Photothermally Controllable Cytosolic Drug Delivery Based On Core

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Photothermally controllable cytosolic drug delivery based on core-shell MoS-porous silica nanoplates 2

Junseok Lee, Jinhwan Kim, and Won Jong Kim Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.6b02944 • Publication Date (Web): 18 Aug 2016 Downloaded from http://pubs.acs.org on August 23, 2016

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Chemistry of Materials

Photothermally controllable cytosolic drug delivery based on coreshell MoS 2 -porous silica nanoplates Junseok Lee†, Jinhwan Kim§, and Won Jong Kim†, § †

Center for Self-assembly and Complexity, Institute for Basic Science (IBS), Pohang 37673, Republic of Korea

§

Department of Chemistry, Pohang University of Science and Technology (POSTECH), Pohang 37673, Republic of Korea

ABSTRACT: Single layered molybdenum sulfide (MoS 2 ) displays strong photothermal properties, but low colloidal stability in aqueous solution prevents its biomedical application as a functional drug delivery carrier. We report a photothermally controllable nanoplate consisting of porous silica-coated, single-layered MoS 2 , modified with polyethylene glycol (PEG). Silica and PEG enhanced stability and maintained the single layer structure of MoS 2 for over a month. A representative anticancer drug, doxorubicin (DOX), was loaded into the silica structure and subsequent exposure to near infrared irradiation facilitated both endosomal escape of the carrier and the release of DOX. DOX-loaded, silica-coated, single layered MoS 2 (DOX-PSMS-PEG) showed better therapeutic effect against liver and colon cancer compared than free DOX did; a result probably attributable to the combined effects of photothermally facilitated endosomal escape and the vulnerability of cancer cells to localized heating. These studies suggest that considerable new opportunities may exist for spatiotemporally controllable drug delivery systems based on single layered MoS 2 .

INTRODUCTION Chemotherapy is the administration of chemical drugs to the human body in order to control disease and is widely applied for the treatment of severe diseases including cancer. However, the systemic administration of anticancer drugs is limited by the low solubility and instability of drug molecules and the possibility of damage to normal tissues leading to side effects.1 Consequently, drug delivery systems (DDSs) have been developed to increase the solubility and the stability of drugs and thus reduce side effects.2, 3 In particular, nanoparticles have received considerable attention for the systemic delivery of anticancer chemotherapeutics since, 1) they readily enter into cells and release drug and 2) it is possible and relatively facile to prepare carriers with various functionalities by choosing suitable nanoparticles.4-7 Whereas conventional DDSs were designed to successfully deliver drug to a therapeutic target, recent interest in DDS design has focused on the regulation of release behavior from the delivered carrier. Consequently, sensitivity to an applied stimulus has been incorporated into the DDS to provide the precisely controllable drug release.8, 9 Among the various possible stimuli, near-infrared (NIR) has the great advantages of deep penetration into tissue and low cytotoxicity.10-13 A majority of recently developed NIRresponsive drug delivery systems exploit the photothermal effect, whereby light energy is converted to thermal energy.14 Interestingly, the photothermal effect is a particularly promising phenomenon for use in nanoparticle based DDS according to several recent reports.15-17 After entering into the cell through the endocytic pathway, most of the anticancer drug should be released quickly into the cytosol to achieve a potent cytotoxic effect. The localized heating caused by a photothermal effect may destabilize the endosomal membrane and thus facilitate escape of the drug and its carrier from the endosome. Various photothermal agents have been developed for application to DDS. Gold- and carbon-based nanostructures are typical

