Multifunctional Nanoflowers for Simultaneous Multimodal Imaging and

Jan 27, 2018 - TEM images of (a,b) Mn-doped Fe3O4 nanoclusters; (c,d) Mn-doped Fe3O4@MoS2 nanoflowers; (e) Mn-doped Fe3O4@MoS2@CS nanoflowers; and (f)...
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Multifunctional Nanoflowers for Simultaneous Multimodal Imaging and High-Sensitive Chemo-Photothermal Treatment Lingjie Meng, xunan jing, zhe zhi, daquan wang, jing liu, and Yongping Shao Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00053 • Publication Date (Web): 27 Jan 2018 Downloaded from http://pubs.acs.org on January 29, 2018

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Bioconjugate Chemistry

Multifunctional Nanoflowers for Simultaneous Multimodal Imaging and High-Sensitive Chemo-Photothermal Treatment Xunan Jing †, Zhe Zhi ‡, Daquan Wang †, Jing Liu †, Yongping Shao ‡, and Lingjie Meng †,§,*



School of Science, State Key Laboratory for Mechanical Behavior of Materials and

MOE Key Laboratory for Nonequilibrium Synthesis and Modulation of Condensed Matter, Xi'an Jiaotong University, Xi'an 710049, P. R. China ‡

Center for Mitochondrial Biology and Medicine, Ministry of Education Key;

Laboratory of Biomedical Information Engineering, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an 710049, P. R. China §

Instrumental Analysis Center of Xi’an Jiaotong University, Xi'an 710049, P. R.

China

* Corresponding author, Email: [email protected] (L. Meng)

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ABSTRACT: Liver cancer is currently among the most challenging cancers to diagnose and treat. It is of prime importance to minimize the side effects on healthy tissues and reduce drug resistance for precise diagnoses and effective treatment of liver cancer. Herein, we report a facile, but high-yield approach to fabricate a multifunctional nanomaterial through the loading of chitosan and metformin on Mn-doped Fe3O4@MoS2 nanoflowers. Mn-doped Fe3O4 cores are used as simultaneous T1/T2 magnetic resonance imaging (MRI) agents for sensitive and accurate cancer diagnosis, while MoS2 nanosheets are used as effective near-infrared photothermal

conversion

agents

for

potential

photothermal

therapy.

The

surface-functionalized chitosan was able, not only to improve the dispersibility of Mn-doped

Fe3O4@MoS2

nanoflowers

in

bio-fluids

and

increase

their

biocompatibility, but also to significantly enhance the photothermal effect. Furthermore, metformin loading led to highly suppression and eradication of hepatoma cells when photothermally sensitized, but exhibited negligible effects on normal liver cells. Due to its excellent combination of T1/T2 MRI properties with sensitive chemotherapeutic and photothermal effects, our study highlights the promise of developing multifunctional nanomaterials for accurate multimodal imaging-guided, and highly sensitive therapy of liver cancer.

INTRODUCTION

Cancers have become the second leading cause of detriment to human health worldwide due to their poor prognosis, high recurrence and high mortality rates.1 In

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contrast to the stable or declining trends for most cancers, the incidence and mortality rates for liver cancer have increased in recent years.2 Furthermore, in most cases, liver cancer patients are merely treated with palliative care and the prognosis is only of 4–6 months on average.1, 2

Despite nanomedicine having delivered great advances in the improvement of diagnoses and treatment efficacy through the enhanced permeability and retention (EPR) effect since 1986,3 challenges remain with regards to the development of early diagnosis methods and new treatment options that are highly selective and effective. Over the past two decades, multimodal imaging has attracted much attention due to its potential in the early detection, screening, diagnosis, and image-guided treatment of life-threatening diseases and cancer.4 Multimodal imaging involves a combination of inter-complementary imaging modalities, such as fluorescence, magnetic resonance imaging (MRI), ultrasound, and X-ray computed tomography, which together provide more precise biological information compared to any single modality.5-8 MRI has been widely used in the clinic due to its provision of in vivo 3D high-resolution images and great imaging depth.9-11 Superparamagnetic iron oxide (Fe3O4) nanoparticles with excellent biocompatibility are often utilized as an effective contrast agent for T2-weighted MRI and in tracking the therapeutic effect of cancer treatments.12, 13 Conversely, Gd3+- or Mn2+- containing nanoparticles or complexes are commonly used for intuitive T1-weighted MRI.14, 15 Nevertheless, when performed individually, T1 or T2 contrast effects and magnetic susceptibility artifacts may lead to a false diagnosis.16-18 Mn-doped Fe3O4 nanoparticles may be used as simultaneous 3 ACS Paragon Plus Environment

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dual-modal probes for T1/T2 MRI, providing complementary imaging information for an early and precise diagnosis.

Chemotherapy is one of the most commonly used modalities in cancer therapy to date; however, the most frequently used anticancer drugs are non-selective compounds that lead to serious side effects for normal tissues. Over the past two decades, a series of tumor targeting drug delivery systems have been developed to improve the therapeutic effect of common chemotherapeutics; nevertheless, fully overcoming the side effects and drug resistance remains a challenge.19-21 Metformin (MET), a commonly used drug for the management of type II diabetes, suppresses gluconeogenesis production in the liver as well as intestinal glucose absorption.22-24 Recent studies have shown that MET exhibits anticancer properties at high doses in non-diabetic patients, with promising preclinical data having recently come to light.25-29 High dose MET was shown to reduce the proliferation and induce the apoptotic and clonogenic death of cancer cells in vitro, as well as to cause cell cycle arrest and reduce the incidence and growth of experimental tumors without side effects in vivo.30, 31 In addition, MET and its macromolecular formulations led to an increased sensitivity to chemotherapy or decreased drug dose requirements when combined

with

other

therapeutic

modalities

(hyperthermia/radiotherapy/chemotherapy) in a variety of cancer cell lines.27,

32-34

Thus, MET may be a promising new drug for the treatment of liver cancer, overcoming the side effects and drug resistance of the methods used to date.

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Photothermal therapy is an alternative cancer treatment that employs a photo-absorber to convert laser radiation to heat to ablate cancer cells. As tumor cells are more sensitive to heat than healthy cells, hyperthermia can kill target cells with minimal side effects.35, 36 Further, a near-infrared (NIR) laser is preferred as it is able to penetrate skin and tissues to a reasonable depth. Over the past two decades, the use of a wide range of photothermal therapy agents, including gold nanomaterials,37 carbon nanomaterials,38,

39

transition metal dichalcogenide nanosheets,40,

41

and

conjugate organic materials,42, 43 has been explored. Through these studies, MoS2 has emerged as an easily prepared 2D nanomaterial with great photothermal effects. MoS2 nanosheets have many superior or similar characteristics compared to its analogous graphene, including strong NIR absorbance for photothermal tumor ablation, ultra-high surface area available for efficient molecular binding and drug loading, and the ability of tailorable surface modification.44, 45 Therefore, the integration of the chemo-photothermal ability of MoS2 nanosheets with the strong magnetic properties of Mn-doped Fe3O4 to construct a “smart” T1/T2 MRI and chemo-photothermal targeting system could provide a unique opportunity for the effective enhancement of cancer diagnosis and therapy.

Herein, we present the construction of a multifunctional nanoplatform through the loading of chitosan (CS) and MET on flower-like Mn-doped Fe3O4@MoS2 nanomaterial for the precise diagnosis and highly sensitive treatment of liver cancers. The Mn-doped Fe3O4 cores serve as simultaneous T1/T2 MRI agents, while the MoS2 nanosheets and MET are used as an effective near-infrared photothermal conversion 5 ACS Paragon Plus Environment

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agent and anticancer drug, respectively. The versatile multifunctional nanoflowers were successfully prepared by a facile, high-throughput and low-cost, two-step microwave-assisted solvothermal method. MoS2 nanosheets were grown in situ on the surface of Mn-doped Fe3O4 nanoclusters to form Mn-doped Fe3O4@MoS2 nanoflowers, and then functionalized with CS and MET. Interestingly, the intrinsic photothermal therapeutic effect of MoS2 nanosheets was greatly improved through CS functionalization,

and

the

photothermally

triggered

release

of

MET

and

thermo-sensitized chemotherapy led to a synergistic treatment. Noticeably, Mn-doped Fe3O4@MoS2@CS@MET was able to sensitively inhibit and kill hepatoma cells, with a negligible effect on normal liver cells.

RESULTS AND DISCUSSION

Scheme 1. Schematic illustration of the synthesis route of Mn-doped Fe3O4@MoS2@CS@MET nanoflowers.

