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Intelligent MoS2 Nanotheranostic for Targeted and Enzyme/ pH/NIR-Responsive Drug Delivery to Overcome Cancer Chemotherapy Resistance Guided by PET Imaging Xinghua Dong, Wenyan Yin, Xiao Zhang, Shuang Zhu, Xiao He, Jie Yu, Jiani Xie, Zhao Guo, Liang Yan, Xiangfeng Liu, Qing Wang, Zhanjun Gu, and Yuliang Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17506 • Publication Date (Web): 10 Jan 2018 Downloaded from http://pubs.acs.org on January 10, 2018

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ACS Applied Materials & Interfaces

Intelligent MoS2 Nanotheranostic for Targeted and Enzyme/pH/NIR-Responsive Drug Delivery to Overcome Cancer Chemotherapy Resistance Guided by PET Imaging Xinghua Donga,b, Wenyan Yina,*, Xiao Zhanga, Shuang Zhua, Xiao Hea, Jie Yua, Jiani Xiea, Zhao Guoa, Liang Yana, Xiangfeng Liub, Qing Wangd, Zhanjun Gua,b*and Yuliang Zhaoa,b* a

Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High

Energy Physics, Chinese Academy of Sciences, Beijing, 100049, China b

College of Materials Science and Optoelectronic Technology, University of Chinese Academy of

Sciences, Beijing, 100049, China c

CAS Center for Excellence in Nanoscience,National Center for Nanoscience and Technology of

China, Chinese Academy of Sciences Beijing 100190, China, Chinese Academy of Sciences, Beijing, 100190, China d

School of Material Science and Engineering Institue of Nano Engineering, Shandong University

of Science and Technology, Qingdao, 266590, China *Corresponding Authors: [email protected], [email protected], [email protected]

ABSTRACT Chemotherapy resistance remains a major hurdle for cancer therapy in clinic due to poor cellular uptake and insufficient intracellular release of drugs. Herein, an intelligent, multifunctional MoS2 nanotheranostic (MoS2-PEI-HA) ingeniously decorating with biodegradable hyaluronic acid (HA) assisted by polyethyleneimine (PEI) is reported to combat drug-resistant breast cancer (MCF-7-ADR) after loading with chemotherapy drug doxorubicin (DOX). The HA can not only target CD44-overexpressing MCF-7-ADR, but also be degraded by hyaluronidase (HAase) that is concentrated in the tumor microenvironment and thus to accelerate doxorubicin (DOX) release. Furthermore, the MoS2 with strong near-infrared (NIR) photothermal 1 ACS Paragon Plus Environment

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conversion ability can also promote the release of DOX in the acidic tumor environment at a mild 808 nm laser irradiation, achieving a superior anti-tumor activity based on the programmed response to HAase and NIR laser actuator. Most importantly, HA targeting combined with mild NIR laser stimuli rather than using hyperthermia can potently down-regulate the expression of drug-resistance related P-glycoprotein (P-gp), resulting in greatly enhanced intracellular drug accumulation, thus achieving drug resistance reversal. After labeled with 64Cu by a simple chelation strategy, the MoS2 was employed for real-time photon emission computed tomography (PET) imaging of MCF-7-ADR tumor in vivo. This multifunctional nanoplatform paves a new avenue for PET imaging guided spatial-temporal controlled accurate therapy of drug-resistant cancer. Keywords: MoS2 nanosheets, Surface modification, Targeting and P-gp inhibition, Controlled therapy, Theranostics 1. Introduction Chemotherapy has been considered as a main strategy for cancer therapy.1 The developing multi-drug resistance (MDR) and subsequent treatment failures to patients continue to be major impediments during cancer chemotherapy.2 The degree of drug resistance generally depends on the type of cancers and various mechanisms of MDR have been proposed.3-4 At present, the excess expression of P-glycoprotein (P-gp), an efflux pump, is the main causes of MDR.5-7 Under selection pressure, P-gp can export anticancer drugs and prevent the uptake of these drugs by cancer cells from reaching therapeutic levels, consequently leading to decreased intracellular drug concentration 2 ACS Paragon Plus Environment

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and gradual MDR. For breast cancer MCF-7 treatment, doxorubicin (DOX) is a widely accepted chemotherapy drug and the MCF-7 cancer cells usually present with the overexpression of P-gp.8-9 Especially, chemotherapy alone always fails to overcome MDR and meet the clinical demand. To this end, combination therapy has been adopted in clinic and considered as the main strategy to improve the curative effect.10 Therefore, how to combine chemotherapy with other therapeutic strategies to reverse drug resistance and consequently improve cancer therapy outcomes has become an urgent problem. With the development of nanotechnology, nanomaterials have emerged as most promising and viable platform for targeted and controlled drug delivery.11-17 The targeted drug delivery systems have the ability to improve the treatment efficiency and reduce toxicity of anticancer drugs.18-21 Till now, multiple stimuli-responsive nanocarriers, which can release drugs in response to internal or external environment stimulus such as pH,22 redox,23 enzyme,24-26 temperature,27 and magnetic and light,28-33 etc. have received considerable attention. Among them, near-infrared (NIR) light (650-900 nm) stimuli-responsive photothermal nanoagents, such as noble metals (Au, Pt),31 carbon-based nanomaterials,11,

28, 34

CuS,35 2D transitional metal

dichalcogenides (TMDCs MoS2, WS2, MoSe2),36-41 and metal oxide (MoOx, WOx),42 etc., can convert light into heat to ablate cancer cells with noninvasive and deep tissue penetration. Meanwhile, some of these nanoagents can not only combine chemotherapy with other therapeutic strategies and control drug release remotely to

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achieve synergetic effect, but also enhance the sensitiveness and bioavailability of chemotherapy drugs to cancer cells to resolve drug resistance.43-44 MoS2, as one of the typical TMDCs, have obtained great attention in fundamental and practical studies because of its impressive electrical, optical, and structural properties. Recent years, nano-sized MoS2, as a rising star, has been explored in the biomedical application for tumor therapy,45-48 antibacterial,49 detection,50 bone tissue engineering and biosensors,51 due to its large surface area for cargo delivery, high NIR photothermal conversion efficiency, easy surface modification, low toxic constituent (S and Mo) as well as specific fluorescence properties.52-54 For example, Chou et al. developed MoS2 as a novel NIR absorbing nanoagent for cancer treatment.46 Some research groups including us employed MoS2 nanosheets for thermo-chemotherapy of cancer.16,

23, 47

Although the rapid

development in the biomedical field, investigation of the MoS2 for accurate tumor therapy through ingenious surface modification is still in its infant. Most previous studies regarding MoS2 therapeutic application mainly focus on tumor passive targeting strategies which are based on enhanced permeability and retention (EPR) effect. However, many preclinical and clinical research works suggest that EPR effect is not as effective as expected.55 To date, there is still no exploration on designing surface functionalized MoS2 as nanotheranostic for actively targeted and multiple stimuli-responsive therapy of DOX-resistant human breast cancer (MCF-7-ADR). Hyaluronic acid (HA), as a biodegradable natural polysaccharide, widely presents in the extracellular matrix which does not trigger body's immune and inflammatory 4 ACS Paragon Plus Environment

