Investigation of Thermally Induced Cellular Ablation and Heat

Feb 23, 2017 - Such promising studies provide the great opportunity to better understand the cellular basis of light-triggered thermal response. Moreo...
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Investigation of Thermally Induced Cellular Ablation and Heat Response Triggered by Planar MoS2‑Based Nanocomposite Shinya Ariyasu,†,‡ Jing Mu,*,†,‡ Xiao Zhang,§ Ying Huang,§ Edwin Kok Lee Yeow,† Hua Zhang,*,§ and Bengang Xing*,†,# †

Division of Chemistry & Biological Chemistry, School of Physical & Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore, 637371, Singapore § Center for Programmable Materials, School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore # Institute of Materials Research and Engineering (IMRE), Agency for Science, Technology and Research (A*STAR), Singapore, 117602, Singapore S Supporting Information *

ABSTRACT: In comparison to conventional tumor treatment methods, photothermal therapy (PTT) is one of the innovative therapeutic strategies that employs light to produce localized heat for targeted ablation of cancer cells. Among the various kinds of heat generation nanomaterials, transition metal dichalcogenide nanosheets, especially molybdenum disulfide (MoS2), have recently been investigated as one of the promising PTT candidates because of their strong absorbance in the near-infrared (NIR) tissue transparency window and excellent photothermal conversion capability. In line with the great potential of MoS2-based nanomaterials in biomedical applications, their intrinsic therapeutic performance and corresponding cellular response are required to be continually investigated. In order to further improve MoS2-based PTT efficacy and dissect the molecular mechanism during heat stimuli, in this study, we successfully designed a novel and effective PTT platform by integration of MoS2 nanosheets with peptide-based inhibition molecules to block the function of heat shock proteins (Hsp90), one type of chaperone proteins that play protective roles in living systems against cellular photothermal response. Such a combined nanosystem could effectively induce cell ablation and viability assays indicated approximately 5-fold higher PTT treatment efficacy (8.8% viability) than that of MoS2 itself (48% viability) upon 808 nm light irradiation. Moreover, different from the case based on MoS2 alone that could cause tumor ablation through the process of necrosis, the detailed mechanism analysis revealed that the inhibition of Hsp90 could significantly increase the photothermally mediated apoptosis, hence resulting in remarkable enhancement of photothermal treatment. Such promising studies provide the great opportunity to better understand the cellular basis of light-triggered thermal response. Moreover, they can also facilitate the rational design of new generations of PTT platforms toward future theranostics.

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efficacy against cancer resistance.4 In line with such promising capabilities in tumor treatment, so far, numerous nanoscale materials including gold (Au) nanostructures,5,6 CuS nanocrystals,7−9 carbon-based nanotubes, and graphene derivatives10,11 have been extensively exploited as functional platforms for photothermal treatment of a variety of cancers in vitro and in vivo. As a new type of 2D material, transition metal dichalcogenides, especially molybdenum disulfide (MoS2) nanosheets, have recently emerged and gained significant interest in material science due to their unique optical and electrical properties such as large intrinsic band gap and high

INTRODUCTION

Despite a variety of conventional strategies including surgery, chemotherapy, and radiotherapy being frequently used to treat cancer patients, cancer is still one of the leading causes of human death. The limited efficacy, adverse effect in healthy tissues, and potential tendency for drug resistance remain huge challenges during the course of the anticancer treatment. Thus, alternative tumor therapy modalities are still highly required to expand treatment options to benefit patients and society. Among various treatment strategies, photothermal therapy (PTT), which employs a beam of light in the visible or nearinfrared (NIR) region to generate heat for ablation of cancer cells, has been recently considered as one promising therapeutic option.1−3 Compared with the conventional modalities, PTT exhibits great advantages owing to its minimal invasiveness, higher precision, spatial-temporal selectivity, and greater © XXXX American Chemical Society

Received: December 27, 2016 Revised: February 21, 2017 Published: February 23, 2017 A

DOI: 10.1021/acs.bioconjchem.6b00741 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Scheme 1. Schematic Illustration of the Preparation of MoS2−CS-Cype and Subsequent Cellular Response under the 808 nm Light Irradiationa

