Marriage of AlbuminâGadolinium Complexes and ... - ACS Publications

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Marriage of Albumin−Gadolinium Complexes and MoS2 Nanoflakes as Cancer Theranostics for Dual-Modality Magnetic Resonance/ Photoacoustic Imaging and Photothermal Therapy Liang Chen,† Xiaojun Zhou,‡ Wei Nie,† Wei Feng,† Qianqian Zhang,† Weizhong Wang,† Yanzhong Zhang,† Zhigang Chen,‡ Peng Huang,*,§ and Chuanglong He*,†,‡ †

College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging, School of Biomedical Engineering, Health Science Center, Shenzhen University, Shenzhen 518060, China ‡ State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, Donghua University, Shanghai 201620, China

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§

S Supporting Information *

ABSTRACT: The construction of safe and stable theranostics is beneficial to realize simultaneous cancer diagnosis and treatment. In this study, bovine serum albumin−gadolinium (BSA−Gd) complexes and MoS2 nanoflakes (MoS2−Gd−BSA) were successfully married as cancer theranostics for dual-modality magnetic resonance (MR)/photoacoustic (PA) imaging and photothermal therapy (PTT). BSA−Gd complexes were prepared by the biomineralization method and then conjugated with MoS2 nanoflakes via an amide bond. The as-prepared MoS2−Gd−BSA possessed a good photostability and photothermal effect. The cytotoxicity assessment and hemolysis assay suggested the excellent biocompatibility of MoS2−Gd−BSA. Meanwhile, MoS2−Gd−BSA could not only achieve in vivo MR/PA dual-modality imaging of xenograft tumors, but also effectively kill cancer cells in vitro and ablate the xenograft tumors in vivo upon 808 nm laser illumination. The biodistribution and histological evaluations indicated the negligible toxicity of MoS2−Gd−BSA both in vitro and in vivo. Thus, our results substantiated the potential of MoS2−Gd−BSA for cancer theranostics. KEYWORDS: cancer theranostics, MoS2 nanoflakes, magnetic resonance imaging, photoacoustic imaging, photothermal therapy

1. INTRODUCTION As a newly emerging therapeutic approach of cancer, photothermal therapy (PTT) has gained burgeoning interest during the past decade owing to its minimal invasiveness and great spatiotemporal selectivity.1,2 Although various photothermal conversion agents (PTCAs), such as gold-based nanomaterials,3−5 carbon-based nanomaterials,6,7 and so on, have been developed for PTT, most of them are nonbiodegradable, and bear poor photostability, low photothermal conversion efficacy, poor pharmacokinetics, or potential longterm toxicity, which limit their widespread biomedical applications.8 Accordingly, those issues invariably impel the researchers to exploit novel PTCAs.9 To obtain precision PTT, the real-time visualization of PTCAs is desirable to guide the in vivo PTT of cancer.10,11 © 2017 American Chemical Society

By offering the soft-tissue morphology and timely feedback information on diseases, magnetic resonance (MR) imaging has been recognized as the favorite diagnostic tool in the clinic.12 Gadolinium (Gd)-chelated complexes, such as Magnevist (Gd− DTPA), are the most commonly used positive contrast agents for clinical MR imaging. Particularly, Gd-based photothermal theranostics were actively explored by using different Gd ion chelators such as 1,4,7,10-tetraazacyclododecane-N,N′,N,N′tetraacetic acid (DOTA) or diethylenetriaminepentaacetic acid (DTPA). For instance, Dai et al. developed gadolinium− chelate (Gd−DOTA)-conjugated polypyrrole nanoparticles Received: March 29, 2017 Accepted: May 9, 2017 Published: May 9, 2017 17786

DOI: 10.1021/acsami.7b04488 ACS Appl. Mater. Interfaces 2017, 9, 17786−17798

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ACS Applied Materials & Interfaces (NPs) for MR/photoacoustic (PA) imaging and PTT.10 However, most of these Gd−chelator agents are limited by high risk of released Gd ion-associated toxicity, which may induce nephrogenic systemic fibrosis (NSF) .13,14 Therefore, it is highly required to develop stable and safe Gd-based photothermal theranostics. As we know, the rigid state of Gd-based NPs effectively prevents the release of Gd ions.15,16 Some studies have demonstrated that bovine serum albumin (BSA) could effectively bind with metal ions and subsequently induce the formation of protein-coated nanoclusters.17,18 In particular, BSA-directed biomineralization of Gd-based NPs with a high relaxation rate has been established for MR imaging.19,20 Recently, Liu et al. explored an albumin-based theranostic agent for dual-modal-imaging-guided PTT to inhibit lymphatic metastasis of cancer.21 Chen et al. fabricated cypate-grafted BSA-biomineralized Gd-based NPs for multimodal imaging and PTT.16 Inspired by those studies, the biomineralization synthesis promises the construction of safe and stable Gdbased photothermal theranostics. Currently, molybdenum disulfide (MoS2) nanomaterials have attracted increasing attention in the biomedical field by virtue of their outstanding photothermal conversion efficacy,22−25 good biocompatibility,26−28 and potential degradability.29 The MoS2-based photothermal theranostics have been examined for simultaneous cancer diagnosis and treatment.30−33 For example, Liu et al. have developed iron oxide NPs decorated and 64Cu-labeled MoS2 nanosheets for multimodal imagingguided cancer phototherapy.34 Chen et al. have prepared MoS2/Bi2S3 composites for PA/CT imaging and combination therapy.35 Yang et al. have grafted upconversion NPs onto MoS2 nanosheets for fluorescence-imaging-guided phototherapy.36 Additionally, Gd−chelator-conjugated MoS2 core−shell magnetic nanomaterials have been synthesized for in vivo MR imaging and exhibit a 4.5-fold longer water proton spin−lattice relaxation time (T1) than that of the commercial Gd−DTPA agent.37 Nevertheless, the potential toxicity of Gd−chelatorbased compounds needs to be more carefully inspected because of the instability of chelating molecules.38 On the other hand, the admirable photothermal property also causes MoS2 nanomaterials to have high potential as contrast agents of PA imaging,39 because they could penetrate tissues more deeply and provide a legible structure and microcosmic information with high sensitivity. Note that the apparent advantages of dual-modal imaging, the combination of PA and MR imaging, is more helpful for precision PTT since MR imaging could quickly locate the diseased region and PA imaging could sensitively afford structural and microscopic information with high resolution. For instance, Zhang and coworkers prepared BSA−Gd/CuS NPs for PA/MR imaging and PTT.40 Chen and co-workers developed Co9 Se 8 -based theranostics for PA/MR-imaging-instructed chemo-photothermal combination therapy.41 Therefore, the marriage of BSA− Gd complexes and MoS2 nanoflakes promises simultaneous PA/MR dual-modal imaging and precision PTT. In this work, the MoS2 nanoflakes were prepared by the onepot hydrothermal method. The surface coating of poly(allylamine hydrochloride) and poly(acrylic acid) (PAH/ PAA) was achieved by the layer-by-layer (LBL) technique.42−45 Meanwhile, the BSA−Gd complexes were synthesized by the mild biomineralization method. Then the BSA−Gd-complexconjugated MoS2 nanoflakes (MoS2−Gd−BSA) were obtained through the amine reaction between amino groups of BSA−Gd