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response.56 Meanwhile, HA can be degraded by hyaluronidase (HAase), which is highly concentrated in the tumor microenvironment and the cells liposomes.25 Besides, HA can specifically combine with CD44 receptor, which usually overexpresses in drug-resistant cancer cells.18 Herein, we design HA functionalized MoS2 assisted by PEI to obtain MoS2-PEI-HA nanosheets as a multifunctional nanoplatform for 1) active targeting of CD44

high

expression

MCF-7-ADR,

2)

delivering

DOX

and

multiple

stimuli-responsive DOX release, 3) reversing drug resistance (Scheme 1). We found that HA targeted MoS2 nanosheets with good biocompatibility can be effectively up-taken by MCF-7-ADR and then act as pH, HAase as well as NIR 808 nm laser stimuli-responsive nanocarriers, resulting in an accelerated DOX release in the cells. This nanocarrier can also remarkably inhibit the expression of P-gp, thereby enhancing the accumulation of DOX in the MCF-7-ADR, consequently improving bioavailability of DOX. Besides therapy, diagnosis is also important. Recently, positron emission tomography (PET) imaging is receiving much attention owing to its higher sensitivity, temporal resolution, and unlimited tissue penetration.57 PET imaging can real-time monitor the accumulation and biodistribution as well as therapeutic response in the tumor-bearing mice because of the successful modification of radioisotope

64

Cu (a positron emitter with a 12.7 h half-life) to the surface of

MoS2-PEI-HA. Therefore, the engineering designed DOX@MoS2-PEI-HA as a multiple-stimuli responsive nanotheranostic provides a promising choice for PET imaging guided spatial-temporal controlled accurate therapy of MDR cancer. 5 ACS Paragon Plus Environment


2. Results and Discussion Scheme 1 illustrates the synthesis process of HA modified MoS2 nanosheets as a multifunctional platform for targeted and multiple stimuli-responsive therapy of MCF-7-ADR cells guided by PET imaging. Firstly, the MoS2 nanosheets were synthesized by a modified oleum-assisted exfoliation process without adding any surfactant reported by our previous study (Scheme 1a).49 Before functionalizing MoS2 with biodegradable HA, a commonly used cationic surface modification agent branched PEI was introduced for the amination of MoS2 through electrostatic interaction. Afterwards, MoS2-PEI-HA nanosheets were synthesized by coupling the -NH2 with -COOH of the HA (Scheme 1b and Scheme S1a). The successful synthesis of MoS2-PEI-HA nanosheets was confirmed by atomic force microscopy (AFM), transmission electron microscopes (TEM), Fourier transform infrared spectroscopy (FT-IR), and Raman spectroscopy. Atomic force microscopy (AFM) images revealed that the thickness of single-layer MoS2 nanosheets (~0.8 nm) (Figure 1a) increased to 5-7 nm (Figure 1d) after being modified with PEI and HA. The increased thickness of MoS2 nanosheets could be attributed to the successful coating of PEI and HA onto the MoS2 nanosheets surface. The average size of single-layer MoS2 nanosheets observed from TEM image was ~30-50 nm (Figure 1c), which was in good agreement with the result obtained by the AFM measurement (Figure 1b). When the tip-ultrasonic time increased from 4 to 8 h, we found that the size of MoS2 nanosheets obviously decreased to ~10 nm (Figure S1), suggesting the size can be easily controlled by changing the tip-ultrasonic time. Raman spectra in Figure 1f showed that the bare 6 ACS Paragon Plus Environment

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MoS2 and MoS2-PEI-HA nanosheets exhibit well-known in-plane E2g1 and out-of-plane A1g peaks of layered 2H-MoS2.16 However, owing to the sonication treatment and modification with HA to the MoS2 nanosheets, a slight shifted Raman bands were observed compared to the bare MoS2 nanosheets, indicating the successful modification of HA onto MoS2. The conjugation of PEI and HA on MoS2 was also qualitatively analyzed using FT-IR (Figure 1g). In the FT-IR spectra, the –OH (3430 cm-1), CH2 (2932 cm-1), and NH2 (1640 cm-1), corresponded to the –OH stretching vibrations, CH2 asymmetric stretching vibrations and bending mode of amino group (-NH2).58 The peak at 2885 cm-1 in MoS2-PEI-HA can be attributed to the C-H stretch of HA.59 The peak centered at 1652 cm-1 in MoS2-PEI-HA sample corresponding to the carbonyl-amide (-CO-NH-) vibration. The zeta potential of MoS2 was about -20 mV, as shown in Figure 1h. After modification with PEI and HA on the surface of MoS2, this zeta potential consequently changed to +20 and -18 mV, respectively. All these results indicated the successful conjugation of HA onto the surface of MoS2 mediated by PEI. Especially, after the HA modification, no obvious aggregation in water and other physiological solution such as IMDM were observed (Figure 1i). The optical absorption spectra of MoS2-PEI and MoS2-PEI-HA aqueous dispersions were investigated using UV–vis–NIR spectroscopy (Figure 2a). The results indicated that the MoS2 nanosheets had remarkable broad band absorption in the NIR region. In order to test their NIR photothermal efficiency, the MoS2-PEI-HA aqueous dispersions were exposed to 808 nm laser (Power density: 1.0 W cm-2). In Figure 2b, temperature of the MoS2-PEI-HA aqueous dispersions rose obviously with 7 ACS Paragon Plus Environment


the increased concentration and exposure time. The temperature elevation of 33, 21, 14, and 7 °C was achieved under the irradiation of NIR laser for 600 s at concentrations of 180, 120, 60, and 30 ppm, respectively (Figure S2). Which is to say, for example, when the concentration of the MoS2-PEI-HA nanosheets is 120 ppm, the temperature can increase to 52 °C within 600 s. In contrast, no significant temperature change was observed when deionized water was exposed to the 808 nm laser, thus verifying the good photothermal efficiency of the MoS2-PEI-HA nanosheets. To further measure the photostability, we also examined the temperature change of MoS2-PEI-HA nanosheets at a concentration of 180 ppm over four laser on/off cycles (1.0 W cm-2). In this process, the temperature had no obvious reduction, which implies that the MoS2-PEI-HA nanosheets possess good photostability (Figure 2c). Multiple stimuli-responsive nanocarriers have received great attention due to their unique advantages. In our experiments, the as-prepared MoS2-PEI-HA nanosheets with 2D nature have unique multiple stimuli-responsive abilities to pH, HAase as well as NIR laser. We choose DOX as an antitumor drug in response to the multiple stimuli. Firstly, the drug loading behaviors in PBS were investigated by impregnating the MoS2-PEI-HA with DOX solution overnight in the dark. Based on UV–vis absorption measurements, the DOX loading ratio of MoS2-PEI-HA at varied pH (pH 5.0, 7.4, and 8.0) were exhibited in Figure S3. It was found that the amount of DOX loaded onto the MoS2-PEI-HA is pH-dependent. The DOX loading ratio at pH 8.0 was as high as 33.6 % in weight. The UV-vis absorption spectra of DOX@MoS2-PEI-HA showed an additional absorption peak centered at ~502 nm, 8 ACS Paragon Plus Environment