(i) Exfoliation process; (ii) thiolated chitosan modification; (iii) adhesion of Cype onto MoS2−CS; (iv) 808 nm irradiation and photothermal treatment. a

carrier mobility.12−16 More importantly, additional attributes in their higher absorbance at the NIR window, as well as excellent photothermal conversion efficiency, enable MoS2 nanosheets to serve as robust PTT agents toward more efficient disease diagnosis and therapy.17−22 Currently, extensive studies have also established that MoS2 nanosheets present good biocompatibility, promising dispersibility in aqueous media, as well as high loading capacity of therapeutic molecules owing to their unique 2D morphologies, which thus open bright prospects for their rational design toward the combination therapy of cancer.23−26 Despite the great potential of light induced photothermal modality in cancer therapies, the thermal treatment efficacy could be compromised by the intrinsic heat shock response originating from heat shock proteins (HSPs) in the cell structures.27 HSPs, a conserved family of ATP-mediated chaperone proteins, are usually induced by the physiological and environmental stimuli (e.g., chemicals, heat shock, or light) and play protective roles in protein refolding, cellular recovery, and anti-apoptotic signaling.28−30 Among these heat shock protein variants, heat shock protein 90 (Hsp90) is one of the most representative molecular chaperones and responsible for assembling and protecting various client proteins from denaturation and aggregation. Once temperature increases, Hsp90 induces heat-shock response and results in strong cytoprotective effect and resistance against photothermal treatment.31,32 As one type of native protective protein against thermal response, Hsp90 has also been found at increased levels in various types of tumor areas, as compared to normal cells and tissues, where the expression of Hsp90 is retained at a rather low level. Hsp90 assists in the maturation and translation process of many oncogenic proteins, which are crucial for tumor cell growth and metastasis.33−35 Hence, the inhibition of such housekeeping proteins, and meanwhile, blocking their relevant heat-shock responses, would not only suppress proliferative signals of tumor cells, but also enhance the tumor sensitivity to the photothermal therapies.36−38 In this work, we present a unique and reliable PTT platform, whose antitumor efficacy can be greatly improved by incorporating the Hsp90 inhibitor into the MoS2 nanosheets. Upon suitable NIR light irradiation, such a combined nanosystem (MoS2−CS-Cype) could remarkably reduce cell

viabilities in cancer cells when compared to the MoS2 nanosheets alone. More importantly, the detailed cellular mechanistic studies reveal that the inhibition of Hsp90 could greatly promote the photothermal-mediated apoptosis, which eventually resulted in the improved treatment effect.

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RESULTS AND DISCUSSION Scheme 1 demonstrated the synthetic procedure for the MoS2inhibitor hybrid nanosystem and 808-nm-induced photothermal effect for cellular ablation. First, MoS2 nanosheets were prepared through an electrochemical intercalation and exfoliation process established by our group previously.39,40 Although the prepared MoS2 nanosheets can disperse in pure water very well, such unmodified nanosheets easily form aggregates and precipitates in the presence of salts (Figure S1). Therefore, in order to enhance their stability and dispersibility in biological context, MoS2 nanosheets were further coated with thiolated chitosan, one type of commonly used linear cationic polysaccharides in pharmaceutics and medicinal chemistry owing to its excellent biocompatible and biodegradable properties.41,42 To investigate the possibility of effective chitosan modification, the formation of chitosan-coated MoS2 nanosheets (MoS2−CS) was first characterized by atomic force microscopy (AFM) (Figure S2). It was found that original MoS2 nanosheets showed an average thickness of ca. 1 nm only, while after chitosan modification, the average thickness of nanosheets increased to ca. 4−6 nm (Figure S2). Moreover, the ninhydrin test for the modified MoS2−CS hybrids further revealed that there was an intense absorption increase at wavelength 570 nm, suggesting the existence of free amino groups after the functionalization of MoS2 with chitosan molecules (Figure S3). All these results clearly verified the successful coating of chitosan layer on MoS2 surface in MoS2− CS. As expected, the MoS2−CS exhibited good dispersibility in both phosphate buffered saline (PBS) and Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) (Figure S1). More importantly, in order to effectively suppress Hsp90 activity, the typical Hsp90 protein inhibitor analog, the cyclic peptide sequence (Cype) that has capability to specifically bind to N-middle domain of Hsp90 to block the protein function, was chosen in our design,38,43,44 and its potent B