The Mn-doped Fe3O4@MoS2 nanoflowers nanoplatform was synthesized through a facile, high-throughput and low-cost, two-step microwave-assisted solvothermal process (Scheme 1). Uniform Mn-doped Fe3O4 nanoclusters were prepared through 6 ACS Paragon Plus Environment

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the control of nucleation with anionic polyelectrolyte PSSMA, allowing clustering-based growth of the colloidal spheres. During this process, primary nucleated nanocrystals aggregated into larger secondary nanoclusters.46,

47

MoS2

nanosheets were then prepared according a similar procedure to that of Mn-doped Fe3O4 nanoclusters. The as-grown MoS2 nanosheets aggregated onto the surface of Mn-doped Fe3O4 nanoclusters, yielding Mn-doped Fe3O4@MoS2 nanoflowers. Compared to the long synthesis time of the traditional hydrothermal method (> 10 h), the microwave-assisted solvothermal reaction is considerably faster (within 2 h), with a high yield (>90%) and satisfactory reproducibility. Finally, CS and MET were sequentially loaded onto the surface of the Mn-doped Fe3O4@MoS2 nanoflowers through physical adsorption. The biodegradable and non-toxic polymer CS was added to enhance the biocompatibility and physiologic stability of the Mn-doped Fe3O4@MoS2 nanoflowers.

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Figure 1. TEM images of (a, b) Mn-doped Fe3O4 nanoclusters; (c,d) Mn-doped Fe3O4@MoS2 nanoflowers;

(e)

Mn-doped

Fe3O4@MoS2@CS

nanoflowers;

and

(f)

Mn-doped

Fe3O4@MoS2@CS@MET nanoflowers.

The structure and morphology of the as-grown nanomaterials were investigated by TEM (Figure 1). Uniform Mn-doped Fe3O4 nanoclusters were observed, with an average diameter of approximately 50 nm (Figure 1a and b) and formed through the aggregation of several nanocrystals 3–5 nm in size (see Supporting information, Figure S1a). The Mn-doped Fe3O4@MoS2 nanoflowers had a petal shape-like profile and a rough and folded surface structure (Figure 1c). The flower-like structure was built from abundant, irregular, and thin curved MoS2 nanosheets, randomly growing 8 ACS Paragon Plus Environment

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Bioconjugate Chemistry

on the Mn-doped Fe3O4 nanocluster surfaces (Figure 1d). Conversely, MoS2 prepared through the same procedure, but without the addition of Mn-doped Fe3O4 nanocluster seeds, had a considerably different agglomerate morphology composed of numerous disordered thin nanosheets (see Supporting information, Figure S1b). To improve the physiological and water-dispersible stability, and biocompatibility of Mn-doped Fe3O4@MoS2 nanoflowers, chitosan (CS), a naturally occurring linear cationic polysaccharide with widespread bioapplications, is introduced during the modification process. Following CS and MET functionalization, the nanoflower surfaces became slightly thicker (Figure 1e, f). However, the direct observation of the organic shell is difficult due to its low electron contrast. The hydrodynamic size of Mn-doped Fe3O4 nanoclustes and modified Mn-doped Fe3O4@MoS2 nanoflowers suspended in water was also carried out by DLS measurement (see Supporting information, Figure S2). Compared with Mn-doped Fe3O4@MoS2 nanoflowers, Mn-doped Fe3O4@MoS2@CS exhibits an excellent dispersibility state in deionized water.

Figure 2. (a) XRD patterns and (b) Raman spectra of Mn-doped Fe3O4 nanoclusters and Mn-doped Fe3O4@MoS2 nanoflowers.

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The crystalline structures and phase composition of the as-synthesized Mn-doped Fe3O4 nanoclusters and Mn-doped Fe3O4@MoS2 nanoflowers were determined by XRD (Figure 2a). The diffraction peaks of Mn-doped Fe3O4 nanoclusters were in good agreement with the face centered cubic structure of magnetite Fe3O4 (JCPDS No. 19-0629) and MnFe2O4 (JCPDS No. 10-0319). The diffraction peaks of the Mn-doped Fe3O4 nanoclusters matched well with those for magnetite, probably due to the low Mn2+ doping amount, albeit with abroadening of the diffraction peaks, indicating the small grain size of the nanocomposites.48 However, two new diffraction peaks emerged, at 32.9° and 58.8°, corresponding to the (100) and (110) planes of pristine 2H-MoS2 (JCPDF No. 37-1492) of Mn-doped Fe3O4@MoS2 nanoflowers. It is noteworthy that all of the peaks for Mn-doped Fe3O4 were significantly weakened following MoS2 nanosheet decorating, suggesting that Mn-doped Fe3O4 nanoclusters were shielded by the MoS2 nanosheets.49 These results indicate the good stability of the MoS2 nanosheets and their successful attachment on the Mn-doped Fe3O4 nanoclusters.

The crystal phase of the Mn-doped Fe3O4 nanoclusters and Mn-doped Fe3O4@MoS2 nanoflowers was also confirmed with Raman spectroscopy (Figure 2b). The Raman spectrum of the as synthesized Mn-doped Fe3O4 nanoclusters exhibited three bands at 360, 500, and 700 cm–1, roughly corresponding to the Eg, T2g, and A1g modes of Fe3O4, respectively. This minor shift in peak position may be attributed to the low amount of Mn2+ in the doped Fe3O4. The Raman spectrum of MoS2 has bands at 368 and 403 cm–1, corresponding to the  and A1g modes, respectively. 10 ACS Paragon Plus Environment

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Bioconjugate Chemistry

Interestingly, Raman peaks observed at 298, 630, and 684 cm–1 could not be indexed to MoS2 or Fe3O4, but could be ascribed to the formation of Mo-O, Mo-O2, and Mo-O3 bonds, thus suggesting the formation of heterojunctions between MoS2 and Mn-doped Fe3O4.50 Clearly, the peak strengths of Mn-doped Fe3O4@MoS2 are weakened compared to those of Mn-doped Fe3O4, indicating that the lateral dimensions of these layers are in the nanoregion and the occurrence of phonon confinement. Additionally, the obvious red-shift of the Raman peaks for Mn-doped Fe3O4@MoS2 further implies the existence of a multilayer structure.51 The elemental composition and distribution of the Mn-doped Fe3O4@MoS2 nanoflowers was further qualitatively investigated by dark-field SEM and corresponding elemental mapping (see Supporting information, Figure S3). An overlap of Fe, Mn, and O element signals with those of Mo and S was observed, further indicating that the Mn-doped Fe3O4 nanoclusters are located at the core of the flower-like MoS2 nanosheets. However, compared to the strength of the S and Mo signals, those of Fe, O, and Mn peaks were much weaker, mainly because of the shielding of MoS2 and the low Mn2+ doping amount.

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Figure 3. (a) XPS survey spectrum of Mn-doped Fe3O4@MoS2 nanoflowers: (b) Mo, (c) S, (d) Fe, (e) Mn, and (f) O elements, together with their corresponding fitting curves (the fitting curves were marked with the dotted lines).

XPS analysis provided further details on the chemical state of Mn-doped Fe3O4@MoS2 (Figure 3a–f). A survey XPS plot of the Mn-doped Fe3O4@MoS2 exhibited peaks for Mo, S, Fe, Mn, and O elements (Figure 3a). Three XPS peaks at 229.2, 232.8, and 235.7 eV were assigned to the Mo3d5/2 (Mo4+), Mo3d3/2 (Mo4+), and Mo3d5/2 (Mo6+) states, respectively, and the lower energy peak at ~226.1 eV to the S2s orbital signal (Figure 3b). Thus, Mo is present mainly in the Mo4+ state and binds to S2–. The two peaks at 161.5 and 163.0 eV were assigned to the 2p3/2 and 2p1/2 of

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divalent sulfide ions (S2–), and a weak peak at 169.3 eV was assigned to the 2p3/2 of hexavalent sulfide atom in sulfate (SO42–) (Figure 3c). The above results further indicate the successful growth of MoS2 on Mn-doped Fe3O4. Additionally, some Mo5+ and SO42– were present as impurities developed during the hydrothermal process, in agreement with previous findings.52 The XPS curve of Fe2p, with a peak at 710.7 eV, shows the typical peak of Fe3O4 (Figure 3d). The two fitted curves with peaks at 708.6 and 709.8 eV demonstrate that a new compound, which is composed of ferric and ferrous iron and divalent sulfur, was formed on the interface of Fe3O4 and MoS2. With regards to the XPS curve of Fe2p, the peaks at 711.3, 712.4, and 713.6 eV were assigned to Fe3+ and those at 709.3 and 710.4 eV were assigned to Fe2+. These are the characteristic peaks of Fe2p3/2 in Fe3O4, suggesting the coexistence of Fe2+ and Fe3+. The peaks assigned to Mn 2p1/2 and Mn 2p3/2 were detected at approximately 653.6 and 642.4 eV (Figure 3e), demonstrating the presence of the Mn2+ state in the synthesized Mn-doped Fe3O4.53 The peaks at 530.3 and 531.6 eV in the O1s curve were attributed to the lattice oxygen of Fe3O4 and the hydroxyl group originating from the surface modification, respectively (Figure 3f). Thus, XRD, Raman, and EDX characterization results confirmed the sucessful synthesis of Mn-doped Fe3O4@MoS2 nanoflowers.