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further suggesting the successful loading DOX onto the MoS2-PEI-HA (Figure S4). Afterwards, the controlled drug release profiles were investigated as shown in Figure 2d-f. At pH 7.4, only ~11.2 % of DOX were released within 6 h. However, in the presence of 0.5 mg mL−1 of HAase, 23.2 %, and 41.6 % of DOX were released at pH 7.4 and pH 5.0, respectively, within 6 h (Figure 2d,f), implying the dramatically accelerated DOX release due to the HA degradation by the HAase. Finally, we investigated the pH value and NIR laser-responsive drug release capability of the DOX@MoS2-PEI-HA nanocomposite (Figure 2e, f). A pH-dependent release behavior was observed as well. Without NIR laser irradiation, ~25.5% of DOX was released within 6 h at the pH 5.0. Especially, a sudden DOX release was found when exposed to 808-nm laser irradiation with power density of 0.6 W cm-2 at an acidic buffer and the ratio of DOX release increased to 35.8 % (Figure 2e, f). Consequently, the cumulative release ratio of DOX based on programmed step (I) HAase and step (II) low power density NIR laser irradiation under acidic condition can reach up to 77.4% (Figure 2f). This result could be very useful for the multiple stimuli-responsive enhancement of DOX release at acidic endo/lysosomal tumor microenvironment. To meet the requirements of biomedical application, we further examined the biocompatibility of MoS2-PEI and MoS2-PEI-HA in vitro and in vivo. Firstly, standard CCK-8 assay and live/dead staining were performed by using A549, MCF-7, and MCF-7-ADR cells to evaluate cell viabilities in vitro. After being incubated with MoS2-PEI-HA or MoS2-PEI nanosheets for 24 h, we found that the three kinds of cells incubated with MoS2-PEI had no obvious cytotoxicity and the cell viabilities in 9 ACS Paragon Plus Environment


all groups were higher than 80 % (Figure 3a-c). In addition, normal HUVEC were incubated with the MoS2-PEI-HA for 24 and 48 h, respectively, and the cell viability was higher than 82% (Figure S5). Especially, the group treated by MoS2-PEI-HA showed better cell viability than that of group treated by the MoS2-PEI, demonstrating the significance of the HA functionalization process. We also investigated the influence of MoS2-PEI-HA on hemolysis of red blood cells (RBCs). Figure 3d shows a negligible hemolysis of the RBCs when the concentration of MoS2-PEI-HA was 800 µg/mL. The live/dead staining of three kinds of cells by calcein AM and propidium iodide (PI) dyes were further used to visually evaluate the biocompatibility, as shown in Figure 3e. All these results indicated that MoS2-PEI-HA has low toxicity at the tested dose. To further ensure the safe use of MoS2-PEI-HA in vivo, healthy female BALB/c mice were intravenously injected with MoS2-PEI-HA (10 mg kg-1). The mice were sacrificed during the tested day. After that, the main organs were excised and stained with hematoxylin and eosin (H&E). The histological analysis of H&E stained slices of the main tissues had no obvious damage and inflammation within 15 days compared with the control group (Figure 4). In addition, we measured the body weights of the mice and found that no weight loss was observed during the treatment (Figure S6), suggesting the good safety of MoS2-PEI-HA. Drug-resistance is a major problem during the chemotherapy. It has been reported that external environmental stimulus such as NIR light induced photothermal effect of nanomedicines have the ability to sensitize drug-resistant cells even prevent 10 ACS Paragon Plus Environment

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drug resistance.44 However, how to use targeted and stimuli-responsive drug delivery nanoplatform to reverse drug resistance and improve cancer therapy outcomes has become an urgent problem. Before the MCF-7-ADR cells killing experiments using the multiple stimuli-responsive MoS2-PEI-HA nanoplatform, we firstly performed the CD44 mediated uptake of FITC-labeled MoS2-PEI-HA based on flow cytometry (FACS) (Figure 5a). This uptake can be confirmed using five different cells with varying expression levels of CD44: KB (low), NIH3T3 (negative), MCF-7 (high+), HeLa (high+), and MCF-7-ADR (high++).60-62 In Figure 5a, the CD44++ positive MCF-7-ADR cells showed a very prominent green fluorescence due to the CD44 mediated uptake. However, green fluorescence was negligible for the CD44-negative NIH3T3 cells. These results confirmed the CD44-specific targeting the HA modified MoS2 nanosheets and enhanced uptake of the nanosheets in the MCF-7-ADR. Thereafter, to evaluate the capability of the DOX@MoS2-PEI-HA for drug reversal in MCF-7-ADR cancer therapy, we investigated whether the MoS2-PEI-HA could inhibit P-gp expression level under 808 nm laser irradiation by using western blot. As can be seen in Figure 5b-c, after treated with MoS2-PEI-HA+NIR, the P-gp expression level can be obviously reduced in comparison to other groups. The inhibited P-gp expression could hold back the efflux of DOX, which may consequently lead to a higher DOX accumulation in the MCF-7-ADR cells, implying that the targeting combined mild PTT can effectively reverse the drug-resistance of MCF-7-ADR and enhance the sensitivity. Thus, we believe that this localized heat under the NIR irradiation combined with the HA targeting plays a unique role. 11 ACS Paragon Plus Environment


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The MCF-7-ADR cellular release and uptake behavior were investigated by FACS and fluorescence imaging (Figure 6a-d). In Figure 6a, the fluorescence intensity of MCF-7-ADR cells incubated with DOX@MoS2-PEI-HA+NIR laser was higher than those incubated with DOX and DOX@MoS2-PEI-HA. This result suggested that the MCF-7-ADR cells incubated with DOX@MoS2-PEI-HA+NIR laser can accumulate much more DOX in comparison to the free DOX and DOX@MoS2-PEI-HA

groups

treated

with

equivalent

DOX

(25

µM).

Correspondingly, in Figure 6d, the MCF-7-ADR treated with DOX@MoS2-PEI-HA under NIR laser exposure contains more bright cells than free DOX (Figure 6b) and DOX@MoS2-PEI-HA (Figure 6c) treated groups. Next, we also employed fluorescence lifetime imaging microscopy (FLIM) to detect the DOX release from the DOX@MoS2-PEI-HA in MCF-7-ADR cells. The fluorescence lifetime of a luminescent material is in accord with the average time from its excited state to the ground state.63-64 The lifetime is 2.2 ns for free DOX and 3.3 ns for MoS2-PEI@DOX (Figure S7). The enhanced fluorescence lifetime of DOX is due to the conjugated DOX to MoS2-PEI. After the cells were incubated with DOX@MoS2-PEI-HA, the fluorescence lifetime distribution of DOX further increased to 4.6 ns (Figure S8). Figure 6e shows the fluorescence lifetime distribution for the MoS2-PEI@DOX, DOX@MoS2-PEI-HA, and DOX in MCF-7-ADR cells after 12 h of incubation. The results obtained from the FLIM clearly corroborated the enhanced DOX release using the DOX@MoS2-PEI-HA nanocomposite. This efficient drug delivery and release could be ascribed to two reasons as follows. Firstly, it has been reported that HAase is 12 ACS Paragon Plus Environment

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highly concentrated in the tumor microenvironment. For the HA modified MoS2 nanosheets, the degradation of HA by HAase was the main reason that led to the enzyme-responsive DOX release in the MCF-7-ADR cells. Then, under the 808 nm laser irradiation, the DOX can also be released from the DOX@MoS2-PEI-HA. All these findings indicated that the HA modified MoS2 nanosheets can target MCF-7-ADR specifically to improve cellular uptake and release behavior of DOX under the multiple stimuli response. Impelled by its low toxicity, multiple stimuli response to drug release, targeting ability and reversing drug-resistance, we evaluated the potential of MoS2-PEI-HA as nanocarrier for combating MCF-7-ADR cells. Firstly, the MCF-7-ADR cells incubated with MoS2-PEI + NIR, MoS2-PEI-HA + NIR (Power density of 808-nm laser: 0.6 W cm−2) for 24 h. In contrast to MoS2-PEI + NIR group, the MoS2-PEI-HA + NIR treated group had a higher killing ability to MCF-7-ADR cells (Figure 7a). For example, 41.5 % and 61.4 % of the MCF-7-ADR cells were killed after being treated with MoS2-PEI+NIR and MoS2-PEI-HA+NIR at the same concentration of 50 µg mL-1. The results indicated that the HA modified MoS2 nanosheets can target MCF-7-ADR specifically and improve the effect of PTT. To further illustrate the potential of the HAase and light-responsive MoS2-PEI-HA nanocarriers for the targeting combined chemo/photothermal therapy, the MCF-7-ADR cells were treated with DOX and DOX@MoS2-PEI-HA without or with 808-nm laser irradiation (Figure 7b). As expected, the half-maximum inhibitory concentration (IC50) values of the DOX@MoS2-PEI-HA (DOX concentration: 41.1 µM) were lower than that of the 13 ACS Paragon Plus Environment