DOI: 10.1021/acs.bioconjchem.6b00741 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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conditions or in cell culture medium (DMEM). The release of inhibitor Cype from MoS2−CS was calculated on the basis of the concentration of Cype in the supernatant before and after incubation under different conditions (Figure 1c). Although there was a slight release of Cype over the prolonged incubation time (e.g., at 48 h), the whole system presents sufficient stability over the duration that can be used for the subsequent photothermal ablation studies. To further verify the ability of photothermally controlled release of Hsp90 inhibitors from the MoS2−CS surface, MoS2− CS-Cype in PBS was exposed to 808 nm laser irradiation with different dosage. As shown in Figure 1d, upon NIR light irradiation with 1 W/cm2 for 10 min, HPLC analysis demonstrated that ∼50% of inhibitor release can be observed. More peptide release (e.g., ∼80%) can be clearly monitored when higher power of NIR light irradiation was applied (2 W/ cm2), suggesting that the photothermal release of inhibitor Cype on MoS2−CS-Cype was a process dependent on the dosage of the laser irradiation. These results distinctly showed that such a designed complex could serve as a highly effective NIR light stimuli-responsive platform for controlled tumor treatment. After the characterization of the performance of MoS2−CSCype as PTT platform, we evaluated its therapeutic effect on tumor cells. The cell viability studies were carried out by using standard MTT assay. In this typical study, the Hsp90overexpressed tumor cell line, HCT-116 cells were chosen as a typical example.38 Basically, HCT-116 cells were incubated with MoS2−CS, Hsp90 inhibitor Cype, and MoS2−CS-Cype conjugates, respectively. As shown in Figure 2a, there were no obvious cytotoxic effects observed in all tested samples without photo irradiation. In contrast, when exposed to NIR light at 1 W/cm2 for 5 min, the cell viability after treatment with MoS2− CS was determined as 47 ± 1.8%; this value is regarded as original photothermal efficacy of MoS2. On the other hand, treatment of HCT-116 cells with Cype in the presence of NIR light irradiation did not induce serious cytotoxicity (viability 88 ± 3.3%). However, the MoS2−CS-Cype with NIR light excitation showed much lower cell viability (8.8 ± 1.1%) than MoS2−CS, suggesting that the MoS2 peptide conjugate exhibited more potent photothermal activity against HCT-116 cells. These amazing results strongly support that the successful incorporation of Hsp90 inhibitor Cype could greatly improve the therapeutic effect as compared to those conventional MoS2based platforms. Moreover, further explorations were performed to uncover the biological pathways regarding how the HCT-116 cells respond to light irradiation and how the Hsp90 inhibitor Cype was involved in the enhancement of PTT efficacy in MoS2−CSCype (Figure 2). So far, numerous nanomaterials such as gold, graphene, and carbon nanotubes have been extensively applied for hyperthermia in cancer treatment, in which most studies proposed that the necrosis pathway would be involved in the cell death, especially under high dosage of laser irradiation or higher temperature treatment.45−47 As a typical process for tumor cell ablation, necrosis has been well characterized by loss of plasma membrane integrity and subsequent release of intracellular contents into the extracellular milieu.48,46 In order to verify the possibility of thermally induced necrosis in HCT116 cells, the standard CytoTox 96 assay was applied to detect leakage of lactate dehydrogenase (LDH) from the cytosol after rupture of the cell membrane caused by necrosis.46 As shown in Figure 2b, there was almost no LDH-leakage detected in HCT-

inhibition effect on the Hsp90 functions was also confirmed by citrate synthase aggregation assay (Figure. S4). Considering the unique high-surface-area characteristic and molecule loading capacities of MoS2 nanosheets, we evaluated the feasibility of whether the cyclic peptide Cype could be effectively loaded on the surface of chitosan modified MoS2 nanosheets. Typically, the Cype peptide was added into the MoS2−CS solution and the mixture was stirred for 24 h. After the reaction, the free cyclic peptides were removed by centrifugation and the supernatant was analyzed by high performance liquid chromatography (HPLC). Upon the HPLC quantification of free peptide (Cype) in the supernatant, the loading ratio of Cype onto MoS2 surface was determined to be 73.2 ± 3.7%. Such high loading capabilities suggested the strong interactions between MoS2 nanosheets and cyclic peptide, which offers great advantages in the construction of optimal combination platforms for PTT use in cancer therapy. We subsequently evaluated the basic physicochemical properties of MoS2−CS-Cype. The UV−vis spectra of original MoS2 nanosheets, MoS2−CS and MoS2−CS-Cype, were shown in Figure 1a. The absorption intensity from the UV to NIR

Figure 1. Physical properties of MoS2−CS-Cype. (a) UV−vis spectra of MoS2, MoS2−CS and MoS2−CS-Cype (10 μg/mL in H2O). (b) Temperature changes of solutions containing MoS2, MoS2−CS, or MoS2−CS-Cype. (c) Stability test of MoS2−CS-Cype under the incubation in PBS buffer (pH 7.4), acetate buffer (pH 5.0), and DMEM. (d) Time-dependent Cype release profile from MoS2−CSCype under photoirradiation of 1 or 2 W/cm2 NIR (808 nm) laser.

