Marriage of AlbuminâGadolinium Complexes and MoS2 Nanoflakes

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Marriage of Albumin-Gadolinium Complexes and MoS 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 ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 09 May 2017 Downloaded from http://pubs.acs.org on May 12, 2017

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ACS Applied Materials & Interfaces

Marriage of Albumin−Gadolinium Complexes and MoS2 Nanoflakes as Cancer Theranostics for Dual-Modality Magnetic

Resonance/Photoacoustic

Imaging

and

Photothermal Therapy Liang Chena, Xiaojun Zhouc, Wei Niea, Wei Fenga, Qianqian Zhanga, Weizhong Wanga, Yanzhong Zhanga, Zhigang Chenc, Peng Huang*b, and Chuanglong He*ac a

College of Chemistry, Chemical Engineering and Biotechnology, Donghua

University, Shanghai 201620, China. b

Guangdong Key Laboratory for Biomedical Measurements and Ultrasound Imaging,

School of Biomedical Engineering, Shenzhen University, Shenzhen 518060, China. c

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials,

Donghua University, Shanghai 201620, China.

* Corresponding authors Email: [email protected] (P. Huang) and [email protected] (C.L He)

ACS Paragon Plus Environment


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ABSTRACT The construction of safe and stable theranostics is beneficial to realize simultaneous cancer diagnosis and treatment. In this study, the 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). The BSA-Gd complexes were prepared by biomineralization method, and then conjugated with MoS2 nanoflakes via amide bond. The as-prepared MoS2-Gd-BSA possessed good photostability and photothermal effect. The cytotoxicity assessment and hemolysis assay suggested the excellent biocompatibility of MoS2-Gd-BSA. Meanwhile, the 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


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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

non-biodegradable, and bear poor photostability, low photothermal conversion efficacy, poor pharmacokinetics, or potential long-term toxicity, which limit their widespread biomedical applications.8 Accordingly, those issues invariably impel the researchers to exploit novel PTCAs.9 To obtain the precision PTT, the real-time visualization of PTCAs is desirable to guide the in vivo PTT of cancer.10,11 By offering the soft-tissue morphology and timely feedback information of 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

tetraazacyclododecane-N,N′,N,N′-tetraacetic

ion

chelators acid

such

as

(DOTA)

1,4,7,10or

diethylenetriaminepentaacetic acid (DTPA). For instance, Dai et al. developed gadolinium chelates (Gd-DOTA) conjugated polypyrrole nanoparticles (NPs) for MR/photoacoustic (PA) imaging and PTT.10 However, most of these Gd-chelator



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agents are limited by high risk of released Gd ions-associated toxicity, which may induce nephrogenic systemic fibrosis (NSF) .13, 14 Therefore, it’s 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 high relaxation rate have been estabilshed 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 Gd-based photothermal theranostics. Currently, molybdenum disulfide (MoS2) nanomaterials have attracted increasing attention in biomedical field in 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 64

Cu-labeled MoS2 nanosheets for multimodal imaging-guided cancer phototherapy.34

Chen et al. prepared MoS2/Bi2S3 composites for PA/CT imaging and combination therapy.35 Besides, Yang et al. have grafted upconversion NPs onto MoS2 nanosheets for fluorescence imaging-guided phototherapy.36 Additionally, Gd-chelator conjugated


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MoS2 core-shell magnetic nanomaterials were synthesized for in vivo MR imaging, which exhibited 4.5-times longer water proton spin−lattice relaxation time (T1) than that of commercial Gd-DTPA agent.37 Nevertheless, the potential toxicity of Gd-chelator based compounds needs to be more carefully inspected because of the instability of chelating molecules.38 On the other hand, the admirable photothermal property also makes MoS2 nanomaterials high potential as contrast agents of PA imaging,39 which could penetrate tissues more deeply and provide legible structure and microcosmic information with high sensitivity. Note that the apparent advantages of dual-model imaging, the combination of PA and MR imaging is more helpful for the 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 co-workers prepared BSA-Gd/CuS NPs for PA/MR imaging and PTT.40 Chen and co-workers developed Co9Se8-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 one-pot hydrothermal method. The surface coating of poly (allylamine hydrochloride) and poly (acrylic acid) (PAH/PAA) was achieved by layer-by-layer (LBL) technique.42-45 Meanwhile, the BSA-Gd complexes were synthesized by mild biomineralization method. Then the BSA-Gd complexes conjugated MoS2 nanoflakes (MoS2-Gd-BSA) were obtained



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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 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 advantages 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 complexes modified MoS2 nanoflakes were investigated as cancer theranostics for MR/PA imaging and PTT.