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DOX group (>80 µM). Furthermore, the cell viability of MCF-7-ADR cells incubated with DOX@MoS2-PEI-HA dramatically decreased under 808 nm laser irradiation (power density: 0.6 W cm

-2

) (Figure 7b and Figure S8). For example, when

MCF-7-ADR cells were incubated with DOX@MoS2-PEI-HA plus NIR irradiation (DOX@MoS2-PEI-HA concentration = 100 µg mL-1, DOX concentration = 25 µM), the cell viability was remarkably reduced to 2.9 %, which is obviously lower than free DOX and MoS2-PEI-HA+NIR group. To further prove the effect of targeting combined multiple stimuli response on MCF-7-ADR cells, fluorescence images were obtained from the live–dead cell staining with Calcein AM/PI dyes. As shown in Figure 7c, the survival status of MCF-7-ADR cells was illustrated, where green and red respectively present the live cells and the red cells. After incubation with DOX@MoS2-PEI-HA for 24 h followed by NIR laser irradiation for 600 s, the combination treatment induced significantly higher cell death than that of the free DOX, DOX@MoS2-PEI-HA, or MoS2-PEI+NIR treated groups, which indicate the enhanced therapy efficiency of this platform. Next, due to the remarkable therapeutic effect of DOX@MoS2-PEI-HA in vitro, in vivo therapy was also performed. After the tumor sizes reached to approximately 200 mm3, the MCF-7-ADR tumor-bearing mice were divided into Group 1: Saline injection; Group 2: Saline + NIR, Group 3: MoS2-PEI-HA, Group 4: free DOX, Group

5: MoS2-PEI-HA+NIR,

Group

6: DOX@MoS2-PEI-HA,

Group

7:

DOX@MoS2-PEI-HA+NIR. Then, we i.v. injected MoS2-PEI-HA (Group 4) and DOX@MoS2-PEI-HA (Group 7) and then exposed 808 nm NIR laser (0.6 W cm-2) for 14 ACS Paragon Plus Environment

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10 min to the mice. In Figure 8a, the temperature of the tumor sites in mice after injection with MoS2-PEI-HA or DOX@MoS2-PEI-HA can quickly increase up to ~45 °C with a mild hyperthermia. However, the control group showed a slight increase of temperature. As shown in Figure 8b, no obvious body weight loss of mice was observed, which indicates a very low toxicity of the MoS2-PEI-HA nanosheets in vivo. In addition, the tumor volumes were also measured during the treatment. In Figure 8c, after 25 days’ treatment, tumors volume in group (1), (2), and (3) exhibited significant increase, which indicates that the MoS2-PEI-HA nanosheets group and NIR laser group had no obvious inhibition to tumor. However, weak tumor inhibition rates of 33.1 %, 35.2 %, and 76.0 % were achieved at those treated with free DOX , MoS2-PEI-HA+NIR, and DOX@MoS2-PEI-HA (Figure S9), suggesting that free DOX, MoS2-PEI-HA+NIR, or DOX@MoS2-PEI-HA alone only can partly inhibit the tumor. The tumor-bearing mice treated with MoS2-PEI-HA+NIR also exhibited an obvious recurrence of tumor after two weeks. In contrast, the tumor-bearing mice treated with DOX@MoS2-PEI-HA+NIR presented very high tumor inhibition ratio, approximately 96 %. In Figure 8d, most of the tumors in group (7) were thoroughly eliminated and no tumor regrowth was observed during the experimental period (Figure S10, S11), indicating the efficient therapy effect of DOX@MoS2-PEI-HA under NIR laser irradiation sensitization. Figure 8e represents the mean weights of excised tumor in each group after therapy. As expected, DOX@MoS2-PEI-HA+NIR laser group exhibited the lightest tumor weight among all the groups. Subsequently, 15 ACS Paragon Plus Environment


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the tumors and major organs were removed, fixed with paraffin, and sectioned into slices, for H&E staining. For those tumors, no obvious differences about the cells and nuclear shapes after the irradiation with 808 nm laser were observed (Figure 8f). In the case of the MoS2-PEI-HA+NIR irradiated tumor and free DOX treated tumor, only

a

few

signs

of

cells

necrosis

were

found.

However,

for

the

DOX@MoS2-PEI-HA+NIR group, most of cells had the changes of shrinkage, loss of contact, eosinophilic cytoplasm as well as nuclear damage, which demonstrates DOX@MoS2-PEI-HA+NIR can effectively inhibit tumor growth. Finally, compared with the control group, the blood biochemistry (Figure S12) and blood routine (Table S1) were measured at the 25th day after the different treatment and no noticeable change were found. In addition, the H&E-stained histological images of the major organs implied no obvious lesion and inflammation after the different treatments (Figure S13). Real-time monitoring of nanomedicine in vivo using PET imaging is particularly demanded because it opens a new avenue to optimize therapeutic protocols and monitor therapeutic response to avoid side effects. In our work, in vivo PET imaging was carried out in tumor-bearing mice. Before the PET imaging, the

64

Cu labeled

MoS2-PEI-HA-NOTA and MoS2-PEI-NOTA were successfully synthesized as shown in Scheme S1b. Then, the mice were divided into two groups and i.v. injected with 64

Cu-NOTA labeled MoS2-PEI-HA and MoS2-PEI, respectively, and PET scans were

obtained at 0.5, 1, 2, and 4 h post-injection to show the tumor targeting efficacy and biodistribution of the two nanosheets in main organs (Figure 9a, b). As shown in 16 ACS Paragon Plus Environment

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Figure 9a, within 2 h, radioactivity signals were mainly detected in the lung, liver, and spleen, which could be attributed to the agglomeration of the samples in the lung and the mononuclear phagocytic system in the liver.57 Especially, after 4 h, the signals in the lung decreased, while both the liver and tumor were clearly visualized in the HA targeted group compared with the no targeting group. No signals were detected in other organs, implying that no free 64Cu2+ ions were dissociated from the 64Cu-NOTA labeled MoS2-PEI and MoS2-PEI-HA samples. The mice after the PET imaging were sacrificed and the quantitative analysis of biodistribution of the MoS2-PEI and MoS2-PEI-HA were obtained by the radioactivity of tissues recorded by a γ-counter. As shown in Figure 9c, the accumulation of 64Cu-NOTA labeled MoS2-PEI-HA in the MCF-7-ADR tumor was 10.486 ID/g at 4 h post injection. In contrast, without the conjugation of HA, uptake of

64

Cu-NOTA labeled MoS2-PEI by the MCF-7-ADR

tumor was found to be only 5.015 ID/g at 4 h post injection, indicating that HA conjugation has high targeting ability for enhancing accumulation of the nanosheets in tumor area. 3. Conclusion In summary, a multifunctional nanotheranostic based on DOX@MoS2-PEI-HA has been successfully constructed, which integrates MDR tumor-targeting, precisely drug release, and PTT functions into a single system. This integrative nanoplatform can be used for active CD44-targeting, multiple stimuli-responsive DOX release to enzyme and mild NIR laser irradiation, to effectively combat CD44-positive MCF-7-ADR cells. The HA targeting modification can improve the cellular 17 ACS Paragon Plus Environment