region is almost identical, indicating that the surface coating of chitosan and loading of Cype would not affect the optical properties of MoS2. To further evaluate photothermal conversion efficiency of MoS2 nanosheets, MoS2−CS and MoS2−CS-Cype, the aqueous solutions containing 10 μg/mL of each MoS2 nanosheets were exposed to an 808 nm continuous-wave laser at 1 W/cm2. As shown in Figure 1b, the temperature of pure water showed rather limited increase after NIR light irradiation for 10 min, whereas the aqueous solutions of all MoS2-containing material in this study were rapidly heated and their temperature reached over 50 °C within 10 min, which was mostly attributed to their strong absorption in the NIR region (Figure 1b). The results demonstrated that MoS2 nanosheets can be treated as an effective platform for PTT and surface modification would not affect its thermal properties. Next, we investigated the stability of cyclic-peptide inhibitor Cype on the MoS2−CS surface under different pH C

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Figure 2. Cellular function studies of MoS2−CS-Cype. (a) Viabilities of HCT-116 cells. (b) Normalized LDH-reslease. (c) Caspase-3 activities. (d) FACS analysis of HCT-116 cells when treated with Cype alone (12 μM), MoS2−CS (10 μg/mL), or MoS2−CS-Cype (10 μg/mL) with or without NIR photoirradiation (1 W/cm2, 5 min). HCT-116 was co-stained by Alexa Fluor 488-labeled AnnV and PI. The symbol * means significant difference (p < 0.05) between MoS2−CS and MoS2−CS-Cype.

from similar treatment with MoS2−CS and Cype alone. The different caspase-3 expression after PTT indicated that the integration of Hsp90 inhibitor with MoS2−CS under these heating conditions could induce obvious cellular apoptosis, thereby contributing to higher PTT potency against tumor cells. To further quantify the ratio of necrotic and apoptotic cells after MoS2-mediated PTT, photothermally treated HCT-116 cells were analyzed by fluorescence activated cell sorting (FACS) (Figure 2d). In this experiment, HCT-116 cells after photothermal treatment with MoS2−CS, Hsp90 inhibitor Cype, or MoS2−CS-Cype were co-stained with Alexa Fluor 488labeled Annexin-V (AnnV) and propidium iodide (PI), which are phosphatidylserine-binding protein and nuclear-staining reagents, typically used for cellular apotosis and necrosis identification.48 Apparently, apoptosis could induce the expoure of phosphatidylserin, enabling biniding to AnnV, whereas PI is unable to go across cellular membranes (AnnV(+)/PI(−)). In contrast, the permeabilization of cell membrane in the process of necrosis allowes both AnnV and PI to interact with surface and cellular targets, respectively (AnnV(+)/PI(+)). Quantative values of the number of live, apoptotic, and necrotic cells were illustrated in Figure 2d. Compared to MoS2−CS and Hsp90 inhibitor Cype, HCT-116 cell treatment with MoS2−CS-Cype presented the lowest values of the live cells, which was consistent with the cell viability results shown in Figure 2a. More importantly, conjugation of MoS2 with Hsp90 inhibitor Cype (MoS2−CS-Cype) could slightly enhance the necrosis process but would dramatically increase the occurrence of appoptosis. On the other hand, Hsp90 inhibitor Cype alone could not induce obvious cell death. These studies demonstrated that Hsp90 inhibition could effectively attenuate tumor cell resistance against thermal stress through the induction of photothermally mediated apoptosis.

116 cells upon phototheraml treatment with Hsp90 inhibitor Cype itself, whereas HCT-116 cells treated with MoS2−CS and MoS2−CS-Cype showed obvious LDH leakage upon effective NIR light irradiation at 808 nm, clearly suggesting that the photoexcitation of such 2D materials could induce the necrosis of HCT-116 cells. Interestingly, compared to the photothermal treatment based on MoS2−CS alone, the increase of LDHleakage levels in HCT-116 cells treated with MoS2−CS-Cype was found to be rather limited (∼0.29-fold increase). Considering the signifcantly enhanced photothermal cytotoxicity induced by MoS2−CS-Cype (∼5.34-fold increasing as shown in Figure 2a), the results implied that such a limited increase in necrosis may not be the main reason to induce the significant improvement of PTT efficacy in HCT-116 cells treated with MoS2−CS-Cype. In an attempt to identify the potential mechanism for such improved photothermal treatment, we further analyzed the possibility of the occurrence of other cellular death pathways (e.g., apoptosis) that might be involved in the process of MoS2−CS-Cype-mediated PTT. In contrast to necrosis, cell membrane integrity could be maintained during apoptosis, and “eat me” signals like phosphatidylserine which resides in the inner plasma membrane will relocate to the extracellular portion of the membrane to mark the cell for phagocytosis. Additionally, in the process of the apoptotic pathway, various enzymes such as caspases-3,-6,-9 will be activated. As one typical apoptosis-effector enzyme, caspase-3 has been frequently used as an apoptosis marker in cell biology studies.48 Therefore, in this typical study, we examined whether caspase-3 would be activated during the cellular photothermal treatment to identify possible occurrence of apoptotisis. As shown in Figure 2c, the apoptosis-associated enzyme function studies indicated that photothermal treatment based on MoS2−CSCype conjugates could lead to significant caspase-3 activity. In contrast, there was no obvious caspase-3 expression observed D