2. EXPERIMENT SECTION 2.1 Materials Thioacetamide (TAA, 99%), poly(vinylpyrrolidone) (MW 58000), poly(acrylic acid) (PAA,

MW 1800)

and N-(3-Dimethylaminopropyl)-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 (Shanghai, China). Triton X-100, poly (allylamine hydrochloride) (PAH, MW 15000) and bovine serum albumin (BSA) were obtained from Sigma-Aldrich Trading Co., Ltd (Shanghai, China). Fetal bovine serum (FBS), penicillin-streptomycin, trypsin and Roswell Park Memorial Institute (RPMI) 1640 medium were obtained


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from Gibco Life Technologies Co. (Grand Island, USA). Cell Counting Kit-8 (CCK-8) was purchased from 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, USA). 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) was mixed in 10 mL deionized water. Thereafter, 75 mg of TAA in 5 mL deionized water was slowly introduced into the mixture, which was vigorously stirred to form transparent solution. The solution was then sealed in reaction kettle and maintained at 180 ºC 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 based on a typical procedure.20 First, BSA (0.25 g) was fully dissolved in 9 mL DI water at 37 ºC in water bath, then 1 mL Gd(NO3)3 solution (50 mM) was dropwise added into the BSA solution under vigorous stirring. After 5 minutes, 1 mL NaOH aqueous solution (2 M) was quickly added into the solution. The mixture was continued at 37 ºC for 12 h. Afterwards, the solution was dialyzed against DI water for one day. The BSA-Gd was harvested and



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kept in 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 MoS2 nanoflakes.47 In detail, 5 mL of MoS2 aqueous dispersion (2 mg/mL) was centrifuged and suspended in 10 mL N, N-dimethyl formamide. After 15 min bath sonication, 2 mL PAH aqueous solution (10 mg/mL) was slowly added. Then the mixture was stirred at 80 ºC for 2 h. Then MoS2-PAH was collected and dispersed in 5 mL DI water. The MoS2-PAH solution was slowly introduced into 10 mL of PAA aqueous solution (2 mg/mL). After 2 h reaction, the MoS2-PAH/PAA was harvested and washed several times with DI water. Subsequently, the pH of MoS2-PAH/PAA dispersion was adjusted to 7.4. Then 6 mg EDC was added within one hour to induce the crosslink reaction for another 12 h. The obtained MoS2-PAH/PAA was repeatedly washed and suspended in 10 mL DI water. Last, 1 mL of BSA-Gd solution was added into the above MoS2-PAH/PAA dispersion for reacting another 24 h. The as-prepared MoS2-Gd-BSA was purified and dispersed in DI water. 2.5 Characterizations The nanoflakes were observed by JEM-2100 transmission electron microscope (TEM, JEOL Ltd., Japan, the operation acceleration voltage of 200 kV with a LaB6 electron gun). The size distribution was tested by dynamic light scattering (DLS) method using a BI-200SM multi-angle dynamic/static laser scattering instrument


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(Brookhaven, USA). Zeta-potential analysis was carried out by a Zetasizer Nano ZS apparatus (Malvern Instruments, UK). Ultraviolet-visible (UV-vis) absorption were recorded by using a Lambda 35 UV-vis spectrophotometer (PerkinElmer, USA) at room temperature (RT) under ambient conditions. The Raman spectra were conducted by using an inVia-Reflex micro-Raman spectroscopy system (Renishaw, UK) with a 633 nm solid laser of 50 mW power at RT. Thermogravimetric (TG) analysis was performed under nitrogen flow from RT to 600 ºC 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 KBr disc technique and recorded on a Nexus 670 spectrometer (Thermo Nicolet, USA). The Leeman Prodigy Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) system (Hudson, NH03051, USA) was used to determine the concentration of materials. 2.6 Detection of Photothermal Performance To investigate the photothermal performance, 0.2 mL of MoS2-Gd-BSA 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 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 was also utilized to verify the photostability of MoS2-Gd-BSA. 2.7 Cell Lines and Cell Culture Mouse fibroblast L929 cell line, mouse leukemic monocyte macrophage RAW 264.7 cell line, murine breast cancer 4T1 cell line and human umbilical vein