The surface functional groups of the Mn-doped Fe3O4@MoS2 nanoflowers were characterized by FT-IR spectra (see Supporting information, Figure S4). The FT-IR spectrum of PSSMA-stabilized Mn-doped Fe3O4@MoS2 exhibited the characteristic sulfonate and carboxylate absorption bands of PSSMA. The bands at 1190 and 1125 13 ACS Paragon Plus Environment

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cm–1 were assigned to the asymmetric stretching vibrations and those at 1040 and 1010 cm–1 to the symmetric stretching vibrations of the sulfonate groups, respectively. The typical carboxylate group bands were observed at 1620 and 1410 cm–1. The peaks at 540–590 cm–1 were assigned to Mn-doped Fe3O4, belonging to the characteristic Mn-O and Fe-O stretching vibrations bands. Mn-doped Fe3O4@MoS2 displayed characteristic absorption bands at approximately 626 and 1080 cm–1, ascribed to the Mo-S bands, in good agreement with the results of XPS, as well as a new peak at 761 cm–1, assigned to Mo–O vibrations due to the presence of manganese oxide. 54 These results indicate the formation of a Mn-doped Fe3O4 layered MoS2 composite. The functionalization of Mn-doped Fe3O4@MoS2 nanoflowers with CS and MET was also elucidated by FT-IR. The peak appearing at 3416 cm–1 was assigned to the stretching vibration of the N-H bond. Both the -CH2- stretching vibration (2863–2946 cm–1) and the internal vibration of the amide bonds (1385–1640 cm–1) of CS molecules were also observed in the FT-IR of Mn-doped Fe3O4@MoS2@CS. These results indicate that the CS polymer was successfully attached onto the surface of Mn-doped Fe3O4@MoS2 nanoflowers by an electrostatic interaction. For Mn-doped Fe3O4@MoS2@CS@MET, the signature peaks of C=N stretching at 1624 cm–1 and N-H stretching at 3418 cm–1 were intensified, indicating the successful loading of MET on Mn-doped Fe3O4@MoS2@CS nanoflowers. ζ potential measurements were performed to evaluate the surface charge of the Mn-doped Fe3O4 nanoclusters and the modified Mn-doped Fe3O4@MoS2 nanoflowers

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Bioconjugate Chemistry

suspended in deionized water (see Supporting information, Figure S5). The Mn-doped Fe3O4 nanoclusters showed a negative potential of –25.9 mV due to the presence of negatively charged carboxyl groups. However, following the coating by MoS2, the Mn-doped Fe3O4@MoS2 nanoflowers showed a slightly increased ζ potential of –21.9 mV. Subsequently, the ζ potential showed a dramatic increase to 24.7 mV following CS modification due to the positively charged amino groups on CS. Finally, the loading of MET hydrochloride decreased the ζ potential to 15.4 mV, due to the molecule’s negative charge. The surface charge of nanomaterials greatly affects their dispersion in water through the underlying electronic interactions.27 Herein, all the nanomaterials stages were highly stable without any precipitation within 5 days being observed in deionized water. However, the Mn-doped Fe3O4@MoS2 nanoflowers inevitably aggregated and precipitated in PBS solution because of the electron screen effect. As expected, Mn-doped Fe3O4@MoS2@CS and Mn-doped Fe3O4@MoS2@CS@MET could well dispersed in both deionized water and PBS, and could be stored for at least 5 days (see Supporting information, Figure S6). Therefore, CS functionalization can effectively ensure the physiologic stability of nanoflowers.

The UV-vis spectra of as-grown nanomaterials were also investigated (see Supporting information, Figure S7). The absorption spectrum of Mn-doped Fe3O4 nanoclusters showed strong absorption in the UV region but low absorption in the visible region. Following MoS2 nanosheets coating, the nanoflowers showed a strong absorption in the complete UV–vis range. In our experiment, in particularly, 15 ACS Paragon Plus Environment

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CS-functionalized nanoflowers showed nearly 2-fold NIR absorbance compared to Mn-doped Fe3O4@MoS2 nanoflowers. The result indicated the presence of CS not only improves the biocompatibility and stability of Mn-doped Fe3O4@MoS2 nanoflowers but also increases the NIR absorption efficiency by preventing the rapid reaggregation driven by the large surface energy of MoS2 nanosheets, suggesting that CS functionalization may improve the NIR photothermal effect. Finally, a new peak at

233

nm,

originating

from

MET,

was

observed

for

Mn-doped

Fe3O4@MoS2@CS@MET, indicating the successful loading of MET onto the nanoflowers.

Figure 4. Photothermal properties of nanoflowers. (a) Photothermal conversion curves of pure water and nanomaterial dispersions (100 µg·mL–1) exposed to an 808 nm laser at 1.5 W·cm–2. (b) Temperature heating curves of Mn-doped Fe3O4@MoS2@CS dispersions at various concentrations (25, 50, 100 and 200 µg·mL–1) under 808 nm laser irradiation at a power density of 1.5 W·cm–2. (c)

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Photothermal heating curves of Mn-doped Fe3O4@MoS2@CS solution at 200 µg·mL–1concentration under 808 nm laser irradiation at various power densities (0.5, 1.0, and 1.5 W·cm–2) as a function of irradiation time. (d) Temperature variations of Mn-doped Fe3O4@MoS2@CS solution at concentration of 100 µg·mL under continuous 808 nm laser irradiation for four on–off cycles.

The photothermal properties of the nanoflowers were assessed to verify their hyperthermic capacity (Figure 4). Water and the Mn-doped Fe3O4 aqueous dispersion showed a negligible photothermal phenomenon due to their lack of absorbance in the NIR region. Conversely, due to the excellent photothermal properties of MoS2 nanosheets, all the synthesized nanoflowers showed an obvious increase in temperature within 8 min (Figure 4a). It is worth noting that the Mn-doped Fe3O4@MoS2@CS nanoflowers exhibited a superior photothermal conversion capacity than that of Mn-doped Fe3O4@MoS2. After modified with CS, the water-dispersibility of Mn-doped Fe3O4@MoS2 was improved and could expose more photothermal conversion spots, which can increases the NIR absorption efficiency to greatly improve the NIR photothermal effect in biomedical applications.55,

56

The

chitosan-functionalized Mn-doped Fe3O4@MoS2 nanoflowers can be engineered as a highly effective NIR stimuli-responsive system for simultaneous chemo-photothermal therapy. In addition, the photothermal conversion capacity of Mn-doped Fe3O4@MoS2@CS displayed obvious concentration- and laser power-dependent effects (Figure 4b, c). These results are not unexpected as the nanoflowers can harvest the NIR photons more effectively at higher concentrations or under higher laser power densities and convert these to environmental heat. 17 ACS Paragon Plus Environment

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A further prerequisite of an effective photothermal agent is the stability of its photothermal effect. In order to assess this, we monitored the temperature of Mn-doped Fe3O4@MoS2@CS aqueous solutions under NIR irradiation on–off cycles (Figure 4d). The Mn-doped Fe3O4@MoS2@CS retained its excellent photothermal effect without any perceivable attenuation following exposure to NIR irradiation even after four cycles. Thus, the resulting Mn-doped Fe3O4@MoS2 nanoflowers are highly suitable for NIR laser-driven photothermal therapy. Because the unsatisfactory biodegradability of inorganic nanomaterials severely clouds their biosafety and biomedical applications, the biodegradable capability of nanomaterials appears particular prominent.57-60 The degradability of Mn-doped Fe3O4@MoS2 nanoflowers in cultures with different pH was investigated (see Supporting Information Figure S8). Mn-doped Fe3O4@MoS2 nanoflower dispersion gradually changed from black to colourless within 5 days at pH 7.4 cultures, while the dispersion at pH 5.5 was slight loss of color. TEM images gave further evidances that the MoS2 nanosheets were markedly degraded at pH 7.4, but changed little at pH 5.5. Meanwhile, a fact particularly worthy of mention was that both dispersion at pH 7.4 and 5.5 appeared no precipitates even after degradation for 5 days, indicating that the degradation products of Mn-doped Fe3O4@MoS2 nanoflowers was highly soluble in cultures. The MoS2 nanosheets might be easily oxidized into water-soluble Mo(VI)-oxide species (e.g., MoO42-) at pH 7.4.57, 60 The pH-dependent degradation rate would favor its application for cancer therapy, we also expected that the soluble degradation products of Mn-doped Fe3O4@MoS2 nanoflowers could alleviate its 18 ACS Paragon Plus Environment

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Bioconjugate Chemistry

redundant retention and toxicity in vivo, thus making it more promising for biomedical application.