and carboxyl groups of MoS2 nanoflakes. In this system, the inner MoS2 nanoflakes acted as an efficient agent for PA imaging and PTT, while the bilayer PAH/PAA and outer BSA− Gd were used as the reactive linker and MR imaging contrast agent, respectively. The biocompatibility of MoS2−Gd−BSA was evaluated in terms of cytotoxicity and hemolysis activity. Most importantly, by taking advantage of the high longitudinal proton relaxivity of BSA−Gd and the excellent photothermal effect of MoS2 nanoflakes, the MR imaging capability, PA imaging ability, and PTT efficacy of the obtained MoS2−Gd− BSA were assessed both in vitro and in vivo. The as-prepared BSA−Gd-complex-modified MoS2 nanoflakes were investigated as cancer theranostics for MR/PA imaging and PTT.

2. EXPERIMENTAL SECTION 2.1. Materials. Thioacetamide (TAA; 99%), poly(vinylpyrrolidone) (Mw = 58000), poly(acrylic acid) (PAA; Mw = 1800), and N-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC) were purchased from Aladdin Industrial Inc. (Shanghai, China). Gd(NO3)3·6H2O, N-hydroxysuccinimide (NHS), and ammonium molybdate tetrahydrate (H24Mo7N6O24·4H2O; 99%) were received from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Triton X-100, poly(allylamine hydrochloride) (PAH; Mw = 15000) and bovine serum albumin (BSA) were obtained from SigmaAldrich Trading Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS), penicillin−streptomycin, trypsin, and Roswell Park Memorial Institute (RPMI) 1640 medium were obtained from Gibco Life Technologies Co. (Grand Island, NY). Cell Counting Kit-8 (CCK-8) was purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Acridine orange (AO), calcein-AM, propidium iodide (PI), and 4′,6-diamidino-2-phenylindole (DAPI) were obtained from BestBio Biotechnology Co., Ltd. (Shanghai, China). Paraformaldehyde was obtained from Shanghai Solarbio Bio-Technology Co., Ltd. (Shanghai, China). The Alexa Fluor 568-conjugated phalloidin was obtained from Molecular Probes (Invitrogen, United States). 2.2. Preparation of MoS2 Nanoflakes. The MoS2 nanoflakes were prepared according to previous literature with some modifications.39 In brief, H24Mo7N6O24·4H2O (88 mg) and PVP (0.1 g) were mixed in 10 mL of deionized water. Thereafter, 75 mg of TAA in 5 mL of deionized water was slowly introduced into the mixture, which was vigorously stirred to form a transparent solution. The solution was then sealed in a reaction kettle and maintained at 180 °C for 18 h. Last, the MoS2 nanoflakes were isolated by centrifugation at 13000 rpm for 30 min. 2.3. Preparation of BSA−Gd Complexes. BSA−Gd complexes were synthesized on the basis of a typical procedure.20 First, BSA (0.25 g) was fully dissolved in 9 mL of deionized (DI) water at 37 °C in a water bath, and then 1 mL of Gd(NO3)3 solution (50 mM) was added dropwise into the BSA solution under vigorous stirring. After 5 min, 1 mL of NaOH aqueous solution (2 M) was quickly added to the solution. The mixture was maintained at 37 °C for 12 h. Afterward, the solution was dialyzed against DI water for 1 day. The BSA−Gd was harvested and kept in a refrigerator. 2.4. Preparation of MoS2−Gd−BSA. To prepare functional MoS2 nanoflakes, BSA−Gd complexes were conjugated onto the surface of MoS2.46 The LBL technique was applied to introduce carboxyl groups onto the MoS2 nanoflakes.47 In detail, 5 mL of MoS2 aqueous dispersion (2 mg/mL) was centrifuged and suspended in 10 mL of N,N-dimethylformamide. After 15 min of bath sonication, 2 mL of PAH aqueous solution (10 mg/mL) was slowly added. Then the mixture was stirred at 80 °C for 2 h. Then MoS2−PAH was collected and dispersed in 5 mL of DI water. The MoS2−PAH solution was slowly introduced into 10 mL of PAA aqueous solution (2 mg/mL). After 2 h of reaction, the MoS2−PAH/PAA was harvested and washed several times with DI water. Subsequently, the pH of the MoS2−PAH/ PAA dispersion was adjusted to 7.4. Then 6 mg of EDC was added within 1 h to induce the cross-linked reaction for another 12 h. The obtained MoS2−PAH/PAA was repeatedly washed and suspended in 17787