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endothelial HUVEC cell line were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The 4T1 and HUVEC cells were grown in RPMI 1640 medium. The L929 and RAW 264.7 cells were cultured in DMEM medium. The cells were cultured in a humidified atmosphere containing 5 % CO2 at 37 ºC. 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, cells were cultured in 96-well plate (1 × 104 cells per well) overnight. Then the cells were co-incubated with fresh medium containing different concentrations of MoS2-Gd-BSA for another 24 h. After that, the cells were rinsed twice with PBS. Then CCK-8 assay was conducted. The cells were further cultured for two hours before subjected to a microplate reader (Multiskan MK3, Thermo). The relative cell viabilities were determined by optical density (OD) at 450 nm divide the value of 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 fresh mice blood was centrifuged to obtain red blood cells (RBCs). The RBCs were purified and resuspended in PBS. Then 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 obtained supernatants was tested by UV−vis spectrophotometer. The hemolysis percentage was obtained according to follow


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equation: Hemolysis % =

ODsample - ODnegative × 100% ODpositive - ODnegative

Where ODsample, ODnegative, and ODpositive represented the value of tested samples, the negative and positive control, 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. Afterwards, the cells of 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 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 density 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 microscope. 2.10 Confocal Laser Scanning Microscopy To verify the in vitro photothermal treatment of MoS2-Gd-BSA, confocal microscopic imaging was carried out on a Carl Zeiss LSM700 confocal laser scanning microscopy (CLSM, 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



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cells of photothermal group were illuminated (808 nm, 1 W/cm2) for 10 min. After 2 h 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. R 568 conjugated phalloidin was used to and DAPI was Subsequently, Alexa Fluor○

used to stain 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 were acted as control. The AO staining was also performed to assess the integrity of lysosomal membrane. After different treatments, the cells were washed and stained with AO for 15 min. The unbounding dyes were removed by PBS and the cells were observed by CLSM. The AO was excited at 488 nm. Besides, the 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 dish (2 × 105 per dish) for 24 h. The cells of PTT group were treated to 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 Committees (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 suspensions (2 × 106 cells) was subcutaneously injected into the right hind leg of Balb/c mouse to generate tumor model.


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After the tumor grown up to ~50 mm3, mice were randomly set as 4 groups: control, PBS plus laser, MoS2-Gd-BSA only, MoS2-Gd-BSA plus laser. For groups of PBS plus laser and MoS2-Gd-BSA plus laser, the tumor-bearing mice were illuminated for 15 min after intratumorally injection with 100 µL of PBS or MoS2-Gd-BSA

suspension

(2

mg/mL).

The

temperature

change

of

MoS2-Gd-BSA-injected tumor sites was recorded by infrared thermal camera (GX-A300, Shanghai Guixin Corporation). Additionally, the volume of tumors was calculated as V = (tumor length) × (tumor width)2/2. The length and width of tumor was measured by electronic caliper. The relative tumor volume was 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 MoS2-Gd-BSA solution was investigated by monitoring the release 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 sealed in a dialysis bag (MW cut-off ~3500). Then the dialysis bag was soaked in 9 mL PBS and maintained in the shaker at 37 ºC. At the specific time points, 5 mL of solution was withdrawn for analysis and the equivalent free PBS was supplemented. The concentration of Gd ions was measured by inductively coupled plasma-atomic emission spectroscopy (ICP-AES). To measure transverse proton relaxation times (T1) and T1-weighted MR imaging of samples, the MoS2-Gd-BSA aqueous solution with different Gd concentrations were analyzed by an NMI20-Analyst NMR Analyzing and Imaging system. The