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internalization of DOX@MoS2-PEI-HA into MCF-7-ADR cells and the degradation of the HA in the acidic condition can bring about an enzyme HAase-responsive drug release followed by a mild NIR 808 nm laser irradiation stimuli to further enhance the DOX release, achieving a programmed multiple stimuli response. The HA targeting combined the NIR photothermal stimuli of DOX@MoS2-PEI-HA can inhibit the expression of drug-resistance related P-gp, resulting in enhanced drug accumulation and high tumor inhibition rate in the MCF-7-ADR cells as well as high sensitiveness to drug. The successful modification radioisotope

64

Cu to the surface of

MoS2-PEI-HA nanosheets endowed them with the function of PET imaging to real-time monitor the HA targeting ability in the MCF-7-ADR tumor site and the therapeutic response. Overall, the as-designed nanoplatform provides an insightful and attractive strategy to overcome drug resistance and increases drug accumulation in tumor for spatial-temporal controlled MDR cancer therapeutic efficacy guided by high-sensitive PET imaging. 4. Experimental Section 4.1 Materials Molybdenum sulfide (MoS2, 99%) and Doxorubicin (DOX, 99%) were purchased from Alfa Aesar. Polyethyleneimine (PEI, Mw = 25000, ALOICH), Hyaluronic

acid

(HA,

molecular

weight:

35

kDa,

Cosdscorm.),

1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide hydrochloride (EDC, 98%, J&K. CHEMICAL., LTD), N-hydroxysuccinimide (NHS, 98%, J&K. CHEMICAL., LTD). Hyaluronidase (HAase) was purchased from Cedarlane. Other reagents were obtained 18 ACS Paragon Plus Environment

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from Beijing Chemical Reagent Co. Deionized water was obtained from an 18 MΩ (SHRO-plus DI) system. Fluorescein isothiocyanate (FITC, 95%) was purchased from Alfa Aesar. 1,4,7-Triazacyclononane-N, N′,N″-triacetic acid (NOTA) was purchased from Macrocyclics Inc. (Dallas, TX).

64

CuCl2 was obtained from the China Institute

of Atomic Energy. 4.2 Preparation of Single-Layer MoS2 Nanosheets Single-layer MoS2 nanosheets were prepared by a modified liquid-phase exfoliation method reported by our previous work

[16]

. Typically, commercial MoS2

powders (60 mg) were ground using a ball milling for ~10 h. The ground MoS2 were washed with deionized water for three times and dried at 60 oC for 24 h. Then, the MoS2 were immersed into 20 mL of oleum, and held in a water bath at 90 ° C for 8-10 h. The redundant oleum was removed by centrifugation and repeated washing with distilled water. Thereafter, the oleum-treated MoS2 were dispersed in 20 mL of deionized water and sonicated using tip ultrasonic meter (Power: 320 W) for 5 h. The suspension was centrifugated at 8000 rpm for 25 min. Finally, this supernatant was gently transferred from the top dispersion and washed thoroughly several times with deionized water. 4.3 Preparation of PEI Modified MoS2 Nanosheets To obtain PEI modified MoS2 nanosheets (MoS2-PEI), 20 mL of PEI (1.0 mg mL-1) was added dropwise into 30 mg of MoS2 nanosheets dispersion while being tip-sonication for 1 h (Power: 320 W). Subsequently, MoS2-PEI nanosheets were collected and washed thoroughly several times using deionized water. 19 ACS Paragon Plus Environment


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4.4 Preparation of HA Functionalized MoS2 Nanosheets HA functionalized MoS2 nanosheets (named as MoS2-PEI-HA) was obtained as follow. Firstly, 30 mg of HA was dissolved in deionized water (15 mL), and 16 mg of EDC in 10 mL deionized water was added. After sonicating this mixture for 15 min, 20 mg of EDC in 10 mL deionized water was added. Then, NHS (60 mg) in deionized water (15 mL, pH 8.5) was added. The mixture was stirred at room temperature for 2 h. Upon adding the solution of MoS2-PEI dropwise to the obtained dispersion, the mixture was stirred for another 12 h in the dark. The resulting MoS2-PEI-HA nanosheets was collected by centrifugation and washing several times with deionized water. 4.5 Synthesis of MoS2-PEI-NOTA and MoS2-PEI-HA-NOTA Nanosheets Briefly, 6 mg of MoS2-PEI and MoS2-PEI-HA were respectively dispersed into 5 mL of Na2CO3 (pH 8.6) aqueous solution. Then, 8 mg of NOTA was added into this aqueous solution. After stirring at room temperature, the MoS2-PEI-NOTA and MoS2-PEI-HA-NOTA were centrifuged, and washed with deionized water. 4.6 Synthesis of 64Cu-NOTA Labeled MoS2 Nanosheets Typically, 5 mg of MoS2-PEI-NOTA and MoS2-PEI-HA-NOTA nanosheets were respectively dispersed into 8 mL of CH3COONa-CH3COOH buffer solution (pH 6.5). Then, 100 µL of mixture

solutions

64

CuCl2 (555 MBq) was added to above solutions. These

were

MoS2-PEI-NOTA-64Cu

stirred

and

for

1

h

at

room

MoS2-PEI-HA-NOTA-64Cu

EDTA-2Na solution for several times to remove free 20 ACS Paragon Plus Environment

temperature. were 64

Finally,

washed

with

Cu. Afterwards, the

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as-obtained products were re-dispersed in phosphate buffered saline (PBS) buffer for further use. 4.7 Radiolabeling Efficiency The radiolabeling efficiency and stability analysis of

64

Cu-NOTA labeled

MoS2-PEI and MoS2-PEI-HA were respectively assessed by radio instant thin layer chromatography (radio-ITLC). Typically, radio-ITLC strips were developed with 4 mM EDTA-2Na in PBS, pH 7.4. These strips were quantified by a TLC imaging scanner.

Then,

the

radiolabeling

stability

was

measured

by

incubating

MoS2-PEI-NOTA-64Cu or MoS2-PEI-HA-NOTA-64Cu in PBS which contains EDTA (4 mM) for 18 h at 37 oC. Free

64

Cu2+ ions can move to the solvent front while the

MoS2 was kept at the original place. 4.8 Morphology and Characterization The morphologies and sizes of the MoS2 were acquired by transmission electron microscopes (TEM, Tecnai G2 20 S-TWIN). Zeta-potential analyses were performed by using zeta-potential analyzer (Nicomp380 ZLS plus ZETADi, PSS, USA). Topologies of the single-layer and functionalized MoS2 nanosheets were examined by atomic force microscopy (AFM) (Agilent 5500, Agilent, USA) under ambient conditions. Raman spectra of samples were obtained by Raman spectrometer (HORIBA LabRAM HR Evolution), with a laser excitation wavelength of 514 nm. Fourier transform infrared spectra (FT-IR) were obtained from a micro-Fourier transform infrared spectrophotometer (iN10-IZ10, Thermal Fisher). UV-vis-NIR absorbance measurements were conducted by a HitachiU-3900 spectrophotometer. 21 ACS Paragon Plus Environment


