 650 nm laser irradiation (50 mW/cm2, 15 min, PDT/PTT group). The dose of injection is 200 μL at a concentration of 200 and 10 μg/mL for BSABi2Te3 and MB, respectively. Tumor size and mice weight were measured every 2 d. The tumor volume was calculated according to the following formula: tumor volume (mm3) = (width2 × length)/2. Relative tumor volume was calculated as V/V0 (V0 is the tumor volume when the treatment was initiated). At 15th day, all the mice were euthanized, and the tumor tissues were excised to evaluate the therapeutic efficacy of the different groups. Histology Evaluation. On the 15th day, the tumors were excised, fixed in 4% neutral buffered formalin, and processed routinely into paraffin. Tumors were sectioned to 4 μm thick slices, stained with hematoxylin and eosin (H&E), and observed by a digital microscope (Leica QWin). Characterization. Transmission electron microscopy (TEM, H600, Hitachi) was applied to characterize the morphologies of Bi2Te3 nanosheets. N2 adsorption−desorption isotherms were operated by an Autosorb iQ Station 2 at 77 K. Atomic force microscopic (AFM) images were taken with a Multimode-V atomic force microscope, operating in tapping mode in air. Dynamic light scatter spectrometer (Malvern Zetasizer Nano ZS90, Malvern Instruments Ltd.) was used to further measure the size and zeta potential of Bi2Te3 nanosheets. Energy-dispersive X-ray elemental mapping images were obtained by a FEI TECNAI G2 high-resolution transmission electron microscope operating with a field-emission gun operating at 200 kV. The optical properties of nanomaterials were measured using a PerkinElmer Lambda 25 UV−vis spectrophotometer with a 1 cm cuvette. Fourier transform infrared (FTIR) spectra were obtained on a Nicolet 6700 FT-IR spectrometer using the KBr pellet method in the 4000−500 cm−1 region. X-ray diffraction (XRD) patterns were obtained on a D8 ADVANCE using Cu Kα radiation. The PTT was performed by using an 808 nm laser (LSR808NL-2W). The PDT was performed with a 650 nm laser (LSR650NL-1.4W). A microplate reader (BioTek Instruments Inc.) was used to measure the cell viability. The confocal fluorescence imaging was recorded using confocal laser scanning fluorescence microscope (CLSM, Leica TCS SP2, Leica Microsystems).

× 100 (1)

where Asample, Anegative, and Apositive are the absorbance of sample, the negative control, and positive control, respectively. Confocal Fluorescence Imaging Analysis of BSA-Bi2Te3 Nanosheets. Prior to confocal microscopy study, HeLa cells were plated onto glass-bottomed dishes (20 mm) at a density of 5.0 × 105 cells per dish. Then, cells were incubated with fluorescein isothioyanate (FITC)-loaded BSA-Bi2Te3 nanosheets for 6 h in DMEM. After incubation, the cells were washed with PBS (pH 7.4) three times and visualized by confocal laser scanning microscopy (CLSM). FITC was excited at 488 nm with an argon ion laser, and the emission was collected from 500 to 555 nm. The images were analyzed with the ImageJ software. In Vitro Combination Therapy Tumor. To evaluate the combination therapy efficacy of BSA-Bi2Te3/MB nanosheets, HeLa cells were seeded into 96-well plates (1 × 104 cells per well) and incubated overnight. Then the cells were incubated with free MB (10 μg/mL), BSA-Bi2Te3 (200 μg/mL), and BSA-Bi2Te3/MB (200 μg/ mL, the concentration of MB was 10 μg/mL) dispersions at 37.0 °C for 12 h, respectively. Thereafter, the dispersions were replaced with fresh medium, and the cells were irradiated with an 808 nm NIR laser (2.0 W/cm2, 5 min) for PTT and/or a 650 nm laser (50 mW/cm2, 15 min) for PDT. After laser irradiation, the cells were further incubated at 37.0 °C for 4 h. Finally, MTT assay was used to measure the cell viability, as indicated above. Also, confocal fluorescence image was used to evaluate the combination therapy efficacy of BSA-Bi2Te3/MB nanosheets. First, HeLa cells (5 × 105) were seeded in the 20 mm culture dish and divided into six groups: control group, alone laser irradiation with 808 nm laser at 2.0 W/cm2 for 5 min, and 650 nm laser at 50 mW/cm2 for 15 min, BSA-Bi2Te3/MB alone, BSA-Bi2Te3/MB with 650 nm laser irradiation at 50 mW/cm2 for 15 min (PDT), BSA-Bi2Te3/MB with 808 nm laser irradiation at 2.0 W/cm2 for 5 min (PTT), BSA-Bi2Te3/ MB with 650 nm laser for 15 min at 50 mW/cm2, and 808 nm laser for 5 min at 2.0 W/cm2 (PDT/PTT). Afterward, the cells were

