Dendrimer-Modified MoS2 Nanoflakes as a Platform for

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Dendrimer-Modified MoS2 Nanoflakes as a Platform for Combinational Gene Silencing and Photothermal Therapy of Tumors Lingdan Kong, Lingxi Xing, Benqing Zhou, Lianfang Du, and Xiangyang Shi ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 25 Apr 2017 Downloaded from http://pubs.acs.org on April 26, 2017

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ACS Applied Materials & Interfaces

Dendrimer-Modified MoS2 Nanoflakes as a Platform for Combinational Gene Silencing and Photothermal Therapy of Tumors Lingdan Kong†,§,Lingxi Xing‡,§,Benqing Zhou†,Lianfang Du*,‡, and Xiangyang Shi*,†,⊥

†

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

Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, P. R. China ‡

Department of Ultrasound, Shanghai General Hospital, School of Medicine, Shanghai Jiaotong

University, Shanghai 200080, P. R. China ⊥

CQM-Centro de Química da Madeira, Universidade da Madeira, Campus da Penteada, 9000-390

Funchal, Portugal

Keywords: MoS2 nanoflakes; dendrimers; photothermal therapy; gene silencing; tumors

*Corresponding authors. E-mail: [email protected] (L. Du) and [email protected] (X. Shi) §

Authors contributed equally to this work.

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Abstract Exploitation of novel hybrid nanomaterials for combinational tumor therapy is challenging. In this work, we synthesized dendrimer-modified MoS2 nanoflakes for combinational gene silencing and photothermal therapy (PTT) of cancer cells. Hydrothermally synthesized MoS2 nanoflakes were modified with generation 5 (G5) poly(amidoamine) dendrimers partially functionalized with lipoic acid via disulfide bond. The formed G5-MoS2 nanoflakes display good colloidal stability and superior photothermal conversion efficiency and photothermal stability. With the dendrimer surface amines on their surface, the G5-MoS2 nanoflakes are capable of delivering Bcl-2 (B-cell lymphoma-2) siRNA to cancer cells (4T1 cells, a mouse breast cancer cells) with excellent transfection efficiency, inducing 47.3% of Bcl-2 protein expression inhibition. In vitro cell viability assay data show that cells treated with the G5-MoS2/Bcl-2 siRNA polyplexes under laser irradiation have a viability of 21.0%, which is much lower than other groups of single mode PTT treatment (45.8%) or single mode of gene therapy (68.7%). Moreover, the super efficacy of combinational therapy was further demonstrated by treating a xenografted 4T1 tumor model in vivo. These results suggest that the synthesized G5-MoS2 nanoflakes may be employed as a potential nanoplatform for combinational gene silencing and PTT of tumors.

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Introduction Conventional cancer treatments such as surgical operation, chemotherapy, and radiotherapy suffer severe drawbacks. For instance, tumor site may not be excised completely, resulting in the metastasis of cancer cells; long-term chemotherapy leads to multidrug resistance of tumors, making the cure of cancer be difficult; in addition, the same treatment for cancer cell killing can also lead to the damage of healthy tissue or organs, causing side effects including nausea, vomiting, baldness and severe fatigue, etc.1-3 Therefore, it is crucial to design multifunctional integrated therapeutic delivery nanoplatforms combining different therapeutic modalities such as gene therapy, chemotherapy, photodynamic therapy, radiotherapy, and photothermal therapy (PTT). In recent years, PTT has been considered to be powerful for cancer treatment due to its high local tumor treatment efficacy and less side effects.4-7 In most of the cases, nanomaterials with superior photothermal conversion efficiency under near-infrared (NIR) laser irradiation have been used for effective tumor PTT. The nanomaterials used display NIR absorption feature, and convert optical energy to heat, thereby inducing local high temperature in the tumor tissue to destroy the cancer cells. For PTT of tumors, different nanomaterials systems have been developed including 1) noble metal NPs such as Au NPs with different shapes (nanorods,8 nanostars,5, 9-10 Au nanomatryoshkas,11 nanochains12), Au-Ag alloy urchin-shaped nanostructure,13 and Pd nanosheets;14-16 2) carbon-based nanomaterials (graphene or graphene oxide,17-19 carbon nanotubes,20 carbon dots;21-23 3) metal sulfide NPs (CuS24-25 and ZnS26-27); and 4) conducting polymer-based NPs.28-30 Owing to the advantages including high specific surface area, easiness to be synthesized, high photothermal conversion efficacy, and good biocompatibility, MoS2 nanoflakes have been used as photothermal agents30 or biosensors.31-32 To render the MoS2 nanoflakes with desired colloidal stability 3



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or functionality, the particles have been surface modified with polymers such as polyethylene glycol (PEG) 7 or polyethylenimine.33-34 Nevertheless, individual treatment via PTT is difficult to guarantee the complete killing of cancer cells. Hence, combinational treatments seem to be crucial and necessary for complete and enhanced tumor killing.35 Small interfering RNA (siRNA)-induced cancer gene therapy has been considered to be an alternative promising cancer treatment means. Via the specific gene silencing of cancer cells induced by the corresponding siRNA, cancer cells can be selectively inhibited or killed.36 However, due to the easy degradability of siRNA, it is necessary to develop a safe and highly efficient gene carrier for siRNA delivery to cancer cells. Previously, we have shown that dendrimer-entrapped gold nanoparticles (Au DENPs) and functional Au DENPs can be used as effective carriers for pDNA37-38 or siRNA36 delivery, which is presumably due to the fact that the entrapment of Au NPs within dendrimers enables the reservation of the 3-dimensional shape of the dendrimers, thereby enhancing the DNA compactness of the vectors.37-39 Further, our prior work has also shown that different inorganic NPs can be modified with dendrimers via either electrostatic self-assembly40-43 or covalent binding.44 These prior work related to the synthesis of MoS2 nanoflakes and the dendrimer modification of NPs leads us to hypothesize that MoS2 nanoflakes formed using a hydrothermal approach can be modified with dendrimers via a covalent reaction, thereby providing a unique platform for siRNA delivery-mediated gene silencing and PTT of cancer cells in vitro and tumors in vivo. The major advantages of the proposed dendrimer-modified MoS2 nanoflakes are that the enhanced gene compaction ability could be rendered by the modified dendrimers and that the versatile dendrimer surface chemistry can be applied to afford different functionalities. In this work, a simple approach was developed to synthesize dendrimer-modified MoS2 nanoflakes for combinational gene silencing and PTT of tumors. MoS2 nanoflakes were first synthesized via a hydrothermal approach.1 Generation 5 (G5) poly(amidoamine) (PAMAM) dendrimers with amine termini were reacted with lipoic acid (LA) via 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide 4