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instrumental parameters were listed as follow: 0.5 T magnet, CPMG sequence, point resolution was 156 mm × 156 mm, 0.6 mm of section thickness, 4000 ms of TR, 60 ms of TE, number of excitation was 1. The transverse relaxivity (r1) was figured out by linear fitting the inverse T1 relaxation time (1/T1) as a function of Gd concentration. For in vivo MR imaging, the tumor-bearing mice were anesthetized. Then the tumor-bearing mouse was 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 after 10 min post-injection. The control group was imaged under the same condition before the injection. For PA imaging, the Vevo LAZR2100 PA imaging system was applied to monitor the tumor sites of mice. The ultrasound (US) and PA signals were all collected before and 10 min post-injection of MoS2-Gd-BSA PBS suspension (2 mg/mL). 2.13 Biodistribution and Histology Examination The biodistribution of MoS2-Gd-BSA in mice was measured by ICP-AES. Briefly, 0.1 mL of MoS2-Gd-BSA PBS suspension (2 mg/mL) was intravenously injected into mice. Then the mice were scarified at certain time intervals and the organs were washed with PBS, lyophilized and weighted. Afterward, the organs were immersed into nitric acid and heated in oil bath until the organs were fully dissolved. The cooled solutions were diluted into 10 mL in volumetric flask for the measurement of ICP-AES.


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To further analyze the in vivo histocompatibility of MoS2-Gd-BSA, hematoxylin and eosin (H&E) staining was also conducted within two weeks. The Balb/c mice were sacrificed at two weeks after intravenously 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 microscope. The healthy mouse was used for control. 2.13 Statistical Analysis The data were displayed as the mean ± standard deviation (SD). The significance of experiment data was evaluated by one-way analysis of variance (one-way ANOVA) was used to evaluate. The statistical significance was considered at *P < 0.05 and **P < 0.01.

3. RESULTS AND DISCUSSION 3.1 Preparation and Characterization of MoS2-Gd-BSA The synthetic process of MoS2-Gd-BSA was illustrated in the Scheme 1. First, MoS2 nanoflakes were prepared according to previous report with some modifications,39 and then the carboxyl group was introduced onto the surface of MoS2 nanoflakes via LBL coating. The BSA-Gd complexes were synthesized by biomineralization approach and conjugated onto MoS2 nanoflakes through the amide reaction. As shown in Figure 1A, the as-prepared MoS2 nanoflakes were composed of several lamellar structures and ultimately form round-shape. The BSA-Gd complexes demonstrated the cluster-like morphology, which is in agreement with previous report



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(Figure S1).20 After conjugated with BSA-Gd complexes, the dense organic layer was apparently emerged on MoS2 nanoflakes, supplying direct evidence of successful modification of BSA-Gd (Figure 1B). The chemical state of MoS2 nanoflakes were 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 present of Mo4+ in MoS2 nanoflakes.50 Besides, the signals of S2s at 226.5 eV and S2p at ~162.5 eV also echoed the successful fabrication of MoS2 nanoflakes (Figure S2). Furthermore, the Raman spectrum of 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 as-prepared MoS2 nanoflakes are slightly broaden and red-shifted compared with the bulk MoS2, this may stem from the lateral dimensions of these layers52 and the multilayer structures of MoS2 nanoflakes.53 To construct the MoS2-Gd-BSA, BSA-Gd was attached onto MoS2 nanoflakes to act as MR imaging contrast agent. First, LBL assembly strategy was employed to introduce carboxyl groups onto MoS2. As shown in Figure 2A, the zeta 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 that the zeta potentials of nanoflakes were 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 MoS2 nanoflakes. Then the amino groups


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of BSA-Gd complexes were reacted with carboxyl groups of MoS2-PAH/PAA to obtain MoS2-Gd-BSA with zeta potential of –26.4 mV, which was in favor of forming stable MoS2-Gd-BSA aqueous dispersion.54 Actually, the MoS2-Gd-BSA could stably disperse in both PBS and cell medium without any aggregation even after 1 d (Figure S3). The dispersibility and size distribution of MoS2-Gd-BSA in PBS and cell culture medium exhibited no unexpected change even the slight increase 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 was 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 be resulted from little microscopic agglomeration56 and hydration shell.57 Moreover, other physicochemical properties of these products were also investigated. Figure 2C shows the FT-IR spectra of pristine MoS2 nanoflakes and MoS2-Gd-BSA. It was found that the FT-IR spectrum of MoS2 nanoflakes was completely different with our previous reported flower-like 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 is due to the residual PVP. After the modification of BSA-Gd, the typical amide II bands of BSA at 1545 cm–1 was emerged on the spectrum of MoS2-Gd-BSA. Additionally, the weight loss of