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RESULTS AND DISCUSSION

Synthesis of BSA-Bi2Te3 NSs. First, the bulk Bi2Te3 was synthesized via a facile solvothermal method according to a reported process.20 To obtain BSA-Bi2Te3 nanosheets (BSABi2Te3 NSs), the bulk Bi2Te3 was exfoliated in water using BSA as the exfoliating agent and stabilizer (Figure 1A). The obtained BSA-Bi2Te3 NSs exhibited a typical 2D sheetlike morphology with the mean size of ∼100 nm and thickness of 15 nm (Figure 1B and Figure S1A), and a well dispersion. However, exfoliated Bi2Te3 in water without BSA clearly showed poor dispersion, resulting in the aggregation of nanosheets with thickness of 80 nm (Figure 1C and Figure S1B), thus indicating that the Bi2Te3 NSs was successfully obtained using BSA as exfoliation agent. The hydrodynamic C

DOI: 10.1021/acs.inorgchem.8b01385 Inorg. Chem. XXXX, XXX, XXX−XXX

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size of BSA-Bi2Te3 NSs as measured with dynamic light scattering (DLS) in PBS solution was ∼110 nm (Figure S2), agreeing with TEM results. The elemental composition of the sample is determined using an energy-dispersive spectroscopy (EDS; Figure S3), which shows that the as-prepared sample contained Bi, Te, C, and O elements. The Bi/Te atomic ratio is almost close to 2:3, that is, the stoichiometric atom ratio of Bi2Te3. To further confirm the successful synthesis of Bi2Te3 NSs, XRD was used to study crystal nature of the samples (Figure S4). The wide-angle XRD pattern for only ultrasonic exfoliation of Bi2Te3 demonstrates that all diffraction peaks are well-indexed to the rhombohedral lattice structured Bi2Te3 with parameters of a = 4.38 Å and c = 30.48 Å (JCPDS No. 150863).21 For BSA-assisted exfoliation of Bi2Te3, only main diffraction peaks are observed, while the rest of the peaks weaken and widen significantly, or even disappear (Figure S4), indicating reduction of the average grain size and the formation of few-layer Bi2Te3 nanosheets. The surface area of BSA-Bi2Te3 NSs was evaluated by N2 adsorption−desorption isotherm as shown in Figure S5, which was found to be 84.35 m2 g−1. However, exfoliated Bi2Te3 in water without BSA shows an insignificant amount of surface area, indicating overlap and stacking of the Bi2Te3 layers. In this study, BSA was used not just as an exfoliating agent for the synthesis of Bi2Te3 NSs but also as a stabilizing agent for improving dispersion and biocompatibility of products. The FTIR spectra were used to confirm surface chemical bonding properties of the prepared Bi2Te3 NSs. The free BSA shows two characteristic peaks at 1650 and 1524 cm−1 belong to amide I and amide II of protein, which were not observed in the spectrum of Bi2Te3. However, note that two new peaks

Figure 1. (A) Synthesis schematic. TEM images of Bi2Te3 NSs with (B) or without (C) BSA as an exfoliating agent. FTIR spectra (D) and UV−vis spectra (E) of BSA, Bi2Te3, and BSA-Bi2Te3. (F) The photographs of Bi2Te3 NSs, BSA-Bi2Te3 NSs, and BSA-Bi2Te3/MB NSs dispersed in H2O, PBS, FBS, and DMEM for 30 d.