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hydrochloride (EDC) coupling chemistry to form G5-LA conjugates. Then the MoS2 nanoflakes were modified with the G5-LA conjugates via disulfide bonding according to the literature to form the G5-MoS2 nanoflakes (Scheme 1).1, 45 Subsequently, the formed G5-MoS2 nanoflakes were used as a vector to condense the Bcl-2 (B-cell lymphoma-2) siRNA through electrostatic interaction. The obtained G5-MoS2 nanoflakes and G5-MoS2/siRNA complexes were thoroughly characterized in terms of their structures, compositions, sizes, shapes, surface potentials, and photothermal conversion efficiency. The cytocompability and gene silencing property of the G5-MoS2/siRNA polyplexes and the use of G5-MoS2/siRNA polyplexes for combinational gene therapy and PTT of cancer cells in vitro and a tumor model in vivo were systematically tested. To the best of our knowledge, this is the first report related to the design of dendrimer-modified MoS2 nanoflakes for the combinational gene silencing and PTT of tumors. Our work uniquely combines G5 dendrimers with excellent gene transfection efficiency and MoS2 nanoflake with high photothermal conversion efficacy, thus providing an excellent nanoplatform for potential combinational gene silencing and photothermal therapy of tumors.

Scheme 1. Schematic illustration of the synthesis of G5-MoS2 nanoflakes.

Experimental Section 5



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Preparation of G5-LA Conjugates. LA (3.996 mg) dissolved in 5 mL DMSO was mixed with EDC (51.81 mg) and NHS (33.18 mg) while stirring. Three hours later, the activated LA was dropped into a G5.NH2 dendrimer solution (50 mg, in 5 mL DMSO) under vigorous magnetic stirring for 24 h. The reaction mixture was subjected to dialysis against water (4 L, 6 times, 3 days) via a dialysis membrane with an MWCO of 14 000. Followed by lyophilization, the product of G5-LA conjugates was acquired. Preparation of G5-MoS2 Nanoflakes. MoS2 nanoflakes were synthesized according to a literature protocol.14 Then G5-LA dendrimers (30 mg, in 3 mL water) were added into the suspension of MoS2 nanoflakes (6 mg, in 3 mL water), and the mixture was sonicated for 30 min and then stirred for another 12 h. The mixture was collected by centrifugation at 10 000 rpm for 5 min and rinsed with water for at least 3 times to remove redundant G5-LA dendrimers, followed by lyophilization to get the black powder of the G5-MoS2 nanoflakes. A portion of the G5-MoS2 nanoflakes was dispersed in PBS (pH = 7.4) or water and stored at 4 ºC before further use. Characterization Techniques. The structure, size, morphology, surface property, and composition of the G5-LA dendrimers and G5-MoS2 nanoflakes were characterized with 1H NMR, UV-vis spectroscopy, transmission electron microscopy (TEM), field emission scanning electron microscopy (FE-SEM), dynamic light scattering (DLS), zeta potential measurements, thermal gravimetric analysis (TGA), Fourier transform infrared (FTIR) spectroscopy, and inductively coupled plasma-optical emission spectroscopy (ICP-OES). The number of the primary amines on the surface of G5-MoS2 nanoflakes was measured on the basis of the manufacturer’s instruction of the PANOPA Kit. The photothermal conversion efficiency of the G5-MoS2 nanoflakes was analyzed using a laser device (Shanghai Xilong Optoelectronics Technology Co. Ltd., Shanghai, China) with a wavelength of 808 nm according to our previous work.5, 9 Preparation and Characterization of G5-MoS2/siRNA Polyplexes. G5-MoS2/siRNA polyplexes were prepared by mixing Bcl-2 siRNA and appropriate amount of G5-MoS2 nanoflakes under different 6


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N/P ratios (the molar ratio of primary amines of the dendrimers to phosphates in the siRNA backbone) in PBS and the mixture was gently votexed and incubated for 30 min at room temperature. For gel retardation assay, G5-MoS2/siRNA polyplexes were prepared using 1 µg siRNA under different N/P ratios and the final volume of each polyplex was set at 20 µL. For the measurements of the hydrodynamic size and surface potential of the G5-MoS2/siRNA polyplexes, Bcl-2 siRNA (5 µg) dissolved in diethyl procarbonate (DEPC) water was condensed by different concentrations of G5-MoS2 in PBS to reach a final volume of 1 mL at an N/P value of 2.5, 5, 10, or 15, respectively to form the polyplexes. Gel retardation, dynamic light scattering (DLS) and zeta potential measurements were used to characterize the G5-MoS2/siRNA polyplexes according to literature protocols.36 Cytotoxicity and Hemolysis Assays. 4T1 cells were normally cultivated in DMEM. Cells were incubated at 37ºC and 5% CO2, and were regularly passaged when reaching an 80-100% confluence. In vitro cytotoxicity of G5-MoS2 nanoflakes ([Mo] = 0 ~ 500 µg/mL) and G5-MoS2/siRNA polyplexes ( [Mo] = 0 ~ 500 µg/mL, 1 µg siRNA for each sample) were carried out by CCK-8 assay of the viability of 4T1 cells according to literature protocols.46 To evaluate the hemocompatibility of G5-MoS2/siRNA complexes, hemolysis assay of mouse red blood cells was performed according to our previous procedures.46-47 In Vitro SiRNA Transfection and Gene Silencing. The Bcl-2 siRNA transfection efficiency using G5-MoS2 as a vector was assessed through flow cytometry. Bcl-2 siRNA labeled with Cy3 dye was compacted with G5-MoS2 in order to detect the Cy3 fluorescence signals in 4T1 cells. The cells with a density of 1.0 × 105 cells/well were seeded on a 12-well plate and incubated with 1 mL DMEM supplemented with 10% FBS at 37 ºC and 5% CO2 overnight. When the cells reached a 60 - 70% confluence, the medium in each well was replaced with 1 mL serum-free DMEM containing G5-MoS2/siRNA polyplexes (in 100 µL PBS, 1 µg siRNA) with different N/P ratios (2.5, 5, 10 and 15, respectively) and the cells were incubated for 4 h. The cells treated with PBS and free siRNA were set as controls. Afterwards, the cells were washed with PBS (pH 7.4) for three times, digested by trypsin, 7