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MoS2-Gd-BSA reached up to 49% at 600 °C according to the TGA curve (Figure S4), while the percentage for bare MoS2 nanoflakes and PAH/PAA coated MoS2 was just 37% and 26% at the same condition, respectively. The gradual increased organic component in nanoflakes confirmed the successful of step-by-step modification. Thus, on the basis of above results and former literatures,46,47 we deduced that BSA-Gd complexes were covalently conjugated onto MoS2 nanoflakes. Besides, the conjugation of BSA-Gd on the MoS2 nanoflakes didn’t reduce the optical absorption of MoS2 in NIR region (Figure 2D), which promises the 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 MoS2-Gd-BSA aqueous solution rapidly increased after irradiation, whereas the temperature of pure water only elevated 0.5 °C (Figure 3A and B). Moreover, the photothermal effects of MoS2-Gd-BSA exhibited obvious concentration and power-dependent tendency. 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 observing any weakening of temperature elevation. Meanwhile, no appreciable absorbance change of MoS2-Gd-BSA was observed in Figure 3D, indicating the good photostability of MoS2-Gd-BSA. 3.3 Biocompatibility of MoS2-Gd-BSA


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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, RAW 264.7, L929 and HUVEC cells, by CCK-8 assay. The cell viabilities were depicted in Figure 4A and B, which demonstrated that the MoS2-Gd-BSA has low cytotoxicity for all kinds of cells. Concretely, the cell viabilities for 4T1 cells, L929 cells, RAW 264.7 cells and HUVEC cells 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 the AO staining. As shown in Figure 4C, the different types of live cells exhibited green fluorescence similar with the control group after incubated with MoS2-Gd-BSA, which further confirmed the excellent cytocompatibility of MoS2-Gd-BSA. The evaluation of 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 corresponding supernatants after hemolysis assay were presented in Figure 5A. Compared with the positive control (water), no distinct absorption peaks at 541 nm were observed for both negative control and samples in a concentration range of 6.25-800 µg/mL. It’s worth noting that absorption value of MoS2-Gd-BSA at concentration of 800 µg/mL was a bit higher than other samples in full wavelength, which probably result from the absorbance of residual MoS2-Gd-BSA in the supernatant. The hemolysis percentages of MoS2-Gd-BSA were also calculated by optical density (OD) at 541 nm. Figure 5B shows that the hemolysis percentages of



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these nanoflakes were all less than 5% except the MoS2-Gd-BSA at concentration of 800 µg/mL, which is due to the nature absorption of the residued MoS2-Gd-BSA. Moreover, a light black appeared in the picture of supernatant for 800 µg/mL MoS2-Gd-BSA solution, while negligible red color was observed for 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). The results indicated that MoS2-Gd-BSA were 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 PTT efficacy of MoS2-Gd-BSA, we further conducted 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 the concentration increase to 100 µg/mL, the cell viability was significantly decreased. The MoS2-Gd-BSA showed the concentration-dependent PTT efficacy of against 4T1 cells. Moreover, different laser power densities were also utilized to evaluate the influence of laser density on PTT efficacy. Obviously, the cell viability was decreased with the stronger power density, which dropped to 8.35% at the power density of 0.8 W/cm2 (Figure 6B). Notably, the cell viability wasn’t affected by NIR laser irradiation alone, suggesting that the applied laser exhibited minimal side effect. Additionally, the PTT efficacy of MoS2-Gd-BSA was further confirmed using


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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, the 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 cytoskeleton protein and substantial cellular death, which manifested that heat diffusion derived from photothermal effect of MoS2-Gd-BSA could induce cancer cell apoptosis. Moreover, the in vitro localized photothermal-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 NIR laser (Figure S6). For cells in the PTT group, almost all cells 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 MoS2-Gd-BSA could efficiently mediate the photothermal destruction of cancer cells and inhibit the cell proliferation in vitro. The photothermal damage on cancer cells was associated with the disruption of subcellular organelles such as lysosomes.24 Thus we further traced the integrity of