4.9 Photothermal Effect To evaluate photothermal effect of the MoS2-PEI-HA, we perpendicularly fixed the 808-nm-laser optical fiber to the vessel having MoS2-PEI-HA nanosheets aqueous dispersions (1 mL) with various concentrations. These dispersions were irradiated under the 808 nm laser at a power density of 1.0 W cm-2. The infrared thermal images of dispersions and the temperature were simultaneously recorded every 15 s. Furthermore, for testing photostability, 180 ppm of MoS2-PEI-HA nanosheets was irradiated for 600 s (Power density: 1.0 W cm-2). Then the laser was switched off for 600 s. This procedure was repeated four times. 4.10 Loading and Releasing of DOX The DOX loading behavior was evaluated by adding different concentrations (50-500 µM) of DOX into MoS2-PEI-HA aqueous suspension (1.0 mg mL-1) under different pH vales (pH 5.0, 7.4, and 8.0). The aqueous suspensions were stirred overnight in the dark. Then unloaded DOX can be removed by centrifugation at 12000 rpm and repeated rinsing for several times. To evaluate the amount of DOX loading, the obtained free DOX in the supernatants were collected and determined at 480 nm based on the absorbance of DOX and the loading amount was calculated. For HAase-responsive DOX release experiment, DOX@MoS2-PEI-HA aqueous dispersions (with equivalent DOX of 25 µM in 1.0 mg mL-1 of DOX@MoS2-PEI-HA) was centrifuged at 12000 rpm and then dissolved with 0.5 mg mL-1 HAase solution which contains PBS buffers (pH 4.5, 6.0, 0.5 mg mL-1) at 37 °C. All the solutions were centrifuged at different time intervals 0, 0.5, 1, 2, 4, and 6 h. The supernatant 22 ACS Paragon Plus Environment

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was collected. Simultaneously, fresh PBS with the same volume as the collected supernatant was added into residual solution. Finally, UV-vis absorbance spectrum at 480 nm was used to measure the release efficiency of DOX. For NIR laser-triggered DOX release experiment, PBS buffers (pH 5.0 and 7.4) containing DOX@MoS2-PEI-HA (with equivalent DOX 25 µM) were respectively stirred at 37 oC. At different time intervals (0, 0.5, 1, 2, 4, and 6 h), the solutions were exposed to 808 nm laser irradiation (0.6 W cm-2) to elevate the temperature eventually to ~45 oC. UV-vis absorbance of the supernatant at 480 nm was used to measure the DOX release efficiency. 4.11 Cellular Uptake Ability of MoS2-PEI-HA Flow cytometry (FACS) analysis was employed to investigate the cellular uptake of MoS2-PEI-HA in NIH3T3 cells (normal mouse embryonic fibroblasts), KB cells (human epithelial carcinoma cell line), HeLa cells (human cervical cancer cell line), DOX-resistant MCF-7 cells (MCF-7-ADR) as well as DOX sensitive MCF-7 cells (human breast cancer cell line). MoS2-PEI-HA nanosheets were linkaged with FITC by a simple process. Typically, 10 mg of MoS2-PEI-HA were dispersed in DMSO (10 mL). Then this solution was mixed with 1.2 mg of FITC followed by stirring in dark for 12 h. Then, the FITC labeled MoS2-PEI-HA (FITC-MoS2-PEI-HA) were centrifugated washed by ethanol for three times. The cells suspension was incubated in 6-well plates for 24 h at 37 oC with 5 % CO2. Besides the controlled ones, 100 µg mL-1 of FITC-MoS2-PEI-HA dispersion was added into the 6-well plates. When these



cells were further incubated for 1-2 h, they were washed and re-suspended using PBS for FACS analysis (flow cytometer, accuri c6, BD, USA). 4.12 Cell Culture and In Vitro Cytotoxicity Study Human lung adenocarcinoma epithelial cell line (A549 cells), and human umbilical vein endothelial cell line (HUVEC) cultured with complete medium Dulbecco's modification of Eagle's medium (DMEM) supplemented with 10% of fetal bovine serum (FBS) upon seeding 1 ×104 cells into 96-well plates/well at 37 oC with 5% CO2 for 24 h. The DOX sensitive MCF-7 and DOX-resistant MCF-7 (MCF-7-ADR) cells were treated with Iscove's Modified Dulbecco's Medium (IMEM) with 10% FBS and seeded into 96-well plates/well at 37oC with 5% CO2 for 24 h. The three cells were exposed to MoS2-PEI or MoS2-PEI-HA with different concentrations from 0, 3, 6, 12, 25, 50, 100, to 200 µg mL-1 for 24 h, and CCK-8 reagent (10%) in serum-free medium was added. After incubation for 1-2 h at 37 oC, the optical densities of each well were measured at 450 nm on a microplate reader (Spectra Max M2MDC, USA). 4.13 Intracellular Release of DOX The MCF-7-ADR intracellular DOX release was testified using FACS analysis, fluorescence imaging, and fluorescence lifetime imaging microscopy (FLIM). For FACS and fluorescence imaging analysis, MCF-7-ADR cells were seeded in a confocal dish with 6000 cells/well and grown for 24 h. Then, the MCF-7-ADR cells were incubated with DOX@MoS2-PEI-HA (25 µM equivalent DOX concentration) or free DOX for 24 h. For the DOX@MoS2-PEI-HA+NIR laser group, after 12 h of 24 ACS Paragon Plus Environment

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incubation, the MCF-7-ADR cells were irradiated with 808 nm laser for 10 min at the power density of 0.6 W cm-2 for incubation another 12 h. Finally, the cells were washed with PBS and suspended in PBS for FACS analysis. At the same time, DOX, DOX@MoS2-PEI-HA, and DOX@MoS2-PEI-HA+NIR laser incubated cells were respectively imaged with confocal microscope. Next, MCF-7-ADR cells were cultured into confocal dish (6000 cells/well) for FLIM analysis. When the MCF-7-ADR cells had grown to 85 % in plates, the cells was incubated with fresh medium containing either DOX@MoS2-PEI-HA (25 µM equivalent DOX concentration) or free DOX at 37 oC. Then, after washing the cells with PBS, FLIM imaging was performed on a LSM-Kit (PicoQuant), which was combined with Nikon A1RSI confocal microscope. The emission was obtained using a 560 nm bandpass filter under the excitation of 485 nm. 4.14 In Vitro Targeting Combined Multiple Stimuli-Responsive Photothermal Ablation To evaluate the therapy effects in vitro, MCF-7-ADR cells were seeded in 96-well plates at the densities of 1×104 cells/well for 24 h at 37 oC under 5% CO2. When the cells grew to 80% in plates, 100 µL of the samples (DOX@MoS2-PEI-HA, free DOX, equivalent DOX concentration of DOX@MoS2-PEI-HA) with different concentrations were added to the cells followed by incubating for 24 h. After washing with PBS, the 10% of CCK-8 was used to test cell killing ability. For combination therapy, the MCF-7-ADR cells were incubated with MoS2-PEI, MoS2-PEI+NIR, MoS2-PEI-HA+NIR,