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and centrifuged at 1 000 rpm for 5 min to collect cells. Finally, the cells were dispersed in 1 mL PBS and analyzed via flow cytometry (FACS Calibur, BD Biosciences, Franklin Lake, NJ). To observe the intracellular localization of G5-MoS2/siRNA polyplexes, confocal laser scanning microscopy (CLSM) analysis was performed. Under the same cell culture conditions as described above for flow cytometry analysis, the cells were seeded onto a 12-well plate with round cover slip sitting at the bottom of each well. The cells were incubated with 1 mL DMEM consisting 100 µL of G5-MoS2/siRNA polyplexes (1 µg siRNA) at different N/P ratios (2.5, 5, 10, and 15, respectively). After 4 h incubation, the cells were then washed with PBS (pH 7.4) for three times, fixed with glutaradehyde (2.5 %, in 500 µL PBS) for 15 min at 4 ºC, washed again with PBS for three times, stained with DAPI (0.1 µg/mL, 500 µL) for 10 min at 37 ºC, and then washed with PBS. Finally, the cells were observed using Zeiss confocal laser scanning microscopy (CLSM, Jena, Germany) equipped with a 63× oil immersion lens. To test the siRNA delivey-associated gene silencing effect, 4T1 cells were seeded into a 12-well plate and treated with the G5-MoS2/siRNA polyplexes (1 µg siRNA for each sample) with an N/P ratio of 10 according to procedures described above. After cultivation for 48 h, the cells were treated with icy cold PBS for three times and lysed with 200 µL lysed buffer in an ice bath for 30 min. The supernatant was collected by centrifugation (12 000 rpm, 5 min) at 4 ºC to remove the cell debris. The protein expression level of Bcl-2 was tested by western blot assay according to the literature.36 Combinational PTT and Gene Therapy In Vitro. We first tested the use of G5-MoS2 nanoflakes for laser ablation of cancer cells in vitro. 4T1 cells were seeded on a 12-well plate with a density of 1.0 × 105 cells/well and cultivated at 37 ºC and 5% CO2 for 24 h. The cells were incubated with 1 mL DMEM containing PBS (100 µL) as a control or PBS solution of G5-MoS2 nanoflakes ([Mo] = 0.1 mg/mL, 100 µL) without laser irradiation or under an 808 nm NIR laser irradiation (1.2 W/cm2) for 5 min. Later, the cells were washed with PBS, fixed with glutaradehyde, washed with PBS again, and

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stained with DAPI according to the above protocols used for confocal microscopy observation before visualization by Zeiss inverted fluorescence microscope (Jena, Germany). To calculate the viability of 4T1 cells treated with the G5-MoS2 nanoflakes with or without laser irradiation, G5-MoS2 nanoflakes with different Mo concentrations ([Mo] = 0 ~ 500 µg/mL) were incubated to cells according to the procedures described above. After 24 h, the cells were irradiated with an 808 nm NIR laser (1.2 W/cm2) for 5 min, and cells without laser irradiation were also tested for comparison. After 4 h incubation, CCK-8 assay of cell viability was performed using protocols mentioned above. To test the efficacy of combinational gene therapy and PTT of cancer cells in vitro, the cells were cultured according to the above conditions and treated with PBS, free siRNA (1 µg siRNA), G5-MoS2 nanoflakes, and G5-MoS2/siRNA complexes (N/P ratio of 15, 1 µg siRNA). After 48 h cultivation, the cells were NIR laser irradiated (808 nm, 1.2 W/cm2) for 5 min, and those cells without laser irradiation were also tested for comparison. CCK-8 assay was used to test the cell viability. In Vivo Photothermal Imaging and Combinational PTT and Gene Therapy of Tumors. All experimental procedures for animal studies were approved by the institutional committee for animal care and also in accordance with the policy of National Ministry of Health. BALB/c nude mice (n = 9, 4-week-old, Shanghai Slac laboratory Animal center, Shanghai, China) were injected with 2 × 106 4T1 cells/mouse on the right front leg. When the volume of tumor reached 300 mm3, G5-MoS2/siRNA polyplexes ([Mo] = 1 mg/mL, 10 µg siRNA, in 0.1 mL NS) were intratumorally injected into the each mouse. For photothermal imaging, 4T1 tumor-bearing BALB/c nude mice were separated into three groups (n = 1 for each group) and intratumorally injected with normal saline (NS, 0.9%, 0.1 mL), G5-MoS2 nanoflakes ([Mo] = 1 mg/mL, in 0.1 mL NS), and G5-MoS2/siRNA polyplexes ([Mo] = 1 mg/mL, 10 µg siRNA, in 0.1 mL NS), respectively for each mouse. The tumor site was NIR laser irradiated (808 nm, 1.2 W/cm2) for 5 min and the dynamic whole-body infrared thermal images were