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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 in green fluorescence of cytosol similar with control group (Figure 6E). However, we clearly noticed that the emergence of orange fluorescence in 4T1 cells of PTT group, revealing that the red fluorescence was leaked from the lysosomes and overlapped with green fluorescence of cytosol because of the disruption of lysosomal membranes. It 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 its kinetic stability. As shown in Figure S7, the release amount of Gd ions from MoS2-Gd-BSA PBS solution was only 0.081 % after 1 week incubation at 37 °C, suggesting the good stability of MoS2-Gd-BSA under physiological condition. Even the MoS2-Gd-BSA was immersed in acidic buffer solution, the release 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 revealed that the intensity of T1-weighted MR images was gradually enhanced with the increase of Gd concentration. The fitting straight line of the 1/T1 as a function


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of Gd concentration was observed in Figure 7B. Consequently, the r1 value of MoS2-Gd-BSA was calculated to be 17.95 mM–1 s–1, which was four times higher than the commercial Gd-DTPA.20,

61

It was worth noting that the intensity 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 NPs. Anyway, the above results confirmed that the MoS2-Gd-BSA is 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 tumor-bearing mouse before and post injection of MoS2-Gd-BSA solution were obtained. As depicted in Figure 7C, compared with original image, the tumor region brightens obviously after the injection of MoS2-Gd-BSA, which was conforming to the typical T1-weighted MR image. The signal enhancement of tumor site after injection was 1.81 times higher than that of control (Figure S8A). 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 and post-injection of MoS2-Gd-BSA were also showed in Figure 7D. Clearly, the enhanced PA signals were detected in the tumor site post-injection of MoS2-Gd-BSA



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and the PA intensity is approximately 4 folds higher than that of pre-injection (Figure S8B), 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 dual-modal imaging, thus providing the 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 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). Tiny temperature change was observed on control mice. Subsequently, 4 groups were set as follows: the PBS group, laser illumination only, MoS2-Gd-BSA only and MoS2-Gd-BSA plus illumination. The in vivo PTT efficacy of MoS2-Gd-BSA was determined by monitoring the tumor volumes of mice in the following 18 days (Figure 8C and Figure S9). It was shown that the relative volume of tumors treated with only laser irradiation or MoS2-Gd-BSA kept growth with time, similar to the PBS group, indicating that the laser illumination or MoS2-Gd-BSA without irradiation have 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 PTT group during the next two weeks. Furthermore, the histological examination of tumor issues corresponding to different


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treatments were 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 with 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. So our results demonstrated that the MoS2-Gd-BSA is an excellent PTCA for in vivo PTT ablation of tumor. 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 neither the MoS2-Gd-BSA injected mice with nor without laser showed 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 were mainly detained in liver and spleen, both of whom are the major organs of the reticuloendothelial system and responsible for the clearance of foreign invaders.63 The amount of Mo in those organs decreased slightly over one week, which demonstrated the gradually clearance of MoS2-Gd-BSA over time Furthermore, the H&E stained histologic section were also



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conducted to evaluate the in vivo toxicity of MoS2-Gd-BSA. As shown in Figure 9C, very similar with the healthy mice, no obvious organ damage and abnormal inflammatory response were observed after the injection of MoS2-Gd-BSA for 7 days. Therefore, the MoS2-Gd-BSA didn’t cause apparent in vivo toxicity at the given dose in the light of our preliminary results.

4. CONCLUSION Overall, a theranostic nanoplatform based on BSA-Gd complexes functionalized MoS2 nanoflakes was successfully fabricated for simultaneously MR/PA imaging and PTT. The as-prepared MoS2-Gd-BSA possessed 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 manifested that no appreciable toxicity in the range of 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 T1-weighted MR imaging and PA dual-modal imaging-guided PTT of tumor. Considering that it’s the first attempt to employ the BSA-Gd complexes to functionalize the surface of nanomaterials, this proof-of-concept design might also be applicable strategy for the preparation of other theranostic agents. Taken all together, this study suggested that


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the as-prepared MoS2-Gd-BSA could probably serve as cancer theranostics.

ASSOCIATED CONTENT Supporting Information TEM image of BSA-Gd complexes; The XPS analysis of S element in as-prepared MoS2

nanoflakes;

Size

distribution

andthe

corresponding

photograph

of

MoS2-Gd-BSA dispersed in different medium. The MoS2-Gd-BSA showed no obvious precipitation after 1 d incubation; The 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, the red line represents the border of laser spot; Release profile of Gd ions from MoS2-Gd-BSA PBS solution measured by ICP-AES; The relative MR signal enhancement and PA signal intensity of tumor site before and after the intratumorally injection of MoS2-Gd-BSA; The tumor volume of tumor-bearing mice during the process of different treatments.