Figure 5. (a) Magnetization loops of Mn-doped Fe3O4 and Mn-doped Fe3O4@MoS2@CS. Inset is a

photo of Mn-doped Fe3O4@MoS2@CS solution placed near a magnet. (b, c) In vitro T1/T2 -weighted MRI and plot of relaxation rates for (d) 1/T1 (r1) and (e) 1/T2 (r2) at varying manganese and iron concentrations in Mn-doped Fe3O4@MoS2@CS.

The saturation magnetization (Ms) values of the Mn-doped Fe3O4 nanoclusters and Mn-doped Fe3O4@MoS2@CS nanoflowers were 51.64 and 35.36 emu·g–1, respectively (Figure 5a). The decreased Ms of Mn-doped Fe3O4@MoS2@CS was attributed to the increased mass of the nanoclusters following the introduction of MoS2 and CS. The two samples were essentially superparamagnetic, with a negligible hysteresis effect. Thus, the strong magnetization and superparamagnetic properties of the nanoflowers allow their facile magnetic manipulation (inset, Figure 5a). The nanoflowers maintained a stable dispersion in deionized water and exhibited a rapid 19 ACS Paragon Plus Environment

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magnetic response under an externally applied magnetic field, while the solids were re-dispersed by mechanical shaking immediately once the magnetic field was removed. Thus, their superparamagnetic properties and high magnetization indicate that the Mn-doped Fe3O4@MoS2@CS nanoflowers have promising potential as an MRI contrast agent for biomedical applications.

Multimodality biomedical imaging techniques play a key role in the diagnostic field. T1 + T2 dual-contrast agents require high r1 and r2 simultaneously, and r2 should be not significantly larger than r1. Thus, the T1- and T2-weighted MRI performance of Mn-doped Fe3O4@MoS2@CS nanoflowers as T1 + T2 dual-contrast agents at different manganese and iron concentrations were investigated (Figure 5b, c). Increasing the manganese or iron concentration of Mn-doped Fe3O4@MoS2@CS nanoflowers led to a large increase in signal intensity in the T1-weighted MR image, while the signal intensity in the T2-weighted MR image was dramatically reduced. The Mn-doped Fe3O4@MoS2@CS nanoflowers were also employed to investigate the r1 and r2 values, calculated from a fitting curve of the transverse relaxation (1/T1, r1) and longitudinal relaxation (1/T2, r2) rates as a function of the manganese and iron concentrations (Fig 5d, e). The r1 and r2 value were calculated to be 18.46 and 63.75 mM–1·s–1, respectively. The good MRI performance of the Mn-doped Fe3O4@MoS2@CS nanoflowers demonstrates their promise as T1/T2 dual-contrast agents (see Supporting information, Table S1).

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Bioconjugate Chemistry

Figure 6. (a) The absorption intensity (at 233 nm) as a function of MET concentration (0 – 20 µg·mL– 1

). (b) The loading ratio of MET to Mn-doped Fe3O4@MoS2@CS as a function of MET concentration

(at pH 8.00, 7.4 and 5.5). (c) In vitro release profiles of Mn-doped Fe3O4@MoS2@CS@MET at different pH values (pH 7.4 and 5.5). (d) Release profiles of MET from Mn-doped Fe3O4@MoS2@CS at pH 5.5 with or without 808 nm NIR laser irradiation. Arrows indicate the time points at which NIR irradiation was applied (1.5 W·cm–2, 8 min).

The characteristics of MET loading and

release from the Mn-doped

Fe3O4@MoS2@CS nanoflowers were investigated (Figure 6). Before the in vitro MET release test, the drug loading content of Mn-doped Fe3O4@MoS2@CS@MET was determined by using the calibration of the absorbance at 233 nm (Figure 6a). The maximum loading ratios gradually increases with the increase of pH values and reaches up to 30.2 % when the added MET concentration reached 0.9 mg·mL–1 at pH 8.00 (Figure 6b), such a high MET loading ratio may have resulted from the ultra-high surface area and the wrinkled surface of the nanoflowers. Interestingly, the release of MET was pH dependent, with the release rate increasing with the decrease 21 ACS Paragon Plus Environment

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in pH. At pH 7.4, only 5.9 % of MET was released over 55 h. Conversely, a much faster release was observed at pH 5.5, reaching a 78.0 % release within the first 55 h (Figure 6c). The effective release of MET in a slightly acidic solution is likely due to the increased hydrophilicity of guanido groups and the higher solubility of MET in acidic

environments.

Therefore,

the

hybrid

Mn-doped

Fe3O4@MoS2@CS

nanoflowers hold great potential for pH-responsive drug delivery, which would be beneficial for enhanced anticancer efficiency. We also examined the effect of the photothermal process on the release of MET (Figure 6d). Remarkably, MET release at pH 5.5 showed a sudden increase when Mn-doped Fe3O4@MoS2@CS@MET was irradiated under 808 nm laser for 8 min, the accumulated release of MET approached 15.0 % after five irradiation cycles compared to that of MET released at pH 5.5 without irradiation. Therefore, the stimulation of NIR light was able to obviously trigger MET release from Mn-doped Fe3O4@MoS2@CS. Indeed, it is well documented that heat promotes drug release.44, 61

Thus, MET is bound on the nanoflower surfaces under normal physiological

conditions (pH 7.4), and released under NIR irradiation or at a reduced pH typical of the micro-environments of intracellular lysosomes or endosomes as well as cancerous tissue (pH 5.5). Such an in-built mechanism for selective drug release with an obvious response for NIR irradiation and acidic environment is a major advantage of our proposed system.

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Figure 7. Cell viability of (a) LO2 and (b) Hep3B cells treated with Mn-doped Fe3O4@MoS2 and

Mn-doped Fe3O4@MoS2@CS at different concentrations (20, 50, 100, 200 µg·mL–1) for 24 and 48 h.

Biocompatibility is an essential concern with regards to the development of nanomaterials for biomedical applications. LO2 and Hep3B cells treated with nanoflowers were shown to be more than 90 % viable as measured with the WST-1 assay (Figure 7). Thus, the prepared nanoflowers had a very limited toxicity at the tested concentrations, even as high as 200 µg·mL–1 for 48 h. Though MoS2 nanosheets might be degraded for a long period, no appreciable cytotoxicity was detected because as the cell viabilities of LO2 and Hep3B cells were still higher than 85 % after exposed to 200 µg·mL–1 of nanoflowers for 5 days, (see Supporting information, Figure S9). Further, the Mn-doped Fe3O4@MoS2@CS nanoflowers showed an improved biocompatibility compared to bare Mn-doped Fe3O4@MoS2, demonstrating the significance of the CS functionalization process. The results indicate that the Mn-doped Fe3O4@MoS2@CS had low cytotoxicity, and thus may be used as a biocompatible nanomaterial for cancer diagnosis and treatment.

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Figure 8. (a) Combination chemotherapy and photothermal therapy on LO2 and Hep3B cells following

incubation with chemotherapeutic (Mn-doped Fe3O4@MoS2@CS@MET), photothermal (Mn-doped Fe3O4@MoS2@CS + NIR) and chemo-photothermal therapeutic (Mn-doped Fe3O4@MoS2@CS@MET + NIR; equal to concentration of MET = 16.5 µg·mL–1) nanoflowers at 100 µg·mL–1 for 24 h with 808 nm laser irradiation at 1.5 W cm–2 for 8 min. (b–e) AO/EB staining of Hep3B cells with different treatments: (b) control, (c) Mn-doped Fe3O4@MoS2@CS@MET,(d) Mn-doped Fe3O4@MoS2@CS + NIR, and (e) Mn-doped Fe3O4@MoS2@CS@MET + NIR laser.

To demonstrate the highly sensitive inhibition and killing ability of the constructed nanoplatform for liver cancer, LO2 cells and Hep3B cells were chosen as the normal and cancer cell models, respectively (Figure 8a). A negligible cytotoxicity of both Mn-doped Fe3O4@MoS2@CS and Mn-doped Fe3O4@MoS2@CS@MET for LO2 cells was observed, even following 808 nm laser irradiation at 1.5 W·cm–2 for 8 min. Conversely, the cell viability of Hep3B cells showed a significant downturn under the same conditions. Therefore, Mn-doped Fe3O4@MoS2@CS@MET was able to highly sensitively inhibit and kill hepatoma cells with negligible side effects.