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ACS Applied Materials & Interfaces 10 mL of DI water. Last, 1 mL of BSA−Gd solution was added to the above MoS2−PAH/PAA dispersion and the resulting mixture was allowed to react for another 24 h. The as-prepared MoS2−Gd−BSA was purified and dispersed in DI water. 2.5. Characterizations. The nanoflakes were observed by transmission electron microscopy (TEM; JEM-2100, JEOL Ltd., Japan, operation acceleration voltage of 200 kV with a LaB6 electron gun). The size distribution was tested by the dynamic light scattering (DLS) method using a BI-200SM multiangle dynamic/static laser scattering instrument (Brookhaven, United States). ζ potential analysis was carried out by a Zetasizer Nano ZS apparatus (Malvern Instruments, United Kingdom). Ultraviolet−visible (UV−vis) absorption spectra were recorded by using a Lambda 35 UV−vis spectrophotometer (PerkinElmer, United States) at room temperature (rt) under ambient conditions. The Raman spectra were obtained by using an inVia-Reflex micro-Raman spectroscopy system (Renishaw, United Kingdom) with a 633 nm solid laser of 50 mW power at rt. Thermogravimetric (TG) analysis was performed under a nitrogen flow from rt to 600 °C with a rate of 10 °C/min by a TG 209 F1 (Netzsch, Germany) analyzer. The Fourier transform infrared (FTIR) spectrum was measured by using the KBr disc technique and recorded on a Nexus 670 spectrometer (Thermo Nicolet, United States). The Leeman Prodigy inductively coupled plasma atomic emission spectroscopy (ICP-AES) system (Hudson, NH03051, United States) was used to determine the concentration of the materials. 2.6. Detection of Photothermal Performance. To investigate the photothermal performance, 0.2 mL of MoS2−Gd−BSA dispersions at various concentrations were illuminated (808 nm, 1 W/cm2, 10 min). The temperature of the dispersions was detected by a thermocouple thermometer. The power density of the near-infrared (NIR) laser was also altered to explore the power-density-dependent photothermal effect of MoS2−Gd−BSA. Moreover, the on−off cycles of 808 nm laser illumination were also utilized to verify the photostability of MoS2−Gd−BSA. 2.7. Cell Lines and Cell Culture. The mouse fibroblast L929 cell line, mouse leukemic monocyte macrophage RAW 264.7 cell line, murine breast cancer 4T1 cell line, and human umbilical vein endothelial cell (HUVEC) line were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The 4T1 and HUVECs were grown in RPMI 1640 medium. The L929 and RAW 264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM). The cells were cultured in a humidified atmosphere containing 5% CO2 at 37 °C. 2.8. In Vitro Cytotoxicity and Hemolytic Assay. The cytotoxicity of MoS2−Gd−BSA was evaluated on four kinds of cells by CCK-8 assay. Typically, the cells were cultured in a 96-well plate (1 × 104 cells per well) overnight. Then the cells were coincubated with fresh medium containing different concentrations of MoS2−Gd−BSA for another 24 h. After that, the cells were rinsed twice with phosphatebuffered saline (PBS). Then the CCK-8 assay was conducted. The cells were further cultured for 2 h before being subjected to a microplate reader (Multiskan MK3, Thermo). The relative cell viabilities were determined by the optical density (OD) at 450 nm divided by the value of the control group (untreated cells), and four parallel experiments were carried out for each group. Hemolysis assay of MoS2−Gd−BSA was performed according to our previous reports.48,49 Typically, 1 mL of fresh mouse blood was centrifuged to obtain red blood cells (RBCs). The RBCs were purified and resuspended in PBS. Then the diluted RBCs were mixed with MoS2−Gd−BSA suspensions with predetermined concentrations. Thereafter, the samples were slightly vortexed at 37 °C for 3 h and then centrifuged for 15 min. Last, the OD at 541 nm of the obtained supernatants was tested with a UV−vis spectrophotometer. The hemolysis percentage was obtained according to the following equation: hemolysis percentage =

ODsample − ODnegative ODpositive − ODnegative

where ODsample, ODnegative, and ODpositive represent the values of the tested samples and the negative and positive controls, respectively. 2.9. In Vitro Photothermal Treatment. To evaluate the in vitro PTT efficacy of MoS2−Gd−BSA, the seeded 4T1 cells were incubated with fresh medium doped with various concentrations of MoS2−Gd− BSA for 3 h. Afterward, the cells of the photothermal group were illuminated (808 nm, 1 W/cm2) for 10 min. Then the cells were all washed with PBS and incubated for 24 h. The CCK-8 assay was conducted to determine the relative cell viability of the different groups. Three parallel experiments were performed for each group. Moreover, the laser-density-dependent PTT efficacy of MoS2−Gd− BSA was also investigated by applying different laser densities to irradiate the cells. Meanwhile, the photothermal effect of MoS2−Gd−BSA was further confirmed by live−dead staining. All cells with different treatments were incubated with live−dead staining solution for 15 min after illumination. The cells were washed and observed by using an inverted fluorescent microscopy. 2.10. Confocal Laser Scanning Microscopy. To verify the in vitro photothermal treatment of MoS2−Gd−BSA, confocal microscopic imaging was carried out with confocal laser scanning microscopy (CLSM; Carl Zeiss LSM700, He−Ne and Ar lasers). Briefly, 4T1 cells were seeded and cultured in 20 mm glass bottom culture dishes (105 per dish) overnight. Thereafter, the cells of the photothermal group were illuminated (808 nm, 1 W/cm2) for 10 min. After 2 h of incubation, the cells were washed and fixed with paraformaldehyde (4%) for 20 min. The fixed cells were then permeabilized by Triton X-100 and blocked by 1% BSA. Subsequently, Alexa Fluor 568-conjugated phalloidin and DAPI were used to stain the F-actin filaments and nucleus, respectively. After removal of unbound dyes by PBS, the cells were subjected to CLSM to observe the cell morphology. The cells without any treatments acted as a control. AO staining was also performed to assess the integrity of the lysosomal membrane. After different treatments, the cells were washed and stained with AO for 15 min. The unbound dyes were removed by PBS, and the cells were observed by CLSM. The AO was excited at 488 nm. DAPI staining was performed to further confirm the PTT efficacy of MoS2−Gd−BSA on cell adhesion. The 4T1 cells were cultured in 20 mm glass bottom culture dishes (2 × 105 per dish) for 24 h. The cells of the PTT group were treated with MoS2−Gd−BSA plus 808 nm laser illumination. Then all cells were rinsed by PBS and stained by DAPI. Last, those samples were immediately observed by CLSM. 2.11. In Vivo Photothermal Treatment. All animal experiments were conducted in compliance with the Institutional Animal Care and Use Committee (IACUC) guidelines. Female Balb/c mice were purchased from SLAC Laboratory Animal Co. Ltd. (Shanghai, China). In a typical process, 0.1 mL of 4T1 cell suspension (2 × 106 cells) was subcutaneously injected into the right hind leg of a Balb/c mouse to generate a tumor model. After the tumor grew to ∼50 mm3, the mice were randomly assigned to four groups: control, PBS plus laser, MoS2−Gd−BSA only, MoS2−Gd−BSA plus laser. For the PBS plus laser and MoS2−Gd− BSA plus laser groups, the tumor-bearing mice were illuminated for 15 min after intratumoral injection with 100 μL of PBS or MoS2−Gd− BSA suspension (2 mg/mL). The temperature change of the MoS2− Gd−BSA-injected tumor sites was recorded with an infrared thermal camera (GX-A300, Shanghai Guixin Corp.). Additionally, the volume of the tumors was calculated as V = [(tumor length)(tumor width)2]/ 2. The length and width of the tumor were measured with an electronic caliper. The relative tumor volume was calculated as V/V0, where V0 represents the initial tumor volume before the treatment. The body weight was also monitored during the treatment. 2.12. MR and PA Imaging. First, the stability of the MoS2−Gd− BSA solution was investigated by monitoring the released amount of Gd ions. Briefly, 0.2 mL of MoS2−Gd−BSA solution was mixed with 0.8 mL of PBS (pH 7.4 and 5.0) and the resulting mixture was sealed in a dialysis bag (Mw cutoff ∼3500). Then the dialysis bag was soaked in 9 mL of PBS and maintained in the shaker at 37 °C. At specific time