Figure 2. (A) Photothermal heating curves of water and different concentrations of BSA-Bi2Te3 NSs suspensions under laser irradiation (808 nm, 2.0 W cm−2, 10 min). (B) Heating curves of BSA-Bi2Te3 NSs suspension for five laser on/off cycles. (C) Heating and cooling curves of the BSABi2Te3 NSs suspension. (D) Linear time data vs −ln θ obtained from the cooling period of Figure 2C. Time constant for heat transfer of the system is k = 243.31 s. D

DOI: 10.1021/acs.inorgchem.8b01385 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 3. (A) UV−Vis spectra of BSA-Bi2Te3 and BSA-Bi2Te3/MB. (B) Consumption of DPBF due to 1O2 generation in different groups: only laser, BSA-Bi2Te3, BSA-Bi2Te3/MB, BSA-Bi2Te3 with laser irradiation, and BSA-Bi2Te3/MB with laser irradiation (650 nm, 50 mW cm−2, 20 min). (C) Confocal images of an intracellular ROS generation (DCFH) in HeLa cells treated with or without only laser, BSA-Bi2Te3, BSA-Bi2Te3/MB, BSA-Bi2Te3 with laser irradiation, and BSA-Bi2Te3/MB with laser irradiation (650 nm, 50 mW cm−2, 20 min). Scale bar = 50 μm.

temperature gauge was used to record real-time temperature change during laser irradiation. Obviously, BSA-Bi2Te3 NSs exhibited a rapid increase of temperature accompanying with the increase of irradiation time or the concentration of the BSA-Bi2Te3 NSs, while pure water showed only a little change (Figure 2A). Moreover, the photothermal stability of BSABi2Te3 NSs was further studied by monitoring temperatures with five laser on/off cycles. No obvious temperature decrease and absorption change are observed (Figure 2B and Figure S7), indicating their good photostability as photothermal agents. According to heating−cooling curves (Figure 2C,D),24 the photothermal conversion efficiency of BSA-Bi2Te3 NSs was calculated to be ∼45.3% (see Supporting Information for details). For comparison, we further investigated photothermal conversion efficiency of the well-known photothermal agent of Au nanorods under the same experimental conditions (Figure S8). The photothermal conversion efficiency of Au nanorods was calculated to be 39.4%, which is much lower than that of as-synthesized BSA-Bi2Te3 NSs. Furthermore, the photothermal conversion efficiency of BSA-Bi2Te3 NSs is also higher than those of previously reported materials including Bi2S3 (18.3%)13 and MoS2 (24.37%),25 and it is similar to that of Cu7.2S4 nanocrystals (56.7%).26 The data clearly indicate that BSA-Bi2Te3 NSs could act as an efficient photothermal agent for killing cancer cells. Extracellular and Intracellular Singlet Oxygen Production. Moreover, the large surface area of BSA-Bi2Te3 NSs makes them highly suitable as drug carrier for drug delivery. In

assigned to amide I and amide II of protein can be observed at 1650 and 1524 cm−1 for the BSA-Bi2Te3 NSs (Figure 1D), which further proved successful conjugation of BSA with Bi2Te3. Also, UV−vis absorption spectrum of BSA-Bi2Te3 NSs shows the absorption characteristic peak of BSA at 278 nm compared with Bi2Te3 (Figure 1E), further indicating that BSA was successfully adsorbed on the surface of Bi2Te3 NSs. In addition, the synthesized BSA-Bi2Te3 NSs exhibit excellent stability in H2O, PBS, FBS, or cell culture medium (DMEM) for 30 d (Figure 1F), which is obviously desirable for the applications of BSA-Bi2Te3 NSs in vivo. The BSA improved the hydrophilicity and stability of Bi2Te3 NSs, which are further confirmed by measuring the surface charge of BSA-Bi2Te3 NSs, yielding a strong negative value of −30.2 mV. The negative surface charge facilitates the transport of BSA-Bi2Te3 NSs in bloodstream. Photothermal Properties of BSA-Bi2Te3 NSs. In addition, BSA-Bi2Te3 NSs with narrow band gap22 show a broad absorption in the NIR region (Figure S6), suggesting that BSA-Bi2Te3 NSs have the capability to convert NIR light into heat. According to Lambert−Beer law, the extinction coefficient of BSA-Bi2Te3 NSs at 808 nm was estimated to be 3.6 L g−1 cm−1 (Figure S6), which is almost comparable to that of Au nanorods (3.9 L g−1 cm−1).23 To determine the potential of BSA-Bi2Te3 NSs as an efficient PTT agent, BSA-Bi2Te3 NSs with different concentrations (10, 20, 30, 50, 70, and 100 μg/ mL) were exposed to an 808 nm NIR laser under a power density of 2.0 W/cm2, and pure water was used as a control. A E