MET is a first-line therapeutic drug for type II diabetes mellitus, and has recently been identified as a potential and attractive anticancer drug for many tumors at large 24 ACS Paragon Plus Environment

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doses.30 Indeed, Mn-doped Fe3O4@MoS2@CS@MET showed a certain toxicity to Hep3B cells at a relatively low dose (ca. 16.5 µg·mL–1), indicating that the nanoflowers could effectively deliver MET into cancer cells. The cell-killing efficiency of Mn-doped Fe3O4@MoS2@CS with the assistance of NIR irradiation was much better than chemotherapy alone, suggesting that Mn-doped Fe3O4@MoS2@CS have excellent photothermal properties. It is not unexpected that hyperthermia can effectively and selectively kill cancer cells, as tumor cells are more sensitive to heat than healthy cells. Perhaps not unexpectedly, chemo-photothermal therapy exhibited the highest cancer cell killing efficiency. On the one hand, the CS-functionalized Mn-doped Fe3O4@MoS2 nanoflowers showed better photothermal properties compared to bare Mn-doped Fe3O4@MoS2 nanoflowers. On the other hand, the photothermal effect was able to accelerate the release of MET and sensitize the cells to chemotherapy. This synergistic effect of photothermal-chemotherapy leads to a much improved treatment effect. The synergistic therapeutic efficiency of Mn-doped Fe3O4@MoS2@CS@MET on Hep3B cells was further visually valuated by AO/EB double staining assay (Figure 8b–e). AO is a weak base dye able to readily penetrate through the cell membrane of both normal and dead cells, while EB is only absorbed by dead cells with damaged membranes leading to a bright red color.62 Thus AO/EB staining results in a green and orange color in the normal and dead cells, respectively. As expected, the control cells without any treatment were all green (Figure 8b). In sharp contrast, compared with 25 ACS Paragon Plus Environment

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the chemo-treatment only group (Figure 8c), a higher rate of cell death was observed in the NIR-irradiated photothermal groups (Figure 8d, e); in particular, Mn-doped Fe3O4@MoS2@CS@MET-treated cells showed a better synergistic effect than chemotherapy or photothermal therapy treatment alone. The results are in good agreement with the WST-1 assay results.

CONCLUSIONS In

summary,

a

highly

effective

multimodal

theranostic

nanoplatform,

Fe3O4@MoS2@CS@MET nanoflowers, has been successfully developed to overcome the limitations of traditional cancer diagnosis and treatment, including false diagnosis, significant side effects, and drug resistance. The nanoflowers were prepared through a simple two-step solvothermal microwave-assisted strategy followed by CS and MET functionalization. The surface functionalized CS was able not only to improve the dispersibility of Mn-doped Fe3O4@MoS2 nanoflowers in bio-fluids and increase the biocompatibility, but also to significantly enhance the photothermal effect. Remarkably, MET release was responsive to NIR irradiation and an acidic environment, providing an in-built mechanism for selective drug release. The synergistic chemo-photothermal therapy exhibited much better cancer cell killing efficiency than that of chemotherapy or photothermal therapies alone. Further, the precisely designed nanoflowers showed effective contrast for T1/T2 MRI and highly selective chemo-photothermal attack on hepatoma cells, but a negligible effect in

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normal cells. Thus, the developed system holds great promise for accurate, multimodal MR imaging-guided, and highly sensitive therapy of liver cancer.

EXPERIMENTAL SECTION Materials. All the chemical reagents used herein were of analytical grade and used without further purification. Ferric chloride hexahydrate (FeCl3·6H2O), manganese chloride tetrahydrate (MnCl2.4H2O), and sodium acetate trihydrate (C2H3NaO2·3H2O, NaAc·3H2O) were purchased from Guangdong GuangHua Technology Co., Ltd. Poly(4-styrenesulfonic acid-co-maleic acid) sodium salt (PSSMA; PSS:MA = 3:1, Mw = 20,000), ammonium molybdate tetrahydrate ((NH4)6Mo7O24·4H2O), thiourea (CN2H4S), ethylene glycol, MET, agarose, acridine orange (AO), and ethidium bromide (EB) were purchased from Aladdin Chemical Reagent Co., Ltd. Fetal bovine serum, Dulbecco’s Modified Eagle’s medium (DMEM), and CS were purchased from Sinopharm Chemical Reagent Co., Ltd. Ultrapure water (18.2 MΩ·cm–1) was obtained from a Millipore Milli-Q purification system and was used in all the experiments. Preparation of Mn-doped Fe3O4 nanoclusters. A multi-mode microwave parallel synthesis system (XH-800S, China) at a frequency of 2.45 GHz was used to synthesize the Mn-doped Fe3O4 nanoclusters.63 In general, PSSMA (1.0 g) was dissolved in ethylene glycol (40 mL) under magnetic stirring to form a clear solution, followed by the addition of FeCl3·6H2O (0.81 g, 3.0 mmol), MnCl2.4H2O (0.2970 g, 1.5 mmol), and sodium acetate (4.2 g). The obtained homogeneous red brown solution was then sealed in a 50 mL microwave reaction kettle and heated at 200 °C for 2 h. 27 ACS Paragon Plus Environment

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After cooling to room temperature, the precipitates were isolated with a magnet, washed several times with deionized water, and dried under vacuum at 60 °C for 24 h. Preparation of Mn-doped Fe3O4@MoS2 nanoflowers. Mn-doped Fe3O4@MoS2 nanoflowers were prepared according to a similar microwave-assisted solvothermal procedure to that of Mn-doped Fe3O4 nanoclusters. Briefly, (NH4)6Mo7O24·4H2O (1.05 g, 0.84 mmol) and thiourea (2.28 g, 30 mmol) were dissolved in 40 mL of distilled water by ultrasound to form a homogeneous solution. Mn-doped Fe3O4 nanoclusters (80 mg) were then added into the solution and dispersed by ultrasound for a further 30 min. The mixture was then transferred into a 50 mL microwave reaction kettle and heated at 180 °C for 1.5 h. Thereafter, the reaction system was allowed to naturally cool to room temperature. The final, dark precipitates were collected by magnetic separation, washed several times with deionized water, and dried at 60 °C under vacuum for characterization. Preparation of CS-functionalized nanoflowers. To improve the physiologic stability and biocompatibility of the prepared nanoflowers, CS was functionalized on the surface of Mn-doped Fe3O4@MoS2. An aqueous solution (10 mL) of Mn-doped Fe3O4@MoS2 nanoflowers (1 mg·mL–1) was firstly sonicated in an ice bath for 0.5 h. Then, 10 mL of a CS solution (0.05 wt%) was added into the mixture, followed by mechanical stirring and sonication using a sonic water-bath at room temperature for 24 h. The resulting hybrid was repeatedly centrifuged at 10,000 rpm for 10 min to remove the unbound CS and washed with deionized water several times. The

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Bioconjugate Chemistry

as-prepared Mn-doped Fe3O4@MoS2@CS nanoflowers were re-dispersed in deionized water and stored at 4 °C for further use. MET loading onto and release from the nanoflowers. MET at different concentrations was loaded onto Mn-doped Fe3O4@MoS2@CS nanoflowers under different pH values (pH 5.5, 7.0, and 8.0) at constant stirring and room temperature for 24 h. Any unbound MET was removed by repeated centrifugation at 10,000 rpm for 10 min. The resulting Mn-doped Fe3O4@MoS2@CS@MET solution (2 mg·mL–1) was stored at 4 °C. The supernatant and the washing solutions were collected together and the amount of non-loaded MET was determined by UV-Vis spectra at a wavelength of 233 nm. The MET loading ratio was then calculated using the following equation:

Loading ratio %=

Weight of loaded MET ×100% (1) Weight of Mn-doped Fe O @MoS2@CS

For in vitro MET release, Mn-doped Fe3O4@MoS2@CS@MET nanoflowers (2 mg) were immersed in 10 mL of PBS buffer at pH 5.5 and pH 7.4 at 37 °C, and shaken slightly. At predetermined time intervals, 4 mL aliquots were withdrawn for analysis and an equivalent aliquot of fresh buffer solution was added back. The supernatant was then diluted by corresponding buffer solution for analysis. The amount of released MET was quantified by UV-Vis spectrometry. The reported values were averaged from three measurements.