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Scheme 1. Schematic Illustration of the Preparation of Bovine Serum Albumin−Gd-Complex-Functionalized MoS2 Nanoflakes as a Theranostic Nanoplatform for Magnetic Resonance/Photoacoustic Imaging and Photothermal Therapy

Figure 1. TEM images of (A) bare MoS2 nanoflakes and (B) MoS2−Gd−BSA. (C) XPS analysis and (D) Raman spectra of the as-prepared MoS2 nanoflakes. points, 5 mL of solution was withdrawn for analysis and an equivalent amount of free PBS was supplemented. The concentration of Gd ions was measured by ICP-AES. To measure the transverse proton relaxation times (T1) and T1weighted MR imaging of the samples, MoS2−Gd−BSA aqueous solutions with different Gd concentrations were analyzed by an NMI20-Analyst NMR analyzing and imaging system. The instrumental parameters were as follows: 0.5 T magnet, CPMG sequence, point resolution 156 mm × 156 mm, 0.6 mm section thickness, 4000 ms

repetition time (TR), 60 ms echo time (TE), one excitation. The transverse relaxivity (r1) was determined by linearly fitting the inverse T1 relaxation time (1/T1) as a function of the Gd concentration. For in vivo MR imaging, the tumor-bearing mice were anesthetized. Then the tumor-bearing mice were imaged by a 3.0 T Signa HDxt superconducting clinical MR system (Chenguang Med Tech, Shanghai, China). Thereafter, 50 μL of the MoS2−Gd−BSA in PBS (2 mg/mL) was intratumorally injected into the tumor sites. Then the corresponding MR image was collected 10 min postinjection. The 17789


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Figure 2. (A) ζ potential of the different MoS2 nanoflakes in aqueous solution. (B) Hydrodynamic diameter of MoS2 and MoS2−Gd−BSA nanoflakes (the inset photograph shows the hybrid nanoflakes MoS2−Gd−BSA in different media). (C) FTIR spectra of MoS2 and MoS2−Gd−BSA. (D) UV−vis spectra of MoS2 and MoS2−Gd−BSA. control group was imaged under the same conditions before the injection. For PA imaging, the Vevo LAZR2100 PA imaging system was applied to monitor the tumor sites of the mice. The ultrasound (US) and PA signals were all collected before injection and 10 min postinjection of the MoS2−Gd−BSA PBS suspension (2 mg/mL). 2.13. Biodistribution and Histology Examination. The biodistribution of MoS2−Gd−BSA in the mice was measured by ICP-AES. Briefly, 0.1 mL of MoS2−Gd−BSA PBS suspension (2 mg/ mL) was intravenously injected into the mice. Then the mice were scarified at certain time intervals, and the organs were washed with PBS, lyophilized, and weighed. Afterward, the organs were immersed into nitric acid and heated in an oil bath until the organs were fully dissolved. The cooled solutions were diluted to 10 mL in a volumetric flask for ICP-AES measurement. To further analyze the in vivo histocompatibility of MoS2−Gd− BSA, hematoxylin and eosin (H&E) staining was also conducted within 2 weeks. The Balb/c mice were sacrificed at 2 weeks after intravenous injection of MoS2−Gd−BSA (2 mg/mL). The major organs were collected, fixed by 10% neutral buffered formalin, and embedded routinely in paraffin. The organs were sectioned into pathological slices, stained with H&E, and finally imaged by using an inverted microscopy. The healthy mouse was used as a control. 2.14. Statistical Analysis. The data are displayed as the mean ± standard deviation (SD). The significance of the experimental data was evaluated by one-way analysis of variance (one-way ANOVA). The statistical significance was considered at P < 0.05 (*) and P < 0.01 (**).