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collected by a thermal imager (FLIR A300, IRS Systems Inc., Shanghai, China) coupled with an infrared camera during laser radiation. To evaluate the G5-MoS2/siRNA polyplexes for combinational PTT and gene silencing in vivo, 4T1 tumor-bearing BALB/c nude mice were randomly divided into six groups (n = 5 for each group): Group I, 0.1 mL NS without laser (NIR-); Group II, 0.1 mL NS under laser irradiation (NIR+); Group III, G5-MoS2 nanoflakes without laser (NIR-, [Mo] = 1 mg/mL, in 0.1 mL NS); Group IV, G5-MoS2 nanoflakes plus laser (NIR+, [Mo] = 1 mg/mL, in 0.1 mL NS); Group V, G5-MoS2/siRNA polyplexes without laser (NIR-, [Mo] = 1 mg/mL, 10 µg siRNA, in 0.1 mL NS); Group VI, G5-MoS2/siRNA polyplexes plus laser (NIR+, [Mo] = 1 mg/mL, 10 µg siRNA, in 0.1 mL NS). The mice were intratumorally injected with the materials and NIR+ groups were given laser (power density = 1.2 W/cm2) for 5 min. The tumor volume was calculated according to the formula of tumor length × (tumor width) 2/2 and the relative tumor volume was calculated according to the formula of V/V0 (V and V0 is the tumor volume of a given time point and initial time point before treatment, respectively). H&E and TUNEL Staining of Tumor Sections. After the tumor mice were treated with NS, G5-MoS2/siRNA polyplexes without laser, G5-MoS2 nanoflakes plus laser, and G5-MoS2/siRNA polyplexes plus laser as described above for 4 days. The mice were killed and the tumors were extracted, sectioned, and H&E or terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) stained according to literature procedures.9-10 The tumor sections were observed by phase contrast microscopy (Leica DM IL LED inverted phase contrast microscope, Wetzlar, Germany). In Vivo Biodistribution and Histological Evaluations of the Major Organs of Mice. G5-MoS2/siRNA polyplexes ([Mo] = 1 mg/mL, 10 µg siRNA, in 0.1 mL NS) were intratumorally injected into each tumor-bearing mouse. The mice were sacrificed at 1 h, 24 h and 48 h post-injection, respectively. The major organs (heart, liver, spleen, lung, kidney and tumor) were harvested and digested with aqua regia. The amount of Mo in these organs was analyzed by ICP-OES.

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The tumor-bearing mice were intratumorally injected with the G5-MoS2/siRNA polyplexes ([Mo] = 1 mg/mL, 10 µg siRNA, in 0.1 mL NS for each mouse). At 7, 14 and 21 days postinjection, the mice were sacrificed and the major organs were collected, fixed, sectioned, and H&E stained. The organ sections were observed by a Leica DM IL LED inverted phase contrast microscope. The mice injected with 0.1 mL NS were used as control.

Results and Discussion Synthesis and Characterization of G5-MoS2 Nanoflakes. In this work, by combining the characteristics of both MoS2 nanoflakes that can be used as a photothermal reagent and dendrimers that can be used for gene delivery, we synthesized dendrimer-modified MoS2 nanoflakes. Hydrothermally synthesized MoS2 nanoflakes were modified with G5 dendrimers partially functionalized with lipoic acid (G5-LA) via disulfide bond (Scheme 1). The materials were well characterized via different methods. First, G5-LA conjugates were synthesized by linking carboxylic acid of LA with the dendrimer terminal amines via EDC coupling (Figure S1, Supporting Information). The structure of G5-LA conjugates was characterized by 1H NMR spectroscopy (Figure S2, Supporting Information). The characteristic methylene proton peaks of LA in the range of 1.25-2.21 ppm and the methylene protons of G5 dendrimers (2.2-3.4 ppm) can be recognized. Through NMR integration, the number of LA moities attached onto each G5 dendrimer can be quantified to be 11.4. MoS2 nanoflakes were synthesized via a known hydrothermal approach reported in the literature.1 The formed MoS2 nanoflakes display apparent strong NIR absorption feature (Figure S3, Supporting Information) in accordance to the literature.1 After modification of G5-LA dendrimers onto the surface of MoS2 nanoflakes, the NIR absorbance feature of the G5-MoS2 nanoflakes does not appreciably 11



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change, implying that the modification of G5 dendrimers does not seem to impact the photothermal conversion behaviors. The grafting efficiency of G5 dendrimers onto the MoS2 nanoflakes was quantified by TGA. At 800 ºC, the weight losses of MoS2 and G5-MoS2 are 21.0% and 29.5%, respectively (Figure S4, Supporting Information). The grafting percentage of G5 dendrimers onto the MoS2 nanoflakes can be calculated to be 8.5%.

Figure 1. TEM (a) and FESEM (b) images of MoS2 nanoflakes (I) and G5-MoS2 nanoflakes (II). Inset of (a) shows the magnified nanoflake(s).