ACKNOWLEDGEMENT This work was financially supported by the National Natural Science Foundation of China (31271028, 31570984, 81401465, 51573096), Open Foundation of State Key Laboratory for Modification of Chemical Fibers and Polymer Materials (LK1416), international Cooperation Fund of the Science and Technology Commission of Shanghai Municipality (15540723400) and Chinese Universities Scientific Fund



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(CUSF-DH-D-2015043). Also, we would like to extend our sincere gratitude to Dr Chen. Peng from Shanghai Tenth People's Hospital (School of Medicine, Tongji University, Shanghai 200072) for her kind help with in vivo MR imaging.

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Li,

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FIGURES

Scheme 1. Schematic illustration of the preparation of bovine serum albumin-Gd complexes functionalized MoS2 nanoflakes as a theranostic nanoplatform for magnetic resonance/photoacoustic imaging and photothermal therapy.


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Figure 1. The TEM images of (A) bare MoS2 nanoflakes and (B) MoS2-Gd-BSA; The (C) XPS analysis and (D) Raman spectra of the as-prepared MoS2 nanoflakes.



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Figure 2. (A) The zeta potential of the different MoS2 nanoflakes in aqueous solution; (B) The hydrodynamic diameter of MoS2 and MoS2-Gd-BSA nanoflakes (the inset photograph is the hybrid nanoflakes MoS2-Gd-BSA in different medium); (C) The FTIR spectra of the MoS2 and MoS2-Gd-BSA; (D) The UV-Vis spectra of the MoS2 and MoS2-Gd-BSA.


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Figure 3. The 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 808 nm NIR laser for 10 min (1 W/cm2); (B) Temperature change of 100 µg/mL MoS2-Gd-BSA solution irradiated by 808 nm NIR laser for 10 min with different power densities; (C) Temperature change of MoS2-Gd-BSA solution (100 µg/mL) irradiated repeatedly by 808 nm NIR laser for on–off cycles; (D) UV-vis absorption spectra of the MoS2-Gd-BSA solution before and after NIR irradiation (inset picture represented the photographs of MoS2-Gd-BSA solution before and after irradiation).



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Figure 4. The cytotoxicity of the MoS2-Gd-BSA nanoflakes. (A) The cell viability of 4T1 and L929 cells treated with MoS2-Gd-BSA suspension at different concentrations; (B) The cell viability of HUVEC and RAW 264.7 cells incubated with different concentrations of MoS2-Gd-BSA suspension for one day; (C) The live-dead cell staining of different cells treated with or without MoS2-Gd-BSA solution (200 µg/mL), the bar is 200 µm.


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Figure 5. Hemolytic activity of MoS2-Gd-BSA nanocomposites at different concentrations. (A) The 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 three hours. The inset images for direct observation of results, suggesting the good biocompatibility of MoS2-Gd-BSA hybrid nanoflakes.



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Figure 6. The in vitro photothermal ablation of 4T1 cells. (A) The cell viability of 4T1 cells under different MoS2-Gd-BSA concentrations with laser power density at 1 W/cm2; (B) The cell viability of 4T1 cells under different laser power density with MoS2-Gd-BSA concentration at 200 µg/mL; (C) The live-dead staining of 4T1 cells under different MoS2-Gd-BSA concentrations with laser power density of 1 W/cm2; (D) CLSM images of 4T1 cells treated by MoS2-Gd-BSA + NIR Laser, blue channel represents DAPI and the green color was Alexa Fluor 488 conjugated phalloidin stained cytoskeleton of 4T1 cells; (E) Confocal laser scanning microscopy images of acridine orange (AO) stained 4T1 cells under different treatments.


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



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Figure 8. (A) The thermal imaging pictures of the mice with tumor injected with PBS or MoS2-Gd-BSA and followed by 10 min NIR laser irradiation, respectively. (B) The temperature alternation of tumor site under NIR irradiation; (C) The relative tumor volume of tumor-bearing mice during the process of different treatments. (D) The H&E staining section of tumor under different treatments.


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



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Marriage of AlbuminâGadolinium Complexes and MoS2 Nanoflakes

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Marriage of AlbuminâGadolinium Complexes and MoS2 Nanoflakes