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(Hsp90) inhibitors for improved photothermal treatment. Basically, such prepared peptide-based inhibitor and 2D material conjugate (MoS2−CS-Cype) not only show favorable solubility and stability in aqueous media, but also have promising NIR absorption and effective photothermal conversion capability. Upon NIR photoirradiation, Hsp90 inhibitor in this MoS2 nanoconjugate can be rapidly released, which can effectively block the protective function of Hsp90, thereby attenuating cell resistance against the hyperthermia stimuli. Detailed investigations of cellular mechanism revealed that inhibition of Hsp90 could effectively increase the photothermally mediated apoptosis, hence resulting in remarkable enhancement of photothermal performance. Therefore, our systemic studies would be beneficial for optimizing the nanomaterial-based photothermal therapies and maximizing their treatment efficacy. More significantly, unraveling the mechanism in the cell death would greatly facilitate the rational design of next generations of PTT platforms toward personalized nanomedicine in the future.

Moreover, in order to gain insight into the molecular events involved in cellular response to Hsp90 inhibitors, relevant signaling protein activities were also analyzed in a more detailed manner. As one of the conserved molecular chaperones, Hsp90 participates in a series of biological events during apoptosis such as protein refolding, assembly, and stabilizing to ensure cell survival. During this process, a variety of co-chaperones such as Aha1, Hip, HOP, and Hsp70 are required to assist in Hsp90’s modulation and protect client proteins from degradation.31,49 Until now, it has been reported that alteration of Hsp90 protein function would potentially affect the expression of Hsp70, which has been considered a wellaccepted pharmacodynamic marker for inhibition of Hsp90.38 In this study, in order to verify the Hsp90 inhibition effect by Cype peptide inhibitor, immunofluoresent staining of Hsp70 in HCT-116 cells was carried out by using confocal laser scanning microscopy (CLSM) and flow cytometry (Figures S5 and S6). As shown in Figure 3, cells treated with MoS2−CS and NIR

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EXPERIMENTAL PROCEDURES Materials and Methods. All the chemical reagents were purchased from Sigma-Aldrich. Commercially available reagents were used without further purification. HCT-116 cells were purchased from American Type Culture Collection (ATCC), USA. Mass spectra (MS) were measured with Thermo LCQ Deca XP Max or Thermo Finnigan MAT 95 XP mass spectrometer for electrospray ionization mass spectra (ESI). Melting points were recorded on a Buchi B-54 melting-point apparatus. Reverse-phase HPLC analysis was performed on a Shimadzu HPLC system using an Alltima C-18 (250 × 10 mm) column at a flow rate of 3.0 mL/min for preparation and a C-18 (250 × 4.6 mm) at a flow rate of 1.0 mL/min for analysis. Fluorescence emission spectra were performed on a Varian Cary Eclipse Fluorescence Spectrophotometer. UV−vis absorption spectra were recorded in a 10 mm path length quartz cell on a Beckman Coulter DU800 spectrometer. OD values in MTT assays were measured by Tecan’s Infinite M200 microplate reader. Fluorescence microscopic imaging and confocal laser scanning microscopic imaging were conducted with a Nikon Eclipse TE2000 Microscope. Preparation of MoS2 Nanosheets. The MoS2 nanosheets were prepared using the electrochemical lithium intercalation and exfoliation method reported previously.39 Briefly, the commercial MoS2 powders were mixed homogeneously with acetylene black and PVDF binder with mass ratio of 8:1:1 dispersed in NMP. The mixture was then uniformly coated on Cu foil disks, and dried in vacuum at 90 °C. After that, the mixture-coated Cu disks were used as cathode for assembling battery cell using Li foil as anode and 1 M LiPF6 as electrolyte. The electrochemical intercalation was operated using galvanostatic discharge at current of 0.025 mA. After the discharge process, the lithium intercalated sample was taken out from the battery cell and washed with acetone. Followed by sonication in 15 mL distilled water, the MoS2 nanosheets were collected from the supernatant by centrifugation (3000 rpm, 15 min). Modification of MoS2 with Thiolated Chitosan (MoS2− CS). Thiolated chitosan was synthesized by modifying the previously reported method.7 Chitosan (medium molecular weight) solution was prepared by dissolving 200 mg chitosan in 20 mL of 1% (v/v) acetic acid solution to which 160 μL of thioglycolic acid was added. Then, 0.2 g of 1-ethyl-3-(3(dimethylamino)propyl) carbodiimide hydrochloride was