The morphology and size of MoS2 nanoflakes before and after dendrimer modification were observed via TEM and FESEM (Figure 1). Clearly, the flake morphology of MoS2 does not significantly change after dendrimer surface modification, instead it seems that after dendrimer modification the aggregated flakes are tightly bound to form closely packed spherical particulate flakes having an average approximate diameter of 150 nm (Figure 1a). The thickness of the prepared MoS2 12


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nanoflakes before dendrimer modification was measured to be 25.1 ± 3.6 nm. Energy dispersive spectroscopy (EDS) analysis of the G5-MoS2 nanoflakes indicated the existence of Mo and S elements that are attributed to the MoS2, and C, N and O elements that are attributed to the backbone composition of the G5 dendrimers (Figure S5, Supporting Information). The morphological change of MoS2 was further confirmed by FESEM (Figure 1b), and the dendrimer modification onto the MoS2 surface seems not to change the aggregated flake structure, but stabilize the flake aggregates to form the close packed particles, in consistence with the TEM results. The hydrodynamic size and surface potential of the MoS2 and G5-MoS2 nanoflakes were also measured (Figure S6a, Supporting Information). DLS data reveal that the hydrodynamic size of MoS2 nanoflakes before and after dendrimer modification is 879 nm and 420 nm, respectively. This suggests that dendrimer modification is able to stabilize the aggregated MoS2 nanoflakes, in accordance with the TEM and FESEM results. It is worth noting that the hydrodynamic sizes of MoS2 and G5-MoS2 nanoflakes are 3-4 times larger than that measured by TEM and FESEM. This might be attributed to fact that DLS measures aggregated or clustered particles in aqueous solution, which may consist of several nanoflake aggregates, whereas TEM or FESEM measures the samples in a dry state and the size of single nanoflake aggregates was measured. Furthermore, zeta potential measurements (Figure S6b, Supporting Information) reveal that the negatively charged MoS2 nanoflakes (-41.0 mV) shifts to be positive after dendrimer modification (34.3 mV). Our results suggest that the dendrimer modification not only stabilize and compact the MoS2 nanoflakes, but also renders the nanoflakes with a positively charged surface potential. The dendrimer modification of MoS2 nanoflakes was further characterized by FTIR spectrum (Figure S7, Supporting Information), where a strong band at 3442 cm-1 is attributed to the N–H stretching vibration of secondary amine groups on the surface of G5 dendrimers hydrated with a slight amount of water.48-49 The absorption bands at 2923 and 2849 cm-1 are observed due to the existence of –CH2 groups, and the peak at 1633 cm-1 infers the presence of amide bond in the dendrimers. Besides, the peaks at 1083, 872, and 479 cm-1 can be assigned to MoS2.50-51 For gene 13



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delivery applications, the amine density of the G5-MoS2 nanoflakes was measured by PANOPA assay. The number of primary amines per G5 dendrimer was measured to be 40.5, thereby providing a great potential for effective siRNA condensation through electrostatic interaction (see below). The colloidal stability of MoS2 (dispersed in PBS), G5-MoS2 nanoflakes (dispersed in PBS) and G5-MoS2 nanoflakes (dispersed in DMEM with 10% FBS) was investigated by measuring the hydrodynamic size of the particles via DLS after they were exposed to different aqueous media for five days (Figure S8, Supporting Information). Clearly, the hydrodynamic size of the particles does not show prominent changes in a given time period, suggesting that the developed G5-MoS2 nanoflakes possess good colloidal stability.

Figure 2. (a) Temperature elevation plot of pure water and the aqueous suspensions of G5-MoS2 nanoflakes at different Mo concentrations (0.1, 0.5, 1.0, 1.5, and 2.0 mg/mL, respectively) under an 808-nm laser irradiation (1.2 W/cm2, 5 min). (b) Temperature change (∆T) of the aqueous suspensions of G5-MoS2 nanoflakes under laser irradiation for 5 min as a function of Mo concentration. (c) Temperature plot of the aqueous suspensions of G5-MoS2 nanoflakes ([Mo] = 0.5 mg/mL) during five cycles of laser on-off (laser on: 300 s; laser off: cool down to room temperature).

Photothermal Property of G5-MoS2 Nanoflakes. To prove the photothermal conversion performance of the G5-MoS2 nanoflakes, the particles in aqueous solution were NIR laser irradiated. As shown in Figure 2a, the trend of temperature increase enhances with the increase of the Mo concentration under the same experimental conditions. As a concrete manifestation, G5-MoS2 14


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nanoflakes with an Mo concentration of 2.0 mg/mL have a solution temperature increase from 22.5 to 74 ºC within 5 min laser irradiation, which is sufficient to kill cancer cells. In contrast, the control group of water shows no distinct temperature variation under the same experimental conditions. The temperature change (∆T) of the solution of G5-MoS2 nanoflakes at various Mo concentrations over a period of 300 s was recorded (Figure 2b). The ∆T at different Mo concentrations can be found to be 5.2, 22.9, 30.6, 39.1, 45.1 and 51.5 ºC, respectively at the Mo concentration of 0.1, 0.5, 1.0, 1.5, and 2.0 mg/mL. To further demonstrate the photothermal conversion stability of the G5-MoS2 nanoflakes, at the fixed Mo concentration (0.5 mg/mL), the photothermal efficiency was tested for five cycles of NIR laser irradiation (808 nm, 1.2 W/cm2, 5 min) and cooling down (Figure 2c). Apparently, the temperature increase does not have any appreciable changes, demonstrating their favorable stability and promising potential as PTT agents for tumor therapy. The photothermal conversion efficiency (η) of the G5-MoS2 nanoflakes under a given concentration (Mo concentration of 0.5 mg/mL) was quantified according to a method reported in the literature9 to be 47.8% (Figure S9, Supporting Information).

Figure 3. Gel retardation assay of G5-MoS2/siRNA polyplexes at different N/P ratios. Lane 1: siRNA alone; Lane 2: N/P = 0. 5 : 1; Lane 3: N/P = 1 : 1; Lane 4: N/P = 2 : 1; Lane 5: N/P =3 : 1; Lane 6: N/P = 4 : 1; Lane 7: N/P = 5 : 1; and Lane 8: N/P = 6: 1.