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To assess the photothermal-triggered drug release from the Mn-doped Fe3O4@MoS2@CS@MET nanoflowers, the samples were dispersed in 10 mL PBS buffer at pH 5.5. At certain time intervals, the solution was irradiated with an 808 nm NIR laser for 8 min. Before and after NIR irradiation, 4 mL aliquots were retrieved and centrifuged to remove any nanoflowers present; a 4 mL aliquot of fresh buffer solution was then replaced. The supernatant was then diluted by corresponding buffer solution for analysis. Measurement of photothermal performance. To evaluate the photothermal effects of the nanoflowers and verify their hyperthermic capacity, 1 mL of the aqueous suspensions were placed in aquartz cuvette (45×12.5×12.5 mm) and the temperature changes were recorded once per second when an 808 nm NIR laser was oriented perpendicular to the cuvette at a distance of 10 cm. Measurement of T1- and T2- weighted imaging. The T1/2 relaxivities (r1/2) were deduced by fitting inverse relaxation times (1/T1/2) as a function of the manganese and iron concentrations. MRI was performed at a magnetic field strength of 0.52 ± 0.05 T using a head coil with T1-weighted (TR = 500 ms, TE = 12 ms, NE = 16, average = 2, FOV= 100×100 mm, slice thickness = 4.0 mm) and T2-weighted (TR = 2000 ms, TE = 20 ms, NE = 16, average = 2, FOV = 100×100 mm, slice thickness = 4.0 mm) spin echo sequences. The samples were suspended in a water solution containing 2 % agarose in plastic vials at various concentrations and placed in a tank. Degradation

Behavior

of

Mn-doped

Fe3O4@MoS2

nanoflowers.

The

degradation behavior of as-prepared hybrid nanoflowers was investigated by 30 ACS Paragon Plus Environment

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incubating Mn-doped Fe3O4@MoS2@CS in cultures with different pH (7.4 and 5.5, respectively) at 37 °C in shaker. In detail, 4 mL of Mn-doped Fe3O4@MoS2@CS nanoflowers culture dispersions with different pH values (5.5 and 7.4) were sealed in Eppendorf tube and maintained at 37 °C shaker with shaking speed of 150 rpm. At specific time points, photographs of dispersions were captured by a digital camera. Also, two kinds of Mn-doped Fe3O4@MoS2@CS colloidal dispersions were diluted by DI-water and dropped on copper grid for TEM observation. Cytotoxicity assay. LO2 cells (human hepatic cell line) and Hep3B cells (human hepatic carcinoma cell line) were obtained from ATCC (Manassas, VA, USA), and incubated according to a previous method.34 Briefly, LO2 and Hep3B cells were grown in DMEM supplemented with 25 mM glucose, 10% fetal bovine serum, 100 U mL–1 penicillin G sodium, and 100 mg mL–1 streptomycin sulfate in 10 cm2 plates at 37 °C in 5 % CO2. For incubation with nanoflowers, cells were seeded overnight at 37 °C in 96-well plates at a density of 5 × 104 cells per well to allow attachment. Nanoflower solutions in fetal bovine serum-free DMEM at varying concentrations (0, 10, 25, 50, 100 and 200 µg·mL-1) were added and the cells were incubated for a further 24 or 48 h. The nanoflower solutions were then discarded and the cells were washed with PBS and cultured in fresh medium. Cell viability was determined by the WST-1 method.61 The optical densities were read at 450 nm using a microplate spectrophotometer (Spectra Max 190, Molecular Devices). Cells cultured without nanoflowers were used as controls.

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Synergistic chemo-photothermal therapy. LO2 cells and Hep3B cells were cultured at a density of 5×103 cells per well in 96-well plates overnight to allow attachment. The cells were then seeded with Mn-doped Fe3O4@MoS2@CS or Mn-doped Fe3O4@MoS2@CS@MET solutions at a concentration of 100 µg·mL-1 for 24 h in duplicate batches. Each group of cells was irradiated with or without a NIR laser (808 nm, 1.5 W cm2) for 8 min; the second batches were not irradiated and served as controls. Following irradiation, the cells were rinsed three times with PBS and cell viability was assessed by WST-1 assay and AO/EB double staining assay.62 Characterization. High resolution-transmission electron microscopy (HR-TEM) was conducted on a JEM-2100 electron microscope at 200 kV (JEOL, Japan). TEM samples were prepared by depositing 5 µL of dilute solution on a copper grid (200-mesh) and then drying at ambient temperature prior to analysis. UV-Vis-NIR absorption spectra were obtained using a Lambda 35 UV-vis spectrophotometer (Perkin Elmer, USA). X-ray diffraction (XRD) patterns were collected on a Bruker D8 ADVANCE X-ray diffractometer using Cu−Kα radiation (λ=1.5418 Å) with an operating voltage of 40 kV and a current of 40 mA. Diffraction patterns were collected from 10° to 80° at a speed of 4°·min−1. X-ray photoelectron spectroscopy (XPS) measurements were performed on a Thermo Fisher ESCALAB-Xi+ spectrometer using a twin-anode Al/Mg Kα (1486.6 eV) X-ray source. All the spectra were calibrated to the binding energy of the adventitious C1s peak at 284.6 eV. Fourier-transform infrared (FT-IR) spectra were recorded on a Paragon 1000 (Perkin Elmer, USA) spectrometer. Samples were dried overnight at 50 ℃ under vacuum 32 ACS Paragon Plus Environment

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and thoroughly mixed and crushed with KBr to fabricate KBr pellets. Zeta (ζ) potentials were measured by a Nano ZS90 Zeta sizer (Malvern, UK). Raman spectra were measured using a Raman microscope equipped with an argon ion laser operating at 514 nm (Renishaw, UK). The magnetic properties of samples were measured on a MPMS-SQUID vibrating magnetometer with a physical property measurement system at 300 K in a magnetic field from –10,000 Oe to 10,000 Oe (MPMS3, USA). The photothermal properties of samples were measured using a NIR laser diode (808 nm, 1.18 W, spot size 0.75 cm2) and a digital thermometer (accuracy 0.1 °C, OMEGA Engineering Inc.). Longitudinal (T1) and transverse (T2) relaxation times were measured on a MiNiMR60-Analyst nuclear magnetic resonance analyzer (22 MHz, 0.52 ± 0.05 T) at 37 °C. An inversion–recovery pulse sequence was used to measure the longitudinal relaxation times and a Carr–Purcell–Meiboom–Gill spin echo sequence was used to measure transverse relaxation times. The iron and manganese concentrations were determined using Inductively Coupled Plasma Mass spectrometer (ICP-MS, NexION 350D, Perkin Elmer).

ASSOCIATED CONTENT

Supporting Information High-resolution TEM image of Mn-doped Fe3O4 nanoclusters and TEM image of MoS2 nanosheets; SEM image and corresponding EDX element mapping images of Mn-doped Fe3O4@MoS2 nanoflowers; Hydrodynamic diameters distribution, Zeta potentials, FT-IR spectra, dispersion stability and hydrodynamic size changes ,and 33 ACS Paragon Plus Environment

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

UV-vis

spectra

of

Mn-doped

Fe3O4,

Mn-doped

Page 34 of 40

Fe3O4@MoS2,

Mn-doped

Fe3O4@MoS2@CS, and Mn-doped Fe3O4@MoS2@CS-MET. Photographs and TEM images of Mn-doped Fe3O4@MoS2 nanoflowers dispersed in cultures with different pH (pH 5.5 and 7.4) at 37 °C for determined time intervals. Cell viability of LO2 and Hep3B cells treated with Mn-doped Fe3O4@MoS2 and Mn-doped Fe3O4@MoS2@CS at different concentrations for 72 and 120 h. Comparison of MR contrast effects of inorganic nanoparticle-based T1 and T1/T2 contrast agents.

AUTHOR INFORMATION

Corresponding Author * Corresponding author, Email: [email protected] (L. Meng).

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21474079, 21674085), the Program for New Century Excellent Talents in University (NCET-13-0453), and the Fundamental Funds for the Central Universities (08143101). We thank the International Center for Dielectric Research (ICDR) and the Instrumental Analysis Center (IAC) of Xi’an Jiaotong University for help with characterization. We also thank Miss Jiaxin Zhang for her help in preparing TOC Figure.