DOX@MoS2-PEI-HA,

DOX@MoS2-PEI-HA+NIR


with


various concentrations of 0, 25, 50, 100, 200 µg mL-1 in IMDM for 12 h without/with 808-nm laser irradiation for 10 min (Power density: 0.6 W cm−2). After the treatment, these cells were further incubated for 12 h followed by washing several times with PBS. Optical absorbance can be recorded at 450 nm after incubation the cells with CCK-8 within the set time. For further intuitively testifying the cellular killing ability, calcein acetoxymethyl ester (Calcein AM)/propidium iodide (PI) live-dead staining of MCF-7-ADR cells after incubating with MoS2-PEI-HA, free DOX, and DOX@MoS2-PEI-HA (equivalent DOX concentration: 25 µM) for 24 h without or with exposure to NIR 808-nm laser at a power density of 0.6 W cm−2 for 10 min. After washing, these samples were placed into fresh medium. PBS (pH 7.4) solution containing calcein AM (2 µM) and PI (4 µM) was added to these cells. These cells were incubated with above mentioned PBS solution for 10 min, washed repeatedly with PBS, and imaged by inverted luminescence microscopy. 4.15 Hemolysis Activity Test Typically, blood of BALB/c mouse (~1 mL) was collected into heparinized tube followed by centrifugation at 2000 rpm for 8 min to obtain red blood cells (RBCs). Then the RBCs were washed with PBS repeatedly. 5% RBCs suspensions diluted with PBS were the negative control and 5% RBCs suspensions diluted with deionized water were positive control. In addition, 5% of RBCs were slightly resuspended with PBS to obtain the MoS2-PEI-HA nanosheets dispersions with different concentrations of 12.5, 25, 50, 100, 200, 400, and 800 µg mL-1. The obtained dispersions were placed 26 ACS Paragon Plus Environment

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at 37 °C for 3 h followed by centrifuging at 8000 rpm for 5 min. The supernatants were used for measuring absorbance of the concentration of free hemoglobin at 540 nm 18. 4.16 In Vivo Toxicity Assessment Female BALB/c mice were purchased from Cancer Institute and Hospital, Chinese Academy of Medical Sciences and intravenously (i.v.) injected with MoS2-PEI-HA (Dosage: 10 mg kg-1, n=4 for each group). Mice were sacrificed at 2, 7, and 15 days. For histology study, the main tissues of the mice were fixed with 10% of formalin, processed into paraffin, and sectioned into slices. Then these slices were stained with hematoxylin & eoxin (H&E). Finally, the slices were imaged by inverted fluorescence microscope (OLYMPUS X-73, JAPAN). The body weights of these mice were recorded. All animals were sacrificed under the protocols approved by the Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Chinese Academy Sciences (CAS). 4.17 In Vivo Infrared Thermal Imaging The MCF-7-ADR tumor-bearing mice were anaesthetized by pentobarbital (1%) and i.v. injected with 200 µL of saline or MoS2-PEI-HA dispersion in saline (1.0 mg mL-1). Afterward, the mice were exposed to 808-nm laser for thermal imaging (Power density: 0.6 W cm-2) and recording the temperature at the tumor sites. 4.18 P-gp Expression Test using Western Blot First, the MCF-7-ADR cells were incubated with IMDM media supplemented with 10% FBS in four 35 mm adherent cell cultures (5×104 per culture) at 37 oC for 27 ACS Paragon Plus Environment


Page 28 of 46

12 h. Then five groups including Control, Control + NIR, MoS2-PEI-HA, MoS2-PEI-HA+NIR, and MoS2-PEI+NIR were tested. After co-incubation for 12 h without or with NIR laser irradiation, the cells were lysed and collected. Then the SDS-PAGE was used to separate the as-obtained cell lysates followed by shifting onto nitrocellulose membranes (PVDF). After that, the PVDF membranes were incubated with western blocking buffer at 37 oC and the diluted primary antibodies for P-gp, and β-actin for 12 h. After the PVDF membranes were washed for three times with detergents, they were incubated with diluted Horseradish Peroxidase (HRP)-labeled secondary antibodies at 37 oC for 2 h. Finally, after the PVDF membranes were stained by ECL detection kit, they were photographed using Chemilumilescent Imaging System (Azure Biosystems, C300). 4.19 Animals Model and Therapeutic Evaluation in vivo BALB/c nude mice with body weight about 20 g were obtained from Beijing Vital River Experimental Animal Technology CO. Ltd,, and housed under standard conditions in Individual Ventilated Cages (IVC). The animals were fed with pure water containing vitamins and sterilized food ad libitum. The BALB/c nude mice were inoculated with 2×106 of MCF-7-ADR cells at fore leg. When the tumors grew to ~200 mm3, we divided the mice as Group 1: Saline (200 µL), Group 2: Saline + NIR, Group 3: MoS2-PEI-HA (200 µL), Group 4: Free DOX

(200

µL,

DOX@MoS2-PEI-HA

25

µM), (in

Group

terms

of

5:

MoS2-PEI-HA+NIR, DOX,

25

µM),

Group Group

6: 7:

DOX@MoS2-PEI-HA+NIR (in terms of DOX, 25 µM). The number in each group is 28 ACS Paragon Plus Environment

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four. The saline, MoS2-PEI-HA were i.v. injected to the mice via tail vein. After 4 h, the tumor regions of groups (2), (5), and (7) were exposed to 808 nm laser (0.6 W cm-2, 10 min). After 25 days, the tumor-bearing mice were sacrificed. The tumors and major organs in each group were peeled off from the mice and weighed. A caliper was used to measure the tumor sizes. The blood were obtained from the fundus artery of mice for blood biochemistry analysis. The hematology indicators were measured. Typically, 1 mL of blood was centrifuged to obtain blood plasma for blood biochemistry analysis. In addition, 100 µL of blood of each mouse was collected into an anticoagulant tube for hematology measurements. The tumors and major tissues were fixed with 4% paraformaldehyde solution, processed into paraffin, and stained with H&E. The slices of each sample were examined using inverted fluorescence microscope. 4.20 Small Animals PET Imaging The details of small-animal PET imaging have been reported before.[51] Typically, the MCF-7-ADR tumor-bearing BALB/c mice were divided into two groups. The mice of the two groups were respectively i.v. injected with 10 mg kg-1 of 64

Cu labeled MoS2-PEI-NOTA and MoS2-PEI-HA-NOTA nanocomposite. Then the

mice were anesthetized with isoflurane. PET scans and imaging analysis were carried out on a small-animal PET scanner developed by the Institute of High Energy Physics, Chinese Academy of Sciences (CAS). The signals were scanned and collected at 0.5, 1, 2, and 4 h, respectively. The time for each scan was 10 min at each time point. After finishing the experiment, the mice were sacrificed and organs were weighed, 29 ACS Paragon Plus Environment


and counted in a gamma counter (CAPRAC-t, Capintec. Inc.). The percentage of injected dose per gram of tissue (% ID g-1) for each tissue sample was calculated by comparing its activity with a standard of the injected dosage. Supporting Information Supporting Information is available free of charge on the ACS Publications website. TEM image of the MoS2 nanodots; Temperature change over a period of 600 s versus MoS2-PEI-HA nanosheets concentration; Quantification of loading ratio of the MoS2-PEI-HA nanosheets versus various DOX concentrations under different pH values; UV-vis absorption spectra of free DOX, MoS2-PEI-HA, and DOX@MoS2-PEI-HA, respectively; The body weight of BALB/c mice i.v. injected with MoS2-PEI-HA; MCF-7-ADR cells were cultured in 96-well plates and imaged by an infrared thermal imager; Corresponding lifetime distribution curve (ns); Tumor inhibition ratio; Photographs of the test mouse on the 23th day; Blood routine measurements of the mice after different treatments; Blood biochemistry in mice; H&E stained images of major organs including heart, liver, spleen, lung, and kidney from tumor-bearing mice after different treatments. Acknowledgements This work was supported by Beijing Natural Science Foundation (2162046), National Natural Science Foundation of China (51772293, 51772292, 11621505, 31571015, and 21320102003), and National Basic Research Programs of China (No. 2016YFA0201600, 2015CB932104, 2014CB931900). Notes 30 ACS Paragon Plus Environment

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The authors declare no competing financial interest. References 1.