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Formation and Characterization of G5-MoS2/siRNA Polyplexes. Since naked siRNA is easily degraded by nuclease in the process of intracellular delivery and it is also difficult for the negatively charged siRNA to cross the negatively charged cell membranes, we then used G5-MoS2 nanoflakes possessing a positive surface potential as a carrier to deliver siRNA. The siRNA compaction capability of the G5-MoS2 nanoflakes through electrostatic interaction was evaluated via gel retardation assay (Figure 3). The migration of siRNA can be completely inhibited by G5-MoS2 at the N/P ratio of 2 or above. Therefore, an N/P ratio higher than 2 was selected for the formation of polyplexes between siRNA and G5-MoS2. An appropriate size and surface charge are extremely important for the transmembrane delivery of the polyplexes. Referring to the consequence of gel retardation assay, different N/P ratios of 2.5, 5, 10, and 15 were chosen to evaluate the hydrodynamic size and surface potential of the polyplexes (Figure 4). It is obvious that the hydrodynamic diameter of the G5-MoS2/siRNA polyplexes is in a range of 240-350 nm, which is a proper dimension for cellular gene delivery (Figure 4a). The mean particle size of the polyplexes generally increases with the N/P ratio, which might be attributable to the fact that at a high N/P ratio, more G5-MoS2 nanoflakes were used. Compared with the G5-MoS2 nanoflakes before siRNA compaction (420 nm), it seems that the formation of the G5-MoS2/siRNA polyplexes shrinks the size of the aggregated G5-MoS2 nanoflakes, further proving the compaction ability of the G5-MoS2 nanoflakes. Additionally, the morphology of the prepared polyplex was further observed by TEM (Figure S10, Supporting Information). At an N/P ratio of 10, the G5-MoS2/siRNA polyplex displayed a mean diameter around 300 nm, showing secondary aggregated structure of G5-MoS2 when compared to that of G5-MoS2 (Figure 1), confirming the formation of the polyplex. The measured size of the G5-MoS2/siRNA polyplex is in accordance with the hydrodynamic size measured by DLS.

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Figure 4. Hydrodynamic particle size (a) and surface potential (b) of the G5-MoS2/siRNA polyplexes under different N/P ratios (mean ± SD, n=3).

The surface potentials of the G5-MoS2/siRNA polyplexes under different N/P ratios were also tested to be around 30 mV (Figure 4b). Interestingly, the change of N/P ratios does not seem to influence the surface potential of the polyplexes. This suggests that although at a high N/P ratio, more G5-MoS2 nanoflakes were participated in the formation of the polyplexes, the overall surface charge density does not have a significant change. Furthermore, the polyplexes appear to have a slightly lower surface potential than the G5-MoS2 nanoflakes before siRNA compaction (34.3 mV), which is reasonable due to the slight neutralization of the dendrimer amines through electrostatic interaction between the particles and the siRNA. With the appropriate hydrodynamic size and positive surface potential, the formed G5-MoS2/siRNA polyplexes could be used for siRNA delivery. Cytotoxicity and Hemolysis Assays. The cytotoxicity of the G5-MoS2 nanoflakes and the G5-MoS2/siRNA polyplexes was tested via CCK-8 assay of the viability of 4T1 cells (Figure 5). After treatment with the G5-MoS2 nanoflakes under different Mo concentrations for 24 h, the cell viability decreases with the Mo concentration (Figure 5a). However, even at the highest concentration tested (500 µg/mL), the cell viability can still be higher than 75%, suggesting the good cytocompatibility of the vector materials. Further, we tested the cytotoxicity of the G5-MoS2/siRNA polyplexes (Figure 5b) 17



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under the same experimental conditions. Results reveal that the viability of cells incubated with the polyplexes is quite similar to that treated with the vector materials, indicating that the G5-MoS2/siRNA polyplexes possess good cytocompatibility, which is important for gene delivery applications.

Figure 5. Viability of 4T1 cells after treated with G5-MoS2 nanoflakes (a) and G5-MoS2/siRNA polyplexes (b) at different Mo concentrations for 24 h (mean ± SD , n = 6).

Next, the hemocompatibility of the G5-MoS2/siRNA polyplexes was tested via hemolysis assay (Figure S11, Supporting Information). The hemolysis percentages of the G5-MoS2/siRNA polyplexes are all below 4% even at the tested highest Mo concentration (500 µg/mL). Taken together, it is safe to conclude that the prepared G5-MoS2/siRNA polyplexes are cytocompatible and hemocompatible in the studied Mo concentration range. In Vitro SiRNA Transfection and Gene Silencing. The performance of siRNA delivery using the G5-MoS2 nanoflakes as a vector was tested by flow cytometry (Figure 6). Clearly, naked siRNA does not have apparent uptake by cells due to the negative charge of siRNA. In contrast, cells treated with the G5-MoS2/Cy3-labled siRNA polyplexes display much stronger Cy3 fluorescence intensity than those treated with naked siRNA, suggesting the apparent uptake of the polyplexes by cells. The fluorescence intensity of cells treated with the polyplexes increases with the N/P ratio. At an N/P ratio of 10, peak fluorescence intensity of the cells can be achieved. Further increasing the N/P ratio to 15:1 might lead to 18


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the toxic effect due to the increased amount of the polycationic vector used, hence the fluorescence intensity of cells decreases. Our data suggest that at an N/P ratio of 10, G5-MoS2 nanoflakes are able to transfect siRNA with the highest efficiency.

Figure 6. Fluorescent intensity of 4T1 cells treated with G5-MoS2/Cy3-labled siRNA polyplexes under different N/P ratios. Cells treated with PBS and naked siRNA were used as controls. Transfection was performed at a dose of 1 µg/well of siRNA for each polyplex. Data are presented as mean ± SD (n = 3).