synthesized by the biomineralization approach and conjugated onto the MoS2 nanoflakes through the amide reaction. As shown in Figure 1A, the as-prepared MoS2 nanoflakes were composed of several lamellar structures and ultimately formed a round shape. The BSA−Gd complexes demonstrated the clusterlike morphology, which is in agreement with a previous report (Figure S1, Supporting Information).20 After conjugation with BSA−Gd complexes, a dense organic layer apparently emerged on the MoS2 nanoflakes, supplying direct evidence of successful modification of BSA−Gd (Figure 1B). The chemical state of the MoS2 nanoflakes was confirmed by X-ray photoelectron spectroscopy (XPS) analysis. As seen in Figure 1C, the characteristic peaks at 232.3 and 229.2 eV could be assigned to the Mo 3d5/2 and Mo 3d3/2 orbital signals, suggesting the presence of Mo4+ in the MoS2 nanoflakes.50 The signals of S2s at 226.5 eV and S2p at ∼162.5 eV also echoed the successful fabrication of the MoS2 nanoflakes (Figure S2, Supporting Information). Furthermore, the Raman spectrum of the MoS2 nanoflakes showed two peaks at 378 and 398 cm−1 corresponding to the typical E2g1 and A1g modes of MoS2 (Figure 1D).51 Actually, the two Raman bands of the asprepared MoS2 nanoflakes are slightly broadened and redshifted compared with those of the bulk MoS2; this may stem from the lateral dimensions of these layers52 and the multilayer structures of the MoS2 nanoflakes.53 To construct MoS2−Gd−BSA, BSA−Gd was attached onto the MoS2 nanoflakes to act as an MR imaging contrast agent. First, the LBL assembly strategy was employed to introduce carboxyl groups onto MoS2. As shown in Figure 2A, the ζ potentials were monitored during the LBL coating process. Since the MoS2 was negatively charged, the nanoflakes were successively coated with cationic polyelectrolyte PAH and anionic polymer PAA via electrostatic binding. It can be seen

3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of MoS2−Gd− BSA. The synthetic process of MoS2−Gd−BSA is illustrated in Scheme 1. First, MoS2 nanoflakes were prepared according to a previous report with some modifications,39 and then the carboxyl group was introduced onto the surface of the MoS2 nanoflakes via LBL coating. The BSA−Gd complexes were 17790


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Figure 3. Photothermal effect of MoS2−Gd−BSA. (A) Temperature change of various concentrations of MoS2−Gd−BSA solution (12.5, 25, 50, 100, and 200 μg/mL) irradiated by an 808 nm NIR laser for 10 min (1 W/cm2). (B) Temperature change of the 100 μg/mL MoS2−Gd−BSA solution irradiated by an 808 nm NIR laser for 10 min with different power densities. (C) Temperature change of the MoS2−Gd−BSA solution (100 μg/mL) irradiated repeatedly by an 808 nm NIR laser for on−off cycles. (D) UV−vis absorption spectra of the MoS2−Gd−BSA solution before and after NIR irradiation (the inset picture shows the MoS2−Gd−BSA solution before and after irradiation).

that the ζ potentials of the nanoflakes changed from −30.2 mV for MoS2 to +29.3 mV for MoS2/PAH, and subsequently decreased to −29.1 mV for MoS2−PAH/PAA, suggesting the successful stepwise polymer coating on the MoS2 nanoflakes. Then the amino groups of the BSA−Gd complexes were reacted with the carboxyl groups of MoS2−PAH/PAA to obtain MoS2−Gd−BSA with a ζ potential of −26.4 mV, which favors the formation of a stable MoS2−Gd−BSA aqueous dispersion.54 Actually, the MoS2−Gd−BSA could be stably dispersed in both PBS and cell medium without any aggregation even after 1 day (Figure S3, Supporting Information). The dispersibility and size distribution of MoS2−Gd−BSA in PBS and cell culture medium exhibited no unexpected change, although a slight increase was observed because of the ion strength and serum protein (Figure S3),55 which suggested the good colloidal stability of the as-prepared MoS2−Gd−BSA. Meanwhile, the hydrodynamic diameter also increased from 205 nm for bare MoS2 nanoflakes to 297 nm for MoS2−Gd− BSA (Figure 2B), revealing the successful functionalization of BSA−Gd complexes. It also should be mentioned that the size measured by DLS was larger than that of TEM observation, which may result from little microscopic agglomeration56 and a hydration shell.57 Moreover, other physicochemical properties of these products were also investigated. Figure 2C shows the FTIR spectra of pristine MoS2 nanoflakes and MoS2−Gd−BSA. It was found that the FTIR spectrum of the MoS2 nanoflakes was completely different from that of our previously reported flowerlike MoS2 NPs.24 Given that the nonionic polymeric surfactant PVP was introduced during the hydrothermal reaction, the absorption bands at 3430 and 1640 cm−1 were assigned to the O−H and CO vibrations, respectively, which

are due to the residual PVP. After the modification of BSA−Gd, the typical amide II band of BSA at 1545 cm−1 emerged on the spectrum of MoS2−Gd−BSA. Additionally, the weight loss of MoS2−Gd−BSA reached 49% at 600 °C according to the TGA curve (Figure S4, Supporting Information), while the percentage for bare MoS2 nanoflakes and PAH/PAA-coated MoS2 was just 37% and 26% at the same conditions, respectively. The gradually increased organic component in the nanoflakes confirmed the successful step-by-step modification. Thus, on the basis of the above results and former literature,46,47 we deduced that BSA−Gd complexes were covalently conjugated onto the MoS2 nanoflakes. In addition, the conjugation of BSA−Gd onto the MoS2 nanoflakes did not reduce the optical absorption of MoS2 in the NIR region (Figure 2D), which promises a sufficient photothermal property of MoS2−Gd−BSA. 3.2. Photothermal Performance of MoS2−Gd−BSA. To validate the photothermal performance of MoS2−Gd−BSA, aqueous dispersions of MoS2−Gd−BSA at various concentrations were illuminated (808 nm, 1 W/cm2). Markedly, the temperature of the MoS2−Gd−BSA aqueous solution rapidly increased after irradiation, whereas the temperature of pure water was only elevated by 0.5 °C (Figure 3A,B). Moreover, the photothermal effects of MoS2−Gd−BSA exhibited obvious concentration- and power-dependent tendencies. Next, we investigated the photostability of MoS2−Gd−BSA. As shown in Figure 3C, the MoS2−Gd−BSA still presented effective photothermal effects even after five on−off cycles of laser irradiation, without observation of any weakening of the temperature elevation. Meanwhile, no appreciable absorbance change of MoS2−Gd−BSA was observed in Figure 3D, indicating the good photostability of MoS2−Gd−BSA. 17791


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Figure 4. Cytotoxicity of the MoS2−Gd−BSA nanoflakes. (A) Cell viability of 4T1 and L929 cells treated with the MoS2−Gd−BSA suspension at different concentrations. (B) Cell viability of HUVECs and RAW 264.7 cells incubated with different concentrations of the MoS2−Gd−BSA suspension for 1 day. (C) Live−dead cell staining of different cells treated with or without the MoS2−Gd−BSA solution (200 μg/mL). The bar represents 200 μm.