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Scheme 1. Schematic illustration of MoS2-PEI-HA nanosheets as a multifunctional platform for targeting and multiple stimuli-responsive therapy of MCF-7-ADR cells guided by PET imaging. (a-b) Synthesis of MoS2 nanosheets using a modified liquid exfoliation process in aqueous solution to produce single-layer MoS2 nanosheets and then modified with HA. (c) DOX loading process to obtain DOX@MoS2-PEI-HA. (d) MoS2-PEI-HA nanosheets for active CD44-targeting DOX delivery and MDR reversal through P-gp protein inhibition. (e) MoS2-PEI-HA functionalized with NOTA-64Cu for PET imaging.


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Figure 1. (a,d) AFM images of MoS2 nanosheets before and after modification with HA. (b,e) Histograms of raw MoS2 and MoS2-PEI-HA nanosheets lateral sizes, respectively. (c) TEM image of a single-layer MoS2 nanosheets. (f) Raman spectra of MoS2 nanosheets before and after HA modification. (g) FT-IR spectra of MoS2 nanosheets before and after functionalized with HA. (h) Zeta potential of MoS2 nanosheets before and after modification with PEI and HA. (i) Photographs of (I-II) DOX@MoS2-PEI-HA and (III-IV) MoS2-PEI-HA nanosheets in water and IMDM for -1

4 h (Concentration: 200 µg mL ).



Figure 2. (a) UV-vis-NIR absorption spectra of MoS2 nanosheets before and after HA modification. (b) Temperature increase of MoS2-PEI-HA nanosheets dispersions with different concentrations as a function of NIR laser irradiation time. (c) Four cycles of NIR laser irradiation for MoS2-PEI-HA nanosheets aqueous solution (Concentration: 180 ppm). Each cycle consisted of 600 s irradiation followed by 600 s cooling phase (Power density: 1.0 W cm-2). (d) Enzyme stimuli-responsive drug release profiles of DOX@MoS2-PEI-HA with or without 0.5 mg mL−1 of HAase at pH 7.4 and 5.0. (e) Drug release profiles form DOX@MoS2-PEI-HA with or without laser irradiation (808 nm, 0.6 W cm-2) at pH 7.4 and 5.0. (f) Statistical data of percentage of cumulative release of DOX from DOX@MoS2-PEI-HA under different conditions including pH value, enzyme, and NIR laser.


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Figure 3. Toxicity experiments in vitro. (a-c) Cell viabilities of (a) A549 cells, (b) MCF-7 cells, and (c) MCF-7-DOX-ADR cells incubated with MoS2-PEI and MoS2-PEI-HA with different concentrations. (d) In vitro hemolytic percent of RBCs incubated with MoS2-PEI-HA at various concentrations. Deionized water (+) and PBS (-) were acted as positive and negative controls, respectively. Inset: Photograph of the direct observation of hemolysis behaviors. (e) Fluorescent images of A549, MCF-7, and MCF-7-ADR cells with live-dead staining.



Figure 4. In vivo toxicity assessment. Histological images of H&E stained organs slices of mice i.v. injected with MoS2-PEI-HA at the dose of 10 mg kg-1. Mice were sacrificed at 2, 7, and 15 days.


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Figure 5. (a) CD44-mediated cell uptake of MoS2-PEI-HA nanosheets. FACS analysis shows that MCF-7-ADR cells with high expression of CD44 have higher cell uptake compared to compared to MCF-7 (CD44 high+), HeLa (CD44 high+), KB(CD44 low), and NIH3T3 (CD44 negative) cells. (b,c) Western blot for the detection of P-gp expression in different groups without or with 808 nm laser irradiation (Power density: 0.6 W cm-2). The asterisks denote statistical differences (*p < 0.05, **p < 0.01).



Page 42 of 46

Figure 6. (a) The fluorescence intensity of DOX obtained from MCF-7-ADR cells after

being

treated

with

free

DOX,

DOX@MoS2-PEI-HA,

and

DOX@MoS2-PEI-HA+NIR laser with equivalent DOX concentration (25 µM). (b-d) Fluorescent images of the MCF-7-ADR cells incubated with (b) free DOX, (c) DOX@MoS2-PEI-HA, and (d) DOX@MoS2-PEI-HA with the equivalent DOX concentrations with or without 808-nm irradiation (Power density: 0.6 W cm-2). The obvious intracellular DOX accumulation was marked with blue arrows. (e) FLIM images showing cell uptake of (I) free DOX, (II) MoS2-PEI@DOX, and (III) DOX@MoS2-PEI-HA using MCF-7-ADR cells after 12 h of incubation. The FLIM images denote the fluorescence lifetimes measured at each pixel and displayed as color contrast image. The corresponding false-color lookup table represents the lifetime distribution.


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Figure 7. (a) Cell viability of MCF-7-ADR cells incubated with MoS2-PEI+NIR and MoS2-PEI-HA for 24 h with or without NIR 808-nm laser irradiation. (b) Cell viability of MCF-7-ADR cells incubated with free DOX and DOX@MoS2-PEI-HA with or without NIR 808-nm laser irradiation for 24 h. (c) Calcein AM/PI live-dead staining of MCF-7-ADR cells after incubation with MoS2-PEI, MoS2-PEI-HA, free DOX, and DOX@MoS2-PEI-HA (equivalent DOX concentration: 25 µM) for 24 h before and after exposure to NIR 808-nm laser (Power density: 0.6 W cm−2, irradiation time: 10 min). The asterisks denote statistical differences (*p < 0.05, **p < 0.01).



Figure 8. In vivo targeting combined multiple stimuli-responsive therapy study. (a) MCF-7-ADR tumor-bearing mice imaged by an infrared thermal imager under 808-nm laser irradiation at 0.6 W cm−2. (b) Body weights of the mice at different groups. (c) Tumor volume growth curves of mice after different treatments. (d) Photograph of tumors after excision. (e) Average tumors weight collected from each group of tumor-bearing mice after therapy. (f) H&E staining images of tumor in different groups after 25 days’ post injection (Group 1: Saline; Group 2: Saline+NIR; Group 3: MoS2-PEI-HA; Group 4: Free DOX; Group 5: MoS2-PEI-HA+NIR; Group 6: DOX@MoS2-PEI-HA; Group 7: DOX@MoS2-PEI-HA+NIR).


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Figure 9. Representative whole-body coronal PET images of MCF-7-ADR tumor-bearing mice at 0.5, 1, 2, and 4 h after i.v. injection of

64

Cu labeled (a)

nontargeting MoS2-PEI-NOTA and (b) targeting MoS2-PEI-HA-NOTA (n = 3 for each group) Circle: tumor area. (c) Corresponding organ biodistribution of

64

Cu

labeled MoS2-PEI and MoS2-PEI-HA in MCF-7-ADR tumor-bearing mice at 4 h after i.v. injection, calculated by γ-counter. The asterisks denote the statistical differences (*p < 0.05, **p < 0.01).



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Table of Contents

Multifunctional

MoS2

nanotheranostic

(MoS2-PEI-HA)

decorating

with

biocompatible hyaluronic acid (HA) were engineering designed. This nanotheranostic integrates MDR tumor-targeting, enzyme/pH/NIR responsive drug release, and reversing drug-resistance functions into one system to defeat drug-resistant breast cancer. The successfully functionalized MoS2-PEI-HA with NOTA-64Cu can be employed for PET imaging to real-time monitor therapeutic response.


pH

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