The intracellular localization of the G5-MoS2/siRNA polyplexes was further examined using CLSM (Figure S12, Supporting Information). Compared to the controls of PBS and free Cy3-labled siRNA that do not have cellular uptake of the siRNA, cells treated with the G5-MoS2/Cy3-labled siRNA polyplexes display obvious red fluorescence spots, which is associated to the siRNA delivered into the cells. At an N/P ratio of 10, the cells show the strongest fluorescence signal, suggesting the most efficient cellular uptake of the polyplexes, in agreement with the flow cytometry analysis. With the successful transfection of Bcl-2 siRNA, corresponding mRNA could bind it and accordingly induced the knockout of the target protein. Western blot assay was performed to detect the corresponding protein expression levels in 4T1 cells, where GAPDH was used as a reference protein (Figure 7). Cells treated with the G5-MoS2/siRNA polyplexes show 47.3% of gene silencing, while 19



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almost 100% protein expression level can be achieved in control and free siRNA groups. In other words, G5-MoS2 nanoflakes have a potential to be used as an siRNA delivery vector to induce specific gene silencing in cancer cells.

Figure 7. (a) Western blot assay of the expression of Bcl-2 gene in 4T1 cells transfected with G5-MoS2/siRNA polyplexes at an N/P ratio of 10, where PBS and naked siRNA were used as control. The GAPDH protein was used as an internal control. (b) Quantitative analysis of relative Bcl-2 protein expression level for the western blot data.

Combinational PTT and Gene Silencing of Cancer Cells In Vitro. With the nice photothermal conversion efficiency, we first checked the ability of the G5-MoS2 nanoflakes to be used for laser ablation of cancer cells in vitro (Figure S13, Supporting Information). It is apparent that in the groups of PBS, G5-MoS2 without laser irradiation, and laser only, 4T1 cells still show a healthy morphology. In 20


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contrast, for the group of G5-MoS2 plus laser, there are no viable cells observed in the region of laser irradiation (marked with white dashed line), and outside of the laser irradiation region, cells are pretty healthy. Our data suggest that only under the circumstance of G5-MoS2 plus laser, cancer cells can be ablated, demonstrating the potential to use G5-MoS2 nanoflakes for cancer cell PTT.

Figure 8. (a) Viability of 4T1 cells treated with the G5-MoS2 nanoflakes at different Mo concentrations for 24 h, followed by laser irradiation (1.2 W/cm2, 5 min). The cells without laser irradiation were also tested for comparison. (b) Viability of 4T1 cells treated with PBS, free siRNA, G5-MoS2 nanoflakes, and G5-MoS2/siRNA polyplexes (1 µg siRNA, N/P ratio of 10) for 48 h with or without laser irradiation (1.2 W/cm2, 5 min). The Mo concentration of the G5-MoS2 nanoflakes was set at 263 µg/mL.

The viability of 4T1 cells treated with the G5-MoS2 nanoflakes at different Mo concentrations (0, 25, 50, 100, 200, and 500 µg/mL) with or without laser irradiation was quantified (Figure 8a). Apparently, cells treated with the G5-MoS2 nanoflakes without laser irradiation display a viability up to 80% or above for all Mo concentrations tested. Under the laser irradiation, the viability of cells decreases with the Mo concentration, especially in the concentration range of 50-500 µg/mL. At the Mo concentration of 500 µg/mL, almost 65.4% cells are killed, showing excellent tumor destruction effect. 21



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To explore the effect of combinational gene silencing and PTT of cancer cells, the viability of cells treated with PBS (as a control), free siRNA, G5-MoS2 nanoflakes, and G5-MoS2/siRNA polyplexes with or without laser irradiation were compared (Figure 8b). As opposed to the groups of PBS and free siRNA with or without laser irradiation, as well as G5-MoS2 without laser irradiation that cells displays good viability, cells treated with G5-MoS2 and G5-MoS2/siRNA polyplexes under laser irradiation show significant reduction of viability. The cell inhibition efficacy follows the order of G5-MoS2/siRNA polyplexes + laser (21.0%) > G5-MoS2 + laser (45.8%) > G5-MoS2/siRNA polyplexes (68.7%). Apparently, the combinational PTT and gene silencing gives rise to a significantly enhanced cancer cell inhibition efficacy. Bcl-2 siRNA can facilitate the low regulation of Bcl-2 protein in most of the cancer cells, which is beneficial for the growth inhibition of cancer cells.36 On the other hand, under the photothermal condition, cancer cells can be ablated due to the hyperthermia-induced destruction of the cell skeleton.5 These two mechanisms can be combined to promote synergistic therapy of cancer cells. Photothermal Imaging and Combinational PTT and Gene Silencing of Tumors in Vivo. The use of G5-MoS2/siRNA polyplexes for photothermal imaging and combinational PTT and gene silencing of tumors in vivo was next explored. The whole body infrared imaging of mice was performed (Figure S14a, Supporting Information). Clearly, the temperature of tumor site injected with the G5-MoS2 nanoflakes and G5-MoS2/siRNA polyplexes reaches 62.8 ºC and 60.2 ºC, respectively after 300 s laser irradiation (Figure S14b, Supporting Information). In contrast, the tumor site injected with NS just display a temperature elevation from 36.9 ºC to 39.0 ºC. The distinct temperature variation enables effective tumor thermal imaging after intratumoral injection of either G5-MoS2 nanoflakes or G5-MoS2/siRNA polyplexes. We next investigated the combinational treatment of a tumor model in vivo. As shown in Figure 9a, the relative tumor volume increases rapidly in control group (NS with or without laser irradiation) and G5-MoS2 group without laser irradiation. On the contrary, G5-MoS2/siRNA polyplexes without laser irradiation reduced the tumor growth rate to some extent, which can be attributed to the independent 22