3.3. Biocompatibility of MoS2−Gd−BSA. As the biocompatibility was the primary requirement for the biomedical applications, the cytotoxicity of MoS2−Gd−BSA was evaluated on four kinds of cells, including 4T1 cells, RAW 264.7 cells, L929 cells, and HUVECs, by CCK-8 assay. The cell viabilities are depicted in Figure 4A,B and demonstrated that the MoS2−Gd−BSA has a low cytotoxicity for all kinds of cells. Concretely, the cell viabilities for 4T1 cells, L929 cells, RAW 264.7 cells, and HUVECs treated with MoS2−Gd−BSA were 91.67%, 91.10%, 91.13%, and 88.34%, respectively, even at 200 μg/mL. Moreover, the excellent biocompatibility of MoS2− Gd−BSA was also verified by AO staining. As shown in Figure 4C, the different types of live cells exhibited green fluorescence similar to the control group after incubation with MoS2−Gd− BSA, which further confirmed the excellent cytocompatibility of MoS2−Gd−BSA. The evaluation of the hemocompatibility of MoS2−Gd−BSA is also an essential prerequisite for its biomedical applications. We assessed the hemocompatibility of MoS2−Gd−BSA by using hemolysis assay. Particularly, the UV−vis spectra of the corresponding supernatants after hemolysis assay are presented in Figure 5A. Compared with the positive control (water), no distinct absorption peaks at 541 nm were observed for both the negative control and samples in a concentration range of 6.25− 800 μg/mL. It is worth noting that the absorption value of MoS2−Gd−BSA at a concentration of 800 μg/mL was a bit higher than that of other samples in full wavelength, which probably resulted from the absorbance of residual MoS2−Gd− BSA in the supernatant. The hemolytic percentages of MoS2− Gd−BSA were also calculated by the OD at 541 nm. Figure 5B shows that the hemolysis percentages of these nanoflakes were all less than 5%, except for the MoS2−Gd−BSA at a concentration of 800 μg/mL, which is due to the natural

Figure 5. Hemolytic activity of MoS2−Gd−BSA nanocomposites at different concentrations. (A) UV−vis spectrum of supernatant solutions of RBCs incubated with different concentrations of MoS2− Gd−BSA. (B) Hemolytic percentages of RBCs treated with different concentrations of MoS2−Gd−BSA solution for 3 h. The inset images are for direct observation of the results, suggesting the good biocompatibility of MoS2−Gd−BSA hybrid nanoflakes.

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Figure 6. In vitro photothermal ablation of 4T1 cells. (A) Cell viability of 4T1 cells under different MoS2−Gd−BSA concentrations with the laser power density at 1 W/cm2. (B) Cell viability of 4T1 cells under different laser power densities with the MoS2−Gd−BSA concentration at 200 μg/ mL. (C) Live−dead staining of 4T1 cells under different MoS2−Gd−BSA concentrations with a laser power density of 1 W/cm2. (D) CLSM images of 4T1 cells treated by MoS2−Gd−BSA plus NIR laser irradiation. The blue color represents DAPI, and the green color represents the Alexa Fluor 48-conjugated phalloidin-stained cytoskeleton of 4T1 cells. (E) Confocal laser scanning microscopy images of AO-stained 4T1 cells under different treatments.

absorption of the residue MoS2−Gd−BSA. Moreover, a light black color appeared in the picture of the supernatant for the 800 μg/mL MoS2−Gd−BSA solution, while negligible red color was observed for the other samples (inset photograph). Therefore, our results demonstrated that MoS2−Gd−BSA possessed good hemocompatibility. Moreover, the cell uptake of MoS2−Gd−BSA by 4T1 cells was also evaluated using bio-TEM (Figure S5, Supporting Information). The results indicated that MoS2−Gd−BSA was mainly located in the cytoplasm, indicating the endocytosis approach, which is in agreement with previous work.30 3.4. In Vitro Photothermal Therapy. To evaluate the PTT efficacy of MoS2−Gd−BSA, we further determined the photothermal cytotoxicity of MoS2−Gd−BSA upon illumination. First, the viability of cells treated with MoS2−Gd−BSA plus illumination was evaluated by CCK-8 assay. With a concentration increase to 100 μg/mL, the cell viability was significantly decreased. The MoS2−Gd−BSA showed concentration-dependent PTT efficacy against 4T1 cells. Moreover, different laser power densities were also utilized to evaluate the influence of the laser density on the PTT efficacy. Obviously, the cell viability decreased with stronger power density, dropping to 8.35% at a power density of 0.8 W/cm2 (Figure 6B). Notably, the cell viability was not affected by NIR laser irradiation alone, suggesting that the applied laser exhibited a minimal side effect. Additionally, the PTT efficacy of MoS2−