Figure 3. Hsp70-expression analysis of HCT-116 cells treated with MoS2−CS, Hsp90 inhibitor Cype alone, or MoS2−CS-Cype upon NIR irradiation at 1 W/cm2. Treated HCT-116 cells were stained with Alexa Fluor 488 labeled anti-Hsp70 antibody. Scale bar = 20 μm. (λex = 488 nm, λem = 515 ± 30 nm.)

light irradiation showed strong fluoresence signals from the staining by Alexa Fluor 488 anti-Hsp70 antibody, indicating an obvious increase of Hsp70 protein expression. Such highly induced Hsp70 expression was derived from the cellular response to the external heat stimuli that was generated by the MoS2−CS nanomaterials. In contrast, HCT-116 cells treated with MoS2−CS-Cype conjugate and NIR light illumination presented very weak fluoresence signals, suggesting that the Hsp70 expression levels were changed and there was low Hsp70 expression observed, which were undoubtedly attributed to the effective inhibition of Hsp90 function by the Cype peptide on the surface of MoS2 nanosheets. Considering the protective roles of Hsp90, these reults clearly demonstrated that the combination of Hsp90 inhibitor with heat generation nanomaterials would greatly promote the apoptosis process, thus leading to the enhanced toxic effect during the PTT treatment.

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CONCLUSIONS In summary, we present a simple and unique PTT platform by integration of MoS2 nanosheets with heat shock protein E

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PBS. The effective loading of Cype onto nanosheets structure was quantified by HPLC. Photothermal Conversion Efficiency of Original MoS2, MoS2−CS, and MoS2−CS-Cype. To measure the photothermal conversion performances of MoS2 materials, a standard method was used in this study.51 808 nm NIR laser was delivered through a quartz cuvette containing aqueous dispersion (3 mL, 10 μg/mL in H2O) of MoS2 samples (original MoS2, MoS2−CS, or MoS2−CS-Cype), and the light source was an external adjustable power (2 W/cm2) 808 nm laser device (EINST Technology). The temperature was monitored by a thermometer and recorded one time per 30 s. Stability Test of MoS2−CS-Cype. To determine drug release kinetics in the absence of NIR irradiation, 50 μL of MoS2−CS-Cype was incubated in different pH buffer (5.0 or 7.4) at 37 °C. After incubation for 6, 12, 24, or 48 h, sample solution was centrifuged (20 000 rpm, 10 min), and the collected supernatant containing released Cype was analyzed by RP-HPLC to determine the concentration of Cype. Photothermal Release Property of Cype from MoS2− CS-Cype. To determine drug release kinetics in the presence of NIR irradiation, 50 μL of MoS2−CS-Cype was exposed to 808 nm NIR laser (1 or 2 W/cm2) for 2.5, 5, 7.5, or 10 min. After NIR irradiation, sample solution was centrifuged (20 000 rpm, 10 min), and the collected supernatant containing released Cype was analyzed by RP-HPLC to determine the concentration of Cype. Photothermal Treatment Efficacy (MTT Assay). HCT116 cells were seeded on 96-well plates containing 5000 cells per well in 100 μL DMEM media and incubated for 24 h. After the incubation with MoS2−CS (10 μg/mL), Cype (12 μM), or MoS2−CS-Cype (corresponding concentration: [MoS2−CS] = 10 μg/mL, [Cype] = 12 μM) at 37 °C for 1 h, the media were removed, and cells were washed with PBS. Each well was exposed NIR irradiation (808 nm) at 1 W/cm2 for 5 min. Upon additional incubation for 24 h, HCT-116 cells were further incubated with cell culture medium containing 20% MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). After incubation at 37 °C for 3.5 h, the medium was removed and the cells were lysed with 100 μL DMSO. Then, the absorbance was measured at 570 nm using the Tecan’s Infinite M200 microplate reader. Each experiment was repeated three times. Cyto Tox 96 Assay. For investigation of necrosis, Cyto Tox 96 assay (Promega) was carried out according to the manufacturer’s instructions.48 HCT-116 cells were seeded on a 96-well plate containing 5000 cells per well in 100 μL DMEM media and incubated for 24 h. After the incubation with MoS2− CS (10 μg/mL), Cype (12 μM), or MoS2−CS-Cype ([MoS2− CS] = 10 μg/mL, [Cype] = 12 μM) at 37 °C for 1 h, the media were removed, and cells were washed with PBS. Each well was exposed NIR irradiation (808 nm) at 1 W/cm2 for 5 min. After additional incubation for 3 h, sample solution was mixed with buffer solution containing substrate of LDH. After incubation in the dark for 30 min, stop solution (with reservoirs to hold Cyto Tox96 reagents) was added into sample solution. Then the absorbance was measured at 490 nm using a Tecan’s Infinite M200 microplate reader. Each experiment was repeated three times. Caspase-3 Activity Assay. Caspase-3 activity assay (Caspase 3 Assay Kit, Fluorimetric, Sigma-Aldrich) was carried out according to the manufacturer’s instructions.48 HCT-116 cells were seeded on 96-well plates containing 5000 cells per