DOI: 10.1021/acs.inorgchem.8b01385 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 4. (A) Relative cell viability after cells treated with various concentrations of BSA-Bi2Te3 and BSA-Bi2Te3/MB for 24 h. (B) Hemolysis percentage of human RBCs upon incubation with different concentrations of BSA-Bi2Te3/MB for 24 h. (C) Confocal images of HeLa cells treated with or without BSA-Bi2Te3−FITC.

exposed to 650 nm light and is suitable as an efficient PDT agent. Encouraged by 1O2 production of BSA-Bi2Te3/MB with laser irradiation, we next investigated intracellular 1O 2 production of BSA-Bi2Te3/MB in HeLa cells. Here, we used dichlorofluorescein diacetate (DCFH-DA) as an intracellular ROS marker. In the presence of ROS, DCFH-DA can be rapidly oxidized to generate fluorescent dichlorofluorescein (DCF) with green fluorescence, and then confocal laser scanning microscopy was used to measure the DCF fluorescence at 488 nm excitation.28 As shown in Figure 3C, no obvious green fluorescence is observed from HeLa cells with or without laser irradiation alone. Cells incubated with BSA-Bi2Te3/MB alone also showed little green fluorescence. It is very clear that either laser irradiation alone or BSA-Bi2Te3/ MB does not induce ROS generation in live cells. While strong green fluorescence is observed in the cells incubated with BSABi2Te3/MB in the presence of 650 nm laser irradiation, indicating the generation of ROS inside the cells during lightmediated BSA-Bi2Te3/MB. This result suggested that the intracellular ROS could be produced by BSA-Bi2Te3/MB inside the cancer cells under light irradiation, which further induce cell death. Biocompatibility and Internalization of BSA-Bi2Te3 NSs. Theranostic safety of nanomaterials for biomedical application is another essential consideration. The in vitro cytotoxicity of BSA-Bi2Te3 and/or BSA-Bi2Te3/MB for HeLa cells (cancer cell line) or HUVEC (normal cell line) was measured by the standard MTT assay. Either cells treated with

this study, MB as photosensitizer was loaded on the surface of BSA-Bi2Te3 NSs with potential ability for PDT (Figure 1A), and sheetlike morphology of BSA-Bi2Te3 NSs has no change during MB loading process (Figure S9). The cationic photosensitizer of MB was assembled with negatively charged BSA-Bi2Te3 NSs via electrostatic attraction to obtain BSABi2Te3/MB with loading content of 101.7 μg mg−1 (Figure S10). The successful incorporation of MB on the surface of BSA-Bi2Te3 NSs (BSA-Bi2 Te3/MB) was confirmed by measuring the characteristic absorption band of MB at 650 nm (Figure 3A).27 BSA-Bi2Te3/MB NSs with excellent stability (Figure 1F) also show suitable photothermal properties similar to those of BSA-Bi2Te3 NSs (Figure S11), indicating that MB does not affect the photothermal properties of BSA-Bi2Te3 NSs. We next evaluated singlet oxygen generating efficiency of BSA-Bi2Te3/MB upon 650 nm irradiation. The singlet oxygen sensor DPBF, whose absorption is quenched by 1O2, was used to measure the in vitro 1O2 generation ability of BSA-Bi2Te3/MB. As shown in Figure 3B, laser irradiation alone, BSA-Bi2Te3 alone, BSA-Bi2Te3/MB alone, or BSA-Bi2Te3 with laser irradiation shows no obvious quenching ability for singlet oxygen, since the absorption intensity of DPBF remained unchanged. However, after BSABi2Te3/MB solution was irradiated using a 650 nm laser, the absorption intensity of DPBF at 410 nm decreased quickly over irradiation times, and 62.4% of DPBF was quenched, indicating effective 1O2 generation ability of BSA-Bi2Te3/MB under light irradiation. These results confirmed that BSABi2Te3/MB could produce high-efficiency singlet oxygen when F