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REFERENCES (1) Siegel, R. L., Miller, K. D., and Jemal, A. (2016) Cancer statistics, 2016. CA Cancer J Clin. 66, 7-30. (2) Noone, A. M., Cronin, K. A., Altekruse, S. F., Howlader, N., Lewis, D. R., Petkov, V. I., and Penberthy, L. (2017) Cancer Incidence and Survival Trends by Subtype Using Data from the Surveillance Epidemiology and End Results Program, 1992–2013. Epidemiol Biomarkers Prev. 26, 632–641. (3) Iyer, A. K., Khaled, G., Fang, J., and Maeda, H. (2006) Exploiting the enhanced permeability and retention effect for tumor targeting. Drug Discov. Today. 11, 812-818. (4) Chen, G., Roy, I., Yang, C., and Prasad, P. N. (2016) Nanochemistry and Nanomedicine for Nanoparticle-based Diagnostics and Therapy. Chem. Rev. 116, 2826-2885. (5) Schwenzer, N. F., Springer, F., Schraml, C., Stefan, N., Machann, J., and Schick, F. (2009) Non-invasive assessment and quantification of liver steatosis by ultrasound, computed tomography and magnetic resonance. J. Hepatol. 51, 433-445. (6) Lyu, Y., Zhen, X., Miao, Y., and Pu, K. (2017) Reaction-Based Semiconducting Polymer Nanoprobes for Photoacoustic Imaging of Protein Sulfenic Acids. ACS Nano 11, 358-367. (7) Rocca, J. D., Liu, D., and Lin, W. (2011) Nanoscale Metal-Organic Frameworks for Biomedical Imaging and Drug Delivery. Accounts Chem Res. 44, 957-968. (8) Teng, Z., Wang, R., Zhou, Y., Kolios, M., Wang, Y., Zhang, N., Wang, Z., Zheng, Y., and Lu, G. (2017) A magnetic droplet vaporization approach using perfluorohexane-encapsulated magnetic mesoporous particles for ultrasound imaging and tumor ablation. Biomaterials. 134, 43-50. (9) Zerda, A. D. L., Bodapati, S., Teed, R., May, S. N. Y., Tabakman, S. M., Liu, Z., Yakub, B. T. K., Chen, X. Y., Dai, H. J, and Gambhir, S. J. S. (2012) Family of Enhanced Photoacoustic Imaging Agents for High-Sensitivity and Multiplexing Studies in Living Mice. ACS Nano 6, 4694-4701. (10) Medarova, Z., Pham, W., Farrar, C., Petkova, V., and Moore, A. (2007) In vivo imaging of siRNA delivery and silencing in tumors. Nat. Med. 13, 372-377. (11) Wilson, K., Homan, K., and Emelianov, S. (2012) Biomedical photoacoustics beyond thermal expansion using triggered nanodroplet vaporization for contrast-enhanced imaging. Nat Commun. 3, 618-627. (12) Yavuz, C. T., Mayo, J. T., Yu, W. W., Prakash, A., Falkner, J. C., Yean, S. j., Cong, L. L., Shipley, H. J., Kan, A., Tomson, M., et al. (2006) Low-Field Magnetic Separation of Monodisperse Fe3O4 Nanocrystals. Sicence 314, 964-967. (13) Hu, F., Macrenaris, K. W., Waters, E. A., Schultz-Sikma, E. A., Eckermann, A. L., and Meade, T. J. (2010) Highly dispersible, superparamagnetic magnetite nanoflowers for magnetic resonance imaging. Chem Commun. 46, 73-75. (14) Johnson, N. J. J., Oakden, W., Stanisz, G. J., Scott Prosser, R., and van Veggel, F. C. J. M. (2011) Size-Tunable, Ultrasmall NaGdF4 Nanoparticles: Insights 35 ACS Paragon Plus Environment

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

Page 36 of 40

into Their T1 MRI Contrast Enhancement. Chem Mater. 23, 3714-3722. (15) Zhang, Y., Lin, J. D., Vijayaragavan, V., Bhakoo, K. K., and Tan, T. T. (2012) Tuning sub-10 nm single-phase NaMnF3 nanocrystals as ultrasensitive hosts for pure intense fluorescence and excellent T1 magnetic resonance imaging. Chem Commun. 48, 10322-10324. (16) Li, Z., Yi, P. W., Sun, Q., Lei, H., Li, Z. H., Zhu, Z. H., Smith, S. C., Lan, M. B., and Lu, G. Q. M., (2012) Ultrasmall Water-Soluble and Biocompatible Magnetic Iron Oxide Nanoparticles as Positive and Negative Dual Contrast Agents. Adv Funct Mater. 22, 2387-2393. (17) Kim, B. H., Lee, N., Kim, H., An, K., Park, Y. I., Choi, Y., Shin, K., Lee, Y., Kwon, S. G., Na, H. B., et al. (2011) Large-scale synthesis of uniform and extremely small-sized iron oxide nanoparticles for high-resolution T1 magnetic resonance imaging contrast agents. J Am Chem Soc. 133, 12624-12631. (18) Hu, F.; and Zhao, Y. S. (2012) Inorganic nanoparticle-based T1 and T1/T2 magnetic resonance contrast probes. Nanoscale 4, 6235-6243. (19) Wong, P. T., and Choi, S. K. (2015) Mechanisms of drug release in nanotherapeutic delivery systems. Chem. Rev. 115, 3388-3432. (20) Allen, T. M., and Cullis, P. R. (2013) Liposomal drug delivery systems: from concept to clinical applications. Adv Drug Deliv Rev. 65, 36-48. (21) Singh, A., and Settleman, J. (2010) EMT, cancer stem cells and drug resistance: an emerging axis of evil in the war on cancer. Oncogene 29, 4741-4751. (22) Graham, G. G., Punt, J., Arora, M., Day, R. O., Doogue, M. P., Duong, J. K., Furlong, T. J., Greenfield, J. R., Greenup, L. C., Kirkpatrick, C. M., et al. (2011) Clinical pharmacokinetics of metformin. Clin Pharmacokinet. 50, 81-98. (23) Foretz, M., Guigas, B., Bertrand, L., Pollak, M., and Viollet, B. (2014) Metformin: from mechanisms of action to therapies. Cell Metab. 20, 953-966. (24) Pulito, C., Sanli, T., Rana, P., Muti, P., Blandino, G., and Strano, S. (2013) Metformin: On Ongoing Journey across Diabetes, Cancer Therapy and Prevention. Metabolites. 3, 1051-1075. (25) Dowling, R. J., Zakikhani, M., Fantus, I. G., Pollak, M., and Sonenberg, N. (2007) Metformin inhibits mammalian target of rapamycin-dependent translation initiation in breast cancer cells. Cancer Res. 67, 10804-10812. (26) Liu, H. Y., Scholz, C., Zang, C., Schefe, J. H., and Habbel, P. (2012) Metformin and the mTOR Inhibitor Everolimus (RAD001) Sensitize Breast Cancer Cells to the Cytotoxic Effect of Chemotherapeutic Drugs In Vitro. Anticancer Res. 32, 1627-1638. (27) Yoo, S. j., Hou, J., Yi, W. H., Li, Y. C., Chen, W. P., Meng, L. J., Si, J. H., and Hou, X. (2017) Enhanced Response of Metformin towards the Cancer Cells due to Synergism with Multi-walled Carbon Nanotubes in Photothermal Therapy. Sci Rep. 7, 1-9. (28) Martin-Castillo, B., Vazquez-Martin, A., Oliveras-Ferraros, C., and Menendez, J. A. (2010) Metformin and cancer: doses, mechanisms and the dandelion and hormetic phenomena. Cell Cycle. 9, 1057-1064. (29) Alimova, I. N., Liu, B., Fan, Z., Edgerton, S. M., Dillon, T., Lind, S. E., and 36 ACS Paragon Plus Environment

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

Bioconjugate Chemistry

Thor, A. D. (2009) Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro. Cell Cycle. 8, 909-915. (30) Xia, C., Chen, R., Chen, J., Qi, Q., Pan, Y., Du, L., Xiao, G., and Jiang, S. (2017) Combining metformin and nelfinavir exhibits synergistic effects against the growth of human cervical cancer cells and xenograft in nude mice. Sci Rep. 7, 43373-43384. (31) Snima, K. S., Jayakumar, R., and Lakshmanan, V. K. (2014) In vitro and in vivo biological evaluation of O-carboxymethyl chitosan encapsulated metformin nanoparticles for pancreatic cancer therapy. Pharm Res. 31, 3361-3370. (32) Lee, H., Park, H. J., Park, C. S., Oh, E. T., Choi, B. H., Williams, B., Lee, C. K., and Song, C. W. (2014) Response of breast cancer cells and cancer stem cells to metformin and hyperthermia alone or combined. PLoS One 9, e87979-e87989. (33) Song, C. W., Lee, H., Dings, R. P., Williams, B., Powers, J., Santos, T. D., Choi, B. H., and Park, H. J. (2012) Metformin kills and radiosensitizes cancer cells and preferentially kills cancer stem cells. Sci Rep. 2, 362-369. (34) Fasih, A., Elbaz, H. A., Huttemann, M., Konski, A. A., and Zielske, S. P. (2014) Radiosensitization of pancreatic cancer cells by metformin through the AMPK pathway. Radiat. Res. 182, 50-59. (35) He, C., Duan, X., Guo, N., Chan, C., Poon, C., Weichselbaum, R. R., and Lin, W. (2016) Core-shell nanoscale coordination polymers combine chemotherapy and photodynamic therapy to potentiate checkpoint blockade cancer immunotherapy. Nat Commun. 7, 12499-12511. (36) Cheng, L., Wang, C., Feng, L., Yang, K., and Liu, Z. (2014) Functional nanomaterials for phototherapies of cancer. Chem. Rev.114, 10869-10939. (37) Liu, Y., Yang, M., Zhang, J., Zhi, X., Li, C., Zhang, C., Pan, F., Wang, K., Yang, Y., Martinez de la Fuentea, J., et al. (2016) Human Induced Pluripotent Stem Cells for Tumor Targeted Delivery of Gold Nanorods and Enhanced Photothermal Therapy. ACS Nano 10, 2375-2385. (38) Zhang, Y., Cui, Z., Kong, H., Xia, K., Pan, L., Li, J., Sun, Y., Shi, J., Wang, L., Zhu, Y., et al. (2016) One-Shot Immunomodulatory Nanodiamond Agents for Cancer Immunotherapy. Adv Mater. 28, 2699-2708. (39) Robinson, J. T., Tabakman, S. M., Liang, Y., Wang, H., Sanchez Casalongue, H., Vinh, D., and Dai, H. (2011) Ultrasmall Reduced Graphene Oxide with High Near-Infrared Absorbance for Photothermal Therapy. J Am Chem Soc. 133, 6825-6831. (40) Cheng, L., Liu, J., Gu, X., Gong, H., Shi, X., Liu, T., Wang, C., Wang, X., Liu, G., Xing, H., et al. (2014) PEGylated WS2 nanosheets as a multifunctional theranostic agent for in vivo dual-modal CT/photoacoustic imaging guided photothermal therapy. Adv Mater. 26, 1886-1893. (41) Xie, H., Li, Z., Sun, Z., Shao, J., Yu, X. F., Guo, Z., Wang, J., Xiao, Q., Wang, H., Wang, Q. Q., et al. (2016) Metabolizable Ultrathin Bi2Se3 Nanosheets in Imaging-Guided Photothermal Therapy. Small 12, 4136-4145. (42) Lin, L. S., Cong, Z. X., Cao, J. B., Ke, K. M., Peng, Q. L., Gao, J. H., Yang, H. H., Liu, G., and Chen, X. Y. (2016) Multifunctional Fe3O4@Polydopamine Core 37 ACS Paragon Plus Environment