added to activate the carboxylic acid moieties of thioglycolic acid. The pH of the whole solution was adjusted to 5.0 using 5 mol/L of NaOH solution. The solution was stirred for 6 h at room temperature. The obtained thiolated chitosan was purified through dialysis followed by lyophilization. The final product was stored at −20 °C. 1 mL of aqueous solution containing MoS2 nanosheets was dispensed into a vial. The aqueous solution containing thiolated chitosan (5 mg/mL, 100 μL) is added to the vial containing MoS2, and the mixture was stirred for 24 h. After 24 h, excess ligands were removed by centrifugation (20 000 rpm, 10 min). The obtained precipitation (MoS2−CS) was dispensed into PBS. Ninhydrin Test of MoS2−CS. The ninhydrin test50 was performed by adding 10 μL of ninhydrin EtOH solution (3.5 mg/mL) to 10 μL of MoS2 or MoS2−CS suspension (10 μg/ mL in H2O) and then heating the reaction solutions in a boiling water bath for 15 min. The reaction solutions were diluted with EtOH, and the UV−vis absorption spectra were measured with the UV−vis spectrometer. The reaction solution of MoS2−CS clearly showed increasing absorption around 600 nm, indicating the existence of chitosan layer in MoS2−CS. Synthesis of Cype. Cyclic peptide, Cype, was prepared according to the reported procedures.43 The liner LKVLF protected Boc group at ε-NH2 in Lys was prepared on resin by a standard Fmoc-solid-phase peptide synthesis procedure. After elongation of the peptide chain, the obtained peptidyl resin was treated with 1% TFA in DCM to yield liner LKVLF protected Boc group at Lys. Next, 1.5 equiv of HATU was dissolved in 3/ 4 of the calculated volume of dry DCM that would give a 0.005−0.007 M overall concentration. The linear peptide was dissolved in the other 1/4 solvent volume of DCM. DIPEA (8 equiv) was then added to the solution containing coupling reagents dissolved in DCM. The protected peptide was slowly added to the bulk solution over 30 min. The reaction mixture was stirred at room temperature for 3 h. The reaction was worked up by washing with water and saturated sodium bicarbonate. The organic layers were combined and concentrated. The obtained crude mixture containing protected Cype was treated with TFA for 3 h to remove the Boc group on Lys residue. After concentration of reaction mixture, reverse-phase HPLC was used for purification of Cype using a gradient of acetonitrile and deionized water with 0.1% TFA. Citrate Synthase Aggregation Assay of Cype. Hsp90 inhibitory activity of Cype was evaluated as reported elsewhere44 by monitoring the thermally induced aggregation of citrate synthase (0.075 μM) in the absence or in the presence of a stoichiometric amount of Hsp90 and 0.3 μM ATP, and with or without a 4-fold molar excess of Cype. Aggregation was initiated by unfolding citrate synthase incubating the protein in 40 mM HEPES-KOH, pH 7.5 at 43 °C. Aggregation was monitored by measuring light scattering with a Varian Cary Eclipse Fluorescence Spectrophotometer. Both the emission and excitation wavelengths were set at 500 nm, and the bandpass was 2 nm. Kinetics traces reported here are the averages of two measurements. Adhesion of Cype onto MoS2−CS (MoS2−CS-Cype). PBS solution containing MoS2−CS was dispensed into a vial. The DMSO solution containing Cype (final concentration 1 mM) was added to the vial containing MoS2−CS, and the mixture was stirred for 24 h. After 24 h, free cyclic peptides were removed by centrifugation (20 000 rpm, 10 min). The obtained precipitation (MoS2−CS-Cype) was dispensed into F