DOI: 10.1021/acs.inorgchem.8b01385 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry BSA-Bi2Te3 and/or BSA-Bi2Te3/MB showed negligible cell toxicity even at a high concentration of 400 μg/mL (Figure 4A and Figure S12), suggesting the excellent biocompatibility and suitability for biomedical application. The excellent biocompatibility of nanocarrier is believed to benefit from the BSA coating agents. In addition, hemolytic activity of BSA-Bi2Te3/ MB NSs against human red blood cells (RBCs) was also tested to evaluate the blood compatibility,29 where deionized water and PBS were denoted as positive and negative controls, respectively (Figure 4B). The hemolytic assay demonstrated that negligible hemoglobin in the supernatant was visualized even at a high concentration of 400 μg/mL (Figure 4B). On the basis of the absorbance of released hemoglobin from RBCs at 541 nm, the hemolysis index of RBCs in the presence of a high BSA-Bi2Te3 concentration of 400 μg/mL was calculated to be 4.8%, which is less than the threshold value of 5%.30 The result suggested that BSA-Bi2Te3 NSs showed ignore hemolysis reaction, indicating the reliable blood compatibility. To evaluate the cell internalization efficiency of BSA-Bi2Te3 NSs, BSA-Bi2Te3 NSs were labeled first with FITC with green fluorescence, denoted as BSA-Bi2Te3/FITC. The confocal imaging analysis demonstrated that strong green fluorescence of FITC was detected on HeLa cells treated with BSA-Bi2Te3/ FITC (Figure 4C), indicating FITC was successfully delivered into cells using BSA-Bi2Te3 as the nanocarrier. As a control, no green fluorescence can be observed in cells without adding nanoparticles. These significant differences indicated that BSABi2Te3/FITC could be readily internalized into the cancer cells. In Vitro Evaluation Synergistic PDT/PTT Therapy. The high photothermal conversion efficiency and singlet oxygen production of the BSA-Bi2Te3/MB NSs impelled us to investigate anticancer effects in vitro by the combination of PTT and PDT. The MTT assay was used to assess therapeutic efficacy (Figure 5A). HeLa cells were incubated with free MB, BSA-Bi2Te3 NSs, and BSA-Bi2Te3/MB NSs and irradiated with a 650 nm laser for PDT and/or an 808 nm laser for PTT, respectively. Negligible cell toxicity was found for cells treated with or without laser irradiation (Figure 5A), demonstrating that laser irradiation alone does not result in any toxicity to HeLa cells. For free MB-treated group, MB with 650 nm laser irradiation caused 34% of cell death, but a negligible cytotoxicity was observed in the presence of 808 nm laser irradiation, indicating that 808 nm irradiation does not show any influence to MB-treated group as expected. For cells treated with BSA-Bi2Te3, BSA-Bi2Te3 exhibited much stronger cytotoxicity upon 808 nm irradiation, compared to BSA-Bi2Te3 alone, confirming the superior thermotherapy efficacy. As expected, BSA-Bi2Te3/MB irradiated with two kinds of laser (808 and 650 nm) was able to induce the most effective cancer cell death, showing an obvious synergistic effect between the PTT from BSA-Bi2Te3 and PDT from the released MB. Next, calcine AM and PI costaining assay was used to visualize the live and dead cells using confocal microscope (Figure 5B). It was found that the cells incubated with BSA-Bi2Te3/MB plus either 808 or 650 nm laser irradiation only showed partial cell death, while almost complete cell death was observed for the cells treated with BSA-Bi2Te3/MB, following 808 and 650 nm laser irradiation, suggesting that combination therapy is more effective than individual PDT or PTT. In Vivo PDT/PTT for Tumor Ablation. On the basis of the effective combination therapy in vitro, we next evaluated the in vivo tumor inhibition efficacy of BSA-Bi2Te3/MB under

Figure 5. (A) Relative viabilities of HeLa cells incubated with DMEM as control, free MB, BSA-Bi2Te3, and BSA-Bi2Te3/MB with or without laser irradiation (650 nm, 50 mW cm−2, 15 min or/and 808 nm, 2.0 W cm−2, 10 min). (B) CLSM of calcein AM and PI costained HeLa cells incubated with DMEM medium (control), laser irradiation alone, BSA-Bi2Te3/MB, BSA-Bi2Te3/MB+650 nm laser (PDT), BSABi2Te3/MB+808 nm laser (PTT), BSA-Bi2Te3/MB+650/808 nm laser (PDT/PTT). Scale bar = 50 μm.