Gd−BSA was further confirmed using live−dead staining. It can be seen in Figure 6C that almost all cells treated with high concentrations of MoS2−Gd−BSA (100 and 200 μg/mL) plus illumination (1 W/cm2, 10 min) were stained with red color. Additionally, CLSM was also utilized to visualize the impact of PTT on the actin cytoskeleton, which is a network of fibers composed of proteins and plays a positive role in the maintenance of cellular generation and homeostasis.58 As depicted in Figure 6D, the actin cytoskeleton and nucleus of 4T1 cells after different treatments were stained by the Alexa Fluor 488 and DAPI, respectively. Apparently, the intact filamentous network of actin proteins was observed for the untreated control cells, indicating the pleasurable cell morphology and structure. In contrast, the cells treated with MoS2−Gd−BSA plus illumination suffered the degradation of the cytoskeleton protein and substantial cellular death, which indicated that heat diffusion derived from the photothermal effect of MoS2−Gd−BSA could induce cancer cell apoptosis. Moreover, the in vitro localized photothermally induced detachment and destruction of cancer cells were also measured by DAPI staining. Likewise, no significant difference in cell density and viability was observed between blank cells and cells exposed to an NIR laser (Figure S6, Supporting Information). Almost all cells in the PTT group were detached from the dish within the laser spot, which was evident from the big dark region in the CLSM image. These results confirmed that 17793


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Figure 7. (A) Original and colored T1-weighted MR images and (B) linear fitting of the 1/T1 of the MoS2−Gd−BSA solution and Gd−DTPA. (C) In vivo MR images of the tumors before and after injection of the MoS2−Gd−BSA solution. The regions in red circles represent the tumor site. (D) In vivo ultrasound/PA images of tumors before and after injection of the MoS2−Gd−BSA solution.

Figure 8. (A) Thermal imaging pictures of the mice with tumors injected with PBS or MoS2−Gd−BSA followed by 10 min of NIR laser irradiation. (B) Temperature alternation of the tumor site under NIR irradiation. (C) Relative tumor volume of tumor-bearing mice during the process of different treatments. (D) H&E staining section of the tumor under different treatments.

in the green fluorescence of cytosol similar to the control group (Figure 6E). However, we clearly noticed an emergence of orange fluorescence in 4T1 cells of the PTT group, revealing that the red fluorescence was leaked from the lysosomes and overlapped with the green fluorescence of the cytosol because of the disruption of lysosomal membranes. This suggested that PTT would contribute to the lysosome rupture process and eventually lead to acute cancer cell death. 3.5. MR/PA Imaging. For the precision PTT, the imaging capacity of MoS2−Gd−BSA was evaluated before in vivo PTT. Foremost, the stability of MoS2−Gd−BSA was investigated as

MoS2−Gd−BSA could efficiently mediate the photothermal destruction of cancer cells and inhibit the cell proliferation in vitro. Photothermal damage on cancer cells was associated with the disruption of subcellular organelles such as lysosomes.24 Thus, we further traced the integrity of lysosomes by using AO, which can generate red fluorescence in intact acidic lysosomes and emit green fluorescence in neutralized cytosol and nuclei.59 Distinctly, no obvious destabilization of lysosomal membranes was observed for cells exposed to MoS2−Gd−BSA or illumination alone, which display some red fluorescence signal 17794


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Figure 9. (A) Body weight of mice during different treatments. (B) Biodistribution of the MoS2−Gd−BSA at different time intervals after injection of MoS2−Gd−BSA determined by ICP-AES measurements of the Mo element. (C) H&E-stained histological slices of mice injected with MoS2− Gd−BSA for 7 days.

tumor site after injection was 2.75 times higher than that of the control (Figure S8A, Supporting Information). Since the good photothermal efficiency enables the MoS2− Gd−BSA as a good PA contrast agent, the in vivo PA imaging was performed. The US and PA signals before injection and postinjection of MoS2−Gd−BSA are also shown in Figure 7D. Clearly, the enhanced PA signals were detected in the tumor site postinjection of MoS2−Gd−BSA, and the PA intensity was approximately 4 times higher than that of preinjection (Figure S8B, Supporting Information), which validated the feasibility of MoS2−Gd−BSA for PA imaging. The results demonstrated that the as-prepared MoS2−Gd−BSA can be used for MR/PA dualmodal imaging, thus providing guidance for in vivo PTT. 3.6. In Vivo Photothermal Therapy. To shed more light on the PTT efficacy of MoS2−Gd−BSA, we further conducted the comparative studies of tumor inhibition effectiveness under different treatments on the 4T1-bearing mice model. The in vivo PTT effect of MoS2−Gd−BSA was first investigated by thermal imaging. Surely, infrared thermal images with high contrast were clearly observed (Figure 8A). The tumor temperature of mice injected with MoS2−Gd−BSA could rapidly increase to over 50 °C upon laser irradiation (Figure 8B). A tiny temperature change was observed on the control mice. Subsequently, four groups were assigned as follows: PBS group, laser illumination only group, MoS2−Gd−BSA only group, and MoS2−Gd−BSA plus illumination group. The in vivo PTT efficacy of MoS2−Gd−BSA was determined by monitoring the tumor volumes of mice over the following 18 days (Figure 8C; Figure S9, Supporting Information). It was shown that the relative volume of tumors treated with only laser irradiation or MoS2−Gd−BSA kept growing with time, similar to the PBS group, indicating that the laser illumination or MoS2−Gd−BSA without irradiation has no inhibition effect on 4T1 tumor growth. However, tumors treated with MoS2− Gd−BSA plus laser irradiation were obviously suppressed. More importantly, no recurrence of tumor was detected for the