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effect of gene silencing. What’s more, the tumor growth is significantly inhibited with the treatment of G5-MoS2 and G5-MoS2/siRNA polyplexes under laser irradiation. It seems that the combinational PTT and gene silencing enables the most effective tumor ablation. The photos of the xenografted tumor-bearing mice under different treatments were taken at different time points (Figure S15, Supporting Information), where the tumors treated with G5-MoS2 (NIR+) and G5-MoS2/siRNA (NIR+) can be completely ablated. The body weight of mice under different treatments at different time points does not display significant changes, implying that the injected G5-MoS2 or G5-MoS2/siRNA materials with or without laser irradiation do not affect the growth status of mice, thus showing no toxicity to the mice (Figure S16, Supporting Information). The survival rate of mice was monitored till 40 days posttreatment (Figure 9b). It is obvious that the average life span of mice for the G5-MoS2/siRNA (NIR+) group was 100% at day 40, which is significantly higher than the groups of G5-MoS2 (NIR+, 60%) and G5-MoS2/siRNA (NIR-, 40%). In contrast, mice in the groups of G5-MoS2 (NIR-), NS (NIR+), and NS (NIR-) all died at 32, 31, and 30 days posttreatment, respectively. The combinational PTT and gene silencing of tumors was further evaluated via H&E and TUNEL staining. H&E staining results (Figure 9c) reveal that only sparse necrosis region can be seen after the treatment of G5-MoS2/siRNA (NIR-), and the necrosis region is slightly larger than the NS control. The tumor necrosis region follows the order of G5-MoS2/siRNA (NIR+) > G5-MoS2 (NIR+) > G5-MoS2/siRNA (NIR-) > NS control. Similarly, TUNEL staining results (Figure 9d) show that the number of apoptotic cells in the G5-MoS2/siRNA (NIR-) group is quite small, while the treatment of G5-MoS2/siRNA (NIR+) and G5-MoS2 (NIR+) leads to the production of a large amount of apoptotic cells. By considering the quantitative analysis of apoptosis rate (Figure S17, Supporting Information), the apoptotic tumor cells follows the order of G5-MoS2/siRNA (NIR+, 68.1%) > G5-MoS2 (NIR+, 58.0%) > G5-MoS2/siRNA (NIR-, 21.9%) > Control (7.7%). Our results suggest that the effect of combinational therapy is superior to single PTT or single gene silencing treatment.

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Figure 9. (a) Relative tumor volume and (b) survival rate of the tumor-bearing mice after different treatments at different time periods. (c) H&E staining and (d) TUNEL staining of 4T1 xenografted tumors at 4 days post intratumoral injection of 0.1 mL NS (I), G5-MoS2/siRNA (NIR-, 10 µg siRNA, [Mo] = 1 mg/mL, in 0.1 mL NS) (II), G5-MoS2 (NIR+, [Mo] = 1 mg/mL, in 0.1 mL NS) (III), and G5-MoS2/siRNA (NIR+, 10 µg siRNA, [Mo] = 1 mg/mL, in 0.1 mL NS) (IV), respectively to each mouse.

In Vivo Biodistribution Study and Histocompatibility Evaluation. To analyze the metabolic status of the G5-MoS2/siRNA polyplexes after intratumoral injection, ICP-OES was performed (Figure S18, Supporting Information). With the time postinjection, the Mo uptake in the tumor region gradually 24


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decreases. What’s more, the amount of Mo element in liver presented a noticeable upward trend with time, while that in kidney did not present an obvious rising trend between 24 h and 48 h and showed a low uptake, implying that the materials are able to be mainly cleared by the reticuloendothelial system (RES) in the liver. The long-term organ compatibility of the G5-MoS2/siRNA polyplexes after intratumoral injection was assessed by H&E staining. Clearly, the slices of heart, liver, spleen, lung and kidney do not show any obvious changes at 7, 14, and 21 days posttreatment (Figure S19, Supporting Information), revealing that the developed G5-MoS2/siRNA polyplexes have good biosafety.

Conclusion In summary, we develop a novel combinational therapy system possessing PTT effect and gene silencing ability based on dendrimer-modified G5-MoS2 nanoflakes. The G5-LA conjugates synthesized were able to be linked onto the surface of MoS2 nanoflakes via disulfide bond, allowing for the generation of the G5-MoS2 nanoflakes. Our study represents the first example to uniquely combine inorganic 2-D materials of G5-MoS2 nanoflakes with synthetic spherical PAMAM dendrimers. The modification of G5 dendrimers renders the MoS2 nanoflakes with compact size and positive surface potential, which is suitable for siRNA delivery without compromising their photothermal conversion capability. The enhanced therapeutic efficacy of the combinational PTT and gene silencing treatment was validated in the treatment of cancer cells in vitro and a xenografted tumor model in vivo. Furthermore, by considering the art of dendrimer chemistry, other imaging agents and drugs may be conveniently linked onto this platform, thus holding a promising potential for theranostics of different types of cancer.

Acknowledgements This research was financially supported by the National Natural Science Foundation of China 25



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(21273032, 81571679, and 81271596), the Fundamental Research Funds for the Central Universities, and the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning. X. Shi acknowledges the support from the FCT-Foundation for Science and Technology (project PEst-OE/QUI/UI0674/2013, CQM, Portuguese Government funds) and funding through the project M1420-01-0145-FEDER-000005 - Madeira Chemistry Center - CQM + (Madeira 14-20).

Supporting Information Additional experimental details and 1H NMR spectrum of G5-LA dendrimers, UV-vis spectroscopy, TGA, EDS, FTIR, TEM, DLS, and zeta potential of MoS2 and G5-MoS2 nanoflakes, photothermal conversion efficiency (η) of G5-MoS2 nanoflakes, hemolysis assays of G5 -MoS2/siRNA polyplexes, the intracellular localization of G5-MoS2/siRNA polyplexes, data of cell adhesion ability after different treatments, in vivo photothermal images and temperature profiles of tumors after different treatments, digital pictures and body weight of tumor-bearing mice after different treatments at different time periods, apoptosis rate of 4T1 cells after different treatments, in vivo biodistribution of G5-MoS2/siRNA polyplexes, and H&E staining images of major organs after intratumoral injection of G5-MoS2/siRNA polyplexes at different time periods. This material is available free of charge via the Internet at http://pubs.acs.org.

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Dendrimer-Modified MoS2 Nanoflakes as a Platform for

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