DOI: 10.1021/acs.bioconjchem.6b00741 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry well and incubated for 24 h. After the incubation with MoS2− CS (10 μg/mL), Cype (12 μM), or MoS2−CS-Cype ([MoS2− CS] = 10 μg/mL, [Cype] = 12 μM) at 37 °C for 1 h, the media were removed, and cells were washed with PBS. Each well was exposed to NIR irradiation (808 nm) at 1 W/cm2 for 5 min. After additional incubation for 3 h, PBS buffer in wells was carefully removed and lysis buffer was added into the wells. After incubation at 0 °C for 20 min, assay buffer containing caspase-3 probe was added. Fluorescence intensity of sample solution at 460 nm was measured with a Varian Cary Eclipse Fluorescence Spectrophotometer at the excitation wavelength of 360 nm. Flow Cytometry of Photothermally Treated HCT-116 Cells. HCT-116 cells were seeded on 96-well plates containing 5000 cells per well in 100 μL DMEM media and incubated for 24 h. After the incubation with MoS2−CS (10 μg/mL), Cype (12 μM), or MoS2−CS-Cype ([MoS2−CS] = 10 μg/mL, [Cype] = 12 μM) at 37 °C for 1 h, the media were removed, and cells were washed with PBS. Each well was exposed NIR irradiation (808 nm) at 1 W/cm2 for 5 min. After additional incubation for 3 h, HCT-116 cells were collected through trypsin treatment and centrifuged to remove medium. The collected HCT-116 cells were stained with Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit (Invitrogen). HCT-116 cells stained with AnnV and PI were analyzed by FACS Calibur flow cytometer (BD Biosciences, USA), and the results were analyzed with FlowJo 7.6.1 software. Hsp70-Expression Analysis of HCT-116 Cells Treated with MoS2−CS-Cype. HCT-116 cells were seeded on the 96well plates containing 5000 cells per well in 100 μL DMEM media and incubated for 24 h. After the incubation with MoS2− CS (10 μg/mL), Cype (12 μM), or MoS2−CS-Cype ([MoS2− CS] = 10 μg/mL, [Cype] = 12 μM) at 37 °C for 1 h, the media was removed, and cells were washed with PBS. Each well was exposed to NIR irradiation (808 nm) at 1 W/cm2 for 5 min. After additional incubation for 3 h, HCT-116 cells were collected through trypsin treatment and centrifuged to remove medium. The collected HCT-116 cells were fixed with 1% paraformaldehyde solution. After fixation for 15 min, HCT-116 cells were washed with PBS and stained with anti-Hsp70 antibody conjugated with Alexa Fluor 488 (Biolegend Alexa Fluor 488 anti-Hsp70) at room temperature for 15 min. Stained HCT-116 cells were washed with PBS and observed with confocal laser scanning microscope (Nikon Eclipse TE2000-E, CFI VC 100× oil immersed optics), using 488 nm laser and 480 ± 25 nm filter.

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ORCID

Edwin Kok Lee Yeow: 0000-0003-0290-4882 Hua Zhang: 0000-0001-9518-740X Bengang Xing: 0000-0002-8391-1234 Author Contributions

‡ Shinya Ariyasu and Jing Mu contributed equally to this manuscript.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge NTU under Start-Up Grant (SUG, M4080469.110.500000, M4081296.070.500000), A*STAR PSF Grant (SERC1121202008), Tier 1 RG35/15, RG64/10, RG11/ 13, COS research collaboration award, MOE under AcRF Tier 2 (ARC 26/13, No. MOE2013-T2-1-034; ARC 19/15, No. MOE2014-T2-2-093; MOE2015-T2-2-057; MOE2016-T2-2103), AcRF Tier 1 (2016-T1-001-147; 2016-T1-002-051, 2016-T1-002-136) and iFood Research Grant (M4081458.070.500000), and Singapore Millennium Foundation in Singapore and the National Natural Science Foundation of China (NSFC 51628201).

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ABBREVIATIONS MoS2, molybdenum disulfide; PTT, photothermal therapy; HSPs, heat shock proteins; AFM, atomic force microscopy; Cype, cyclic peptide sequence; LDH, leakage of lactate dehydrogenase; FACS, fluorescence activated cell sorting; CLSM, confocal laser scanning microscopy; HPLC, high performance liquid chromatography

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00741. Stability testing, AFM characterization, optical absorption spectra, aggregation kinetics, Hsp70-expression analysis, flow cytometry (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. G

DOI: 10.1021/acs.bioconjchem.6b00741 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.bioconjchem.6b00741 Bioconjugate Chem. XXXX, XXX, XXX−XXX