its kinetic stability. As shown in Figure S7 (Supporting Information), the released amount of Gd ions from MoS2− Gd−BSA PBS solution was only 0.081% after 1 week of incubation at 37 °C, suggesting the good stability of MoS2− Gd−BSA under physiological conditions. Even when the MoS2−Gd−BSA was immersed in acidic buffer solution, the released content of Gd ions was only ∼2.8%, which can be attributed to the high affinity of BSA molecules and Gd ions.60 Next, the T1 relaxation time of MoS2−Gd−BSA with different Gd concentrations was measured, and the transverse relaxivity r1 was calculated. Figure 7A reveals that the brightness of T1weighted MR images was gradually enhanced with an increase of the Gd concentration. Straight line fitting of 1/T1 as a function of the Gd concentration is observed in Figure 7B. Consequently, the r1 value of MoS2−Gd−BSA was calculated to be 17.95 mM−1 s−1, which was 4 times higher than that of the commercial Gd−DTPA.20,61 It is worth noting that the brightness of MoS2−Gd−BSA was significantly stronger than that of Gd−DTPA at the same Gd concentrations. According to the previous report,62 the possible reason for the stronger MR imaging capability of MoS2−Gd−BSA than that of Gd− DTPA is the decrease of the tumbling rate of the paramagnetic metal complexes and the improved interaction between water and the Gd complexes by grafting them onto the surface of the NPs. Anyway, the above results confirmed that the MoS2−Gd− BSA is a good MR contrast agent. The low leakage of Gd ions and stronger MR imaging capability promise the MoS2−Gd− BSA as a good MR contrast agent. Inspired by the in vitro T1 contrast enhancement, the in vivo MR imaging was also explored. The MR images of tumorbearing mice before and after injection of MoS2−Gd−BSA solution were obtained. As depicted in Figure 7C, compared with the original image, the tumor region obviously brightens after the injection of MoS2−Gd−BSA, conforming to the typical T1-weighted MR image. The signal enhancement of the 17795


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prepared MoS2−Gd−BSA could probably serve as a cancer theranostic.

PTT group during the next 2 weeks. Furthermore, the histological examination of tumor issues corresponding to different treatments was also performed to elucidate the impact of the PTT effect (Figure 8D). Similarly, it turns out that the tumor regions of mice treated with laser illumination alone or MoS2−Gd−BSA alone have no obvious difference compared with those of mice in the PBS control group in terms of cell size, density, and necrosis, while the typical signs of thermal cell damage, such as nuclear damage, loss of contact, and cell shrinkage, were observed in the tumor tissues treated with MoS2−Gd−BSA plus laser irradiation. Therefore, our results demonstrated that the MoS2−Gd−BSA is an excellent PTCA for in vivo PTT ablation of tumors. 3.7. Biodistribution and Histological Assessment. Apart from the in vivo PTT, the toxicity of MoS2−Gd−BSA was also evaluated. Correspondingly, the body weights of mice under different treatments were monitored during the process of PTT. Figure 9A revealed that the MoS2−Gd−BSA-injected mice with or without laser did not show detectable weight loss in all the time compared to the PBS-treated mice, suggesting that the injection of MoS2−Gd−BSA and the implemented therapeutic procedure would not induce obvious side effects. For tracing the in vivo distribution of MoS2−Gd−BSA, the Mo amount in major organs was detected by ICP-AES. Figure 9B shows the Mo concentration at different time points in those organs. Notably, the MoS2−Gd−BSA was mainly detained in the liver and spleen, both of which are major organs of the reticuloendothelial system and are responsible for the clearance of foreign invaders.63 The amount of Mo in those organs decreased slightly over 1 week, which demonstrated the gradual clearance of MoS2−Gd−BSA over time. Furthermore, the H&E-stained histologic sections were also studied to evaluate the in vivo toxicity of MoS2−Gd−BSA. As shown in Figure 9C, very similar to the healthy mice, no obvious organ damage and abnormal inflammatory response was observed after the injection of MoS2−Gd−BSA for 7 days. Therefore, the MoS2−Gd−BSA did not cause apparent in vivo toxicity at the given dose in light of our preliminary results.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b04488. TEM image of BSA−Gd complexes, XPS analysis of the S element in as-prepared MoS2 nanoflakes, size distribution and the corresponding photograph of MoS2−Gd−BSA dispersed in different media, weight loss percentage of MoS2, MoS2−PAH/PAA, and MoS2− Gd−BSA nanoflakes determined by thermogravimetric analysis, TEM images of 4T1 cells taken up with MoS2− Gd−BSA and the corresponding enlarged images, CLSM images of DAPI-stained 4T1 cells after different treatments, release profile of Gd ions from MoS2−Gd− BSA PBS solution measured by ICP-AES, relative MR signal enhancement and PA signal intensity of the tumor site before and after the intratumoral injection of MoS2− Gd−BSA, and tumor volume of tumor-bearing mice during the process of different treatments (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Email: [email protected]. *Email: [email protected]. ORCID

Peng Huang: 0000-0003-3651-7813 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (Grants 31271028, 31570984, 81401465, and 51573096), Open Foundation of the State Key Laboratory for Modification of Chemical Fibers and Polymer Materials (Grant LK1416), International Cooperation Fund of the Science and Technology Commission of Shanghai Municipality (Grant 15540723400), and Chinese Universities Scientific Fund (Grant CUSF-DH-D-2015043). Also, we extend our sincere gratitude to Dr. Chen. Peng from the Shanghai Tenth People’s Hospital (School of Medicine, Tongji University, Shanghai) for her kind help with in vivo MR imaging.

4. CONCLUSION Overall, a theranostic nanoplatform based on BSA−Gdcomplex-functionalized MoS2 nanoflakes was successfully fabricated for simultaneous MR/PA imaging and PTT. The as-prepared MoS2−Gd−BSA possessed a desirable photothermal effect and photostability as well as excellent biocompatibility. In addition, the results of a series of experiments also confirmed the high efficacy of MoS2−Gd− BSA for efficiently ablating cancer cells, while neither the MoS2−Gd−BSA nor the NIR laser alone could significantly suppress the growth of cancer cells. Furthermore, the biodistribution and histological evaluation of MoS2−Gd−BSA indicated no appreciable toxicity in the range of the study dosages. The strong NIR absorbance also renders this agent with good PA imaging capability. Most importantly, the biomineralization BSA−Gd complexes on the surface of the hybrid nanoflakes could render them with relatively high r1 relaxivity, thereby making MoS2−Gd−BSA suitable for T1weighted MR and PA dual-modal-imaging-guided PTT of tumors. Considering that this is the first attempt to employ the BSA−Gd complexes to functionalize the surface of nanomaterials, this proof-of-concept design might also be an applicable strategy for the preparation of other theranostic agents. Taking all the results together, this study suggested that the as-

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REFERENCES

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