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

Page 38 of 40

Shell Nanocomposites for Intracellular mRNA Detection and Imaging-Guided Photothermal Therapy. ACS Nano 8, 3876-3883. (43) Chen, R., Wang, X., Yao, X., Zheng, X., Wang, J., and Jiang, X. (2013) Near-IR-triggered photothermal/photodynamic dual-modality therapy system via chitosan hybrid nanospheres. Biomaterials. 34, 8314-8322. (44) Liu, T., Wang, C., Gu, X., Gong, H., Cheng, L., Shi, X., Feng, L., Sun, B., and Liu, Z. (2014) Drug delivery with PEGylated MoS2 nano-sheets for combined photothermal and chemotherapy of cancer. Adv Mater. 26, 3433-3440. (45) Chou, S. S., Kaehr, B., Kim, J., Foley, B. M., De, M., Hopkins, P. E., Huang, J., Brinker, C. J., and Dravid, V. P. (2013) Chemically exfoliated MoS2 as near-infrared photothermal agents. Angew Chem Int Ed Engl. 52, 4160-4164. (46) Ge, J., Hu, Y., Biasini, M., Beyermann, W. P., and Yin, Y. (2007) Superparamagnetic magnetite colloidal nanocrystal clusters. Angew Chem Int Ed Engl. 46, 4342-4345. (47) Liu, J., Sun, Z., Deng, Y., Zou, Y., Li, C., Guo, X., Xiong, L., Gao, Y., Li, F., and Zhao, D. (2009) Highly water-dispersible biocompatible magnetite particles with low cytotoxicity stabilized by citrate groups. Angew Chem Int Ed Engl. 48, 5875-5879. (48) Yang, H., Zhang, C., Shi, X., Hu, H., Du, X., Fang, Y., Ma, Y., Wu, H., and Yang, S. (2010) Water-soluble superparamagnetic manganese ferrite nanoparticles for magnetic resonance imaging. Biomaterials. 31, 3667-3673. (49) Xie, J., Zhang, J., Li, S., Grote, F., Zhang, X., Zhang, H., Wang, R., Lei, Y., Pan, B., and Xie, Y. (2013) Controllable Disorder Engineering in Oxygen-Incorporated MoS2 Ultrathin Nanosheets for Efficient Hydrogen Evolution. J Am Chem Soc. 135, 17881-17888. (50) Zhang, X., Ding, P., Sun, Y., Wang, Y., Wu, Y., and Guo, J. (2017) Shell-core MoS2 nanosheets@Fe3O4 sphere heterostructure with exposed active edges for efficient electrocatalytic hydrogen production. J Alloys Compd. 715, 53-59. (51) Yu, J., Yin, W., Zheng, X., Tian, G., Zhang, X., Bao, T., Dong, X., Wang, Z., Gu, Z., Ma, X., et al. (2015) Smart MoS2/Fe3O4 Nanotheranostic for Magnetically Targeted Photothermal Therapy Guided by Magnetic Resonance/Photoacoustic Imaging. Theranostics 5, 931-945. (52) Liu, Y., Yu, Y.-X., and Zhang, W.-D. (2013) MoS2/CdS Heterojunction with High Photoelectrochemical Activity for H2 Evolution under Visible Light: The Role of MoS2. J Phys Chem C. 117, 12949-12957. (53) Cai, W., Lai, T., Dai, W., and Ye, J. (2014) A facile approach to fabricate flexible all-solid-state supercapacitors based on MnFe2O4/graphene hybrids. J Power Sources. 255, 170-178. (54) Zhang, Y., Chen, P., Wen, F., Yuan, B., and Wang, H. (2016) Fe3O4 nanospheres on MoS2 nanoflake: Electrocatalysis and detection of Cr (VI) and nitrite. J Electroanal Chem 761, 14-20. (55) Yin, W. Y., Yan, L., Yu, J., Tian, G., Zhou, L. J., Zheng, X. P., Zhang, X., Yong, Y., Li, J., Gu, Z. J., et al. (2014) High-Throughput Synthesis of SingleLayer MoS2 Nanosheets as a NearInfrared Photothermal-Triggered Drug Delivery for 38 ACS Paragon Plus Environment

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Bioconjugate Chemistry

Effective Cancer Therapy. ACS Nano 8, 6922-6933. (56) Guan, G. J., Zhang, S. Y., Liu, S. H., Cai, Y. Q., Low, M., Teng, C. P., Phang, I. Y., Cheng, Y., Duei, K. L., Srinivasan, B. M., et al. (2015) Protein Induces Layer-by-Layer Exfoliation of Transition Metal Dichalcogenides. J Am Chem Soc. 137, 6152-6155. (57) Hao, J. L., Song, G. S., Liu, T., Yi, X., Yang, K., Cheng, L., and Liu, Z. (2017) In Vivo Long-Term Biodistribution, Excretion, and Toxicology of PEGylated Transition-Metal Dichalcogenides MS2 (M = Mo, W, Ti) Nanosheets. Adv. Sci. 4, 1600160 -1600108. (58) Kurapati, R., Muzi, L., de Garibay, A. P. R., Russier, J., Voiry, D., Vacchi, I. A., Chhowalla, M., and Bianco, A. (2017) Enzymatic Biodegradability of Pristine and Functionalized Transition Metal Dichalcogenide MoS2 Nanosheets. Adv. Funct. Mater. 27, 1605176-1605187. (59) Domenico, C., Melissa, S., Valentina, C., Stefano, L., Giovanni, S., and Valerio, V. (2016) Biodegradable Passion Fruit-Like Nano-Architectures as Carriers for Cisplatin Prodrug. Part. Part. Syst. Charact. 33, 818-824. (60) Chen, Liang., Feng, Y. H., Zhou, X. J., Zhang, Q. Q., Nie, W., Wang, W. Z., Zhang, Y. Z., and He, C. L. (2017) One-Pot Synthesis of MoS2 Nanoflakes with Desirable Degradability for Photothermal Cancer Therapy. ACS Appl. Mater. Interfaces. 9, 17347−17358. (61) Meng, L., Zhang, X., Lu, Q., Fei, Z., and Dyson, P. J. (2012) Single walled carbon nanotubes as drug delivery vehicles: targeting doxorubicin to tumors. Biomaterials. 33, 1689-1697. (62) Kumar, C. S., Raja, M. D., Sundar, D. S., Antoniraj, M. G., and Ruckmani, K. (2015) Hyaluronic acid co-functionalized gold nanoparticle complex for the targeted delivery of metformin in the treatment of liver cancer (HepG2 cells). Carbohydr Polym. 128, 63-74. (63) Jing, X. N., Liu, T. H., Wang, D. Q., Liu, J., and Meng, L. J. (2017) Controlled Synthesis of Water-Dispersible and Superparamagnetic Fe3O4 Nanomaterials by Microwave-assisted Solvothermal Method: From Nanocrystals to Nanoclusters. CrystEngComm. 19, 5089-5099.

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