the 650 and 808 nm laser irradiation. Mice bearing U14 tumors were divided into five groups (n = 10 per group): (1) saline control, (2) BSA-Bi2Te3/MB alone, (3) BSA-Bi2Te3/MB with 650 nm laser irradiation (PDT), (4) BSA-Bi2Te3/MB with 808 nm laser irradiation (PTT), and (5) BSA-Bi2Te3/MB NSs with 650 and 808 nm laser irradiation (PDT/PTT). Tumor sizes and body weight were monitored every 2 d after treatment (Figure 6A,B). Tumors in the control group and BSA-Bi2Te3/MB alone group grew rapidly, indicating that BSA-Bi2Te3/MB with high biocompatibility does not affect the tumor growth. Compared to the control group, the tumors treated with BSA-Bi2Te3/MB NSs plus 650 nm laser irradiation (PDT) or BSA-Bi2Te3/MB NSs plus 808 nm laser irradiation (PTT) showed a moderate growth inhibition effect, suggesting that the PDT or PTT alone of the nanocomposites is insufficient to inhibit tumor growth. In marked contrast, the tumor growth on mice treated with BSABi2Te3/MB plus 650 and 808 nm laser irradiation (PDT/PTT) was greatly inhibited, confirming the obvious advantage and synergistic effect of combination therapy in comparison to monotherapy (Figure 6C,D). Moreover, mice in each group showed increased body weights after treatment (Figure 6B), indicating that the combination therapy introduced by BSABi2Te3/MB exerted no significant side effects in vivo. To further validate the therapeutic effect, H&E staining was performed to compare the lesion degree of the tumor tissue after treatments (Figure 6E). As expected, the tumor treated with BSA-Bi2Te3/MB NSs plus two kinds of laser irradiation G

DOI: 10.1021/acs.inorgchem.8b01385 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

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Calculation of the photothermal conversion efficiency, AFM and TEM images, plotted data including EDS data, hydrodynamic size distribution bar graph, XRD patterns, adsorption-desorption isotherms, absorption spectra, photothermal heating curves, cell viability bar graph (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Xiue Jiang: 0000-0002-0194-1553 Notes

The authors declare no competing financial interest.

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Figure 6. Relative tumor volume (A) and mice body weight (B) of different groups of tumor-bearing mice after treatment. (C) Representative photographs of tumors from mice injected with saline (a), BSA-Bi2Te3/MB (b), BSA-Bi2Te3/MB+650 nm laser irradiation (PDT, c), BSA-Bi2Te3/MB+808 nm laser irradiation (PTT, d) and BSA-Bi2Te3/MB+650/808 nm laser irradiation (PDT/PTT, e). (D) Representative photographs of different groups of tumor-bearing mice after treatment. (E) H&E staining of the excised tumor sections after different treatment.

ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (21505130, 21675149, and 21705146), the Key Research Program of Frontier Sciences, CAS (QYZDY-SSW-SLH019), the Science and Technology Development Program of Jilin Province (20170414037GH), and the K. C. Wong Education Foundation.

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exhibits an evident nucleus deformation and shrinks,31 indicating a significant cell apoptosis of PDT/PTT combination therapy. And the tumor treated with BSA-Bi2Te3/MB NSs plus 650 nm laser or BSA-Bi2Te3/MB plus 808 nm laser shows an irregular morphologic change with moderate apoptosis. However, little apoptosis with intact and plump tumor cell nucleus was observed in the BSA-Bi2Te3/MB and control groups. Overall, the above results demonstrated that BSABi2Te3/MB may act as an effective therapeutic agent for PDT/ PTT synergistic therapy applications.

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CONCLUSIONS In conclusion, the novel BSA-Bi2Te3 nanosheets were successfully prepared via liquid exfoliation strategy by using BSA as exfoliation agent and stabilizer. It was found that BSABi2Te3 nanosheets have a strong NIR absorption and excellent photothermal stability. In addition, BSA-Bi2Te3 nanosheets also act as a potential nanocarrier for photosensitizer MB loading through electrostatic interaction. The obtained BSABi2Te3/MB was demonstrated to generate 1O2 in tumor tissues under 650 nm laser with a high efficiency. Importantly, this nanosystem exhibits a high colloidal stability, excellent hemo/ biocompatibility, preferable cellular uptake, and low toxicity, which thus offers an opportunity for nanomaterials to be utilized in cancer therapy. After accumulation within the tumor tissue, combination of PDT/PTT provided by BSA-Bi2Te3/ MB showed the more efficient cancer cell-killing capacity than PDT or PTT alone both in vitro and in vivo, demonstrating the advantage of synergetic PDT/PTT therapy over monotherapy. Our research brings new hope for designing theranostic agents against cancer through photothermal and photodynamic synergistic therapy.

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DOI: 10.1021/acs.inorgchem.8b01385 Inorg. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.inorgchem.8b01385 Inorg. Chem. XXXX, XXX, XXX−XXX