examples of materials that have been investigated for biomedical applications.18-22 In particular, graphene, a two-dimensional carbon nanosheet, has attracted attention as a drug delivery carrier due to its large surface area, ready functionalization, and excellent biocompatibility.23-25 However, structural limitations to the types of drug molecules that are compatible with these carriers and poor colloidal dispersity after drug loading are still challenging obstacles to extensive biomedical application.26, 27 In order to overcome such problems, modification of the graphene surface using polymers or silica has been investigated and both drug content loading and colloidal stability were considerably enhanced.27, 28 Recently, transition metal dichalcogenide (TMDC) nanosheets that resemble the 2D structure of graphene have been synthesized.29 Like graphene, TMDC also produces a remarkable photothermal effect when dispersed in monolayer form in an aqueous medium. In particular, since molybdenum (Mo) acts as an important cofactor in a number of biological systems and due to its relatively low toxicity, molybdenum sulfide (MoS 2 ) has drawn attention for biomedical applications.30-36 Single layered MoS 2 may be readily produced as a dispersion in an aqueous solution, suitable for biomedical use, from bulk MoS 2 by exfoliation using lithium intercalation.37 However, newly synthesized single layer MoS 2 aggregates easily in aqueous solution by the exchange of stabilizing lithium ions and therefore further modification to stabilize the dispersion is an important prerequisite for biomedical applications.38-40

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a confocal Raman spectroscope (Alpha 300R, WITec) on silicon wafer. Atomic force microscopy images (AFM) were obtained using a scanning probe microscopy (SPM) system (VEECO Dimension 3100, VEECO). Gas sorption isotherms were measured with a BELSORP-max and Autosorp-iQ volumetric adsorption equipment. Hydrodynamic size and zeta potential were measured using a Zetasizer instruments (Nano S90 and Nano Z, Malvern) at 0.1 mg/mL in Dulbecco’s phosphate buffered saline (DPBS). Confocal laser scanning microscopy (CLSM) was performed with an Olympus FV-1000 and analyzed by OLYMPUS FLUOVIEW ver. 1.7 Viewer software.

Scheme 1. Schematic illustration of single layered MoS 2 based nanoplate as a NIR-responsive system. Both drug release and endosomal escape are facilitated by photothermal effect under expose to near infrared (NIR), thus an enhanced anticancer effect is expected. A photothermally responsive drug delivery system based on silica-coated graphene oxide was recently reported by our group.41 In the current study, drawing inspiration and experience from our previous report, a single layered MoS 2 was covered with a porous silica shell and an additional PEG layer was introduced (PSMSPEG) to enhance the colloidal dispersity and stability in aqueous suspension. A representative anticancer drug, doxorubicin (DOX), was loaded into the porous silica layer (DOX-PSMS-PEG) utilizing the PSMS-PEG as an NIR-responsive drug delivery vehicle. Following NIR irradiation of cells after endocytosis of DOX-PSMSPEG, the photothermal effect facilitated both drug release and endosomal escape (Scheme 1). As DOX exhibits its anticancer effect in the nucleus, an enhanced anticancer effect was observed in vitro, in comparison with free DOX treatment. The results of this research indicate the outstanding potential of PSMS-PEG as a photothermally controllable drug delivery system. EXPERIMENTAL SECTION

Preparation of exfoliated molybdenum sulfide (eMoS 2 ). Singlelayered molybdenum sulfide was prepared by a previously reported method.39 MoS 2 flake (2 g) was immersed in 2 mL of 1.6 M BuLi hexane solution and incubated for 48 h. The suspension was filtered and the solid was thoroughly washed with hexane several times. The pellet was dispersed in DW and ultrasonicated for 1 h at 0 oC. The suspension was centrifuged (3,000 rpm, 10 min) and the supernatant was dialyzed against DW for 2 days. The concentration of eMoS 2 in suspension was quantified by UV-vis absorption was measured at 750 nm as diluted in DMSO. Preparation of porous silica coated and surface aminefunctionalized MoS 2 (PSMS-NH 2 ). PSMS-NH 2 was prepared by following a synthetic procedure for mesoporous silica coated gold nanorods with some modifications.42 A suspension of eMoS 2 (25 mL, 0.1 mg/mL) was prepared and 50 mg of CTAB and 250 μL of 0.1 N NaOH was added with stirring. After 30 min, 20 μL of TEOS was added three times at intervals of 30 minutes and 10 μL of APTES was also added at the third addition. The mixture was stirred at 30 °C for 24 h and the product obtained by centrifugation (13,000 rpm, 30 min) following more than five washes with MeOH to remove CTAB and unreacted precursors. The PSMS-NH 2 was analyzed by TEM, HR-TEM, DLS, for zeta potential and FT-IR. The weight fraction of MoS 2 in PSMS-NH 2 was estimated by UVvis absorption at 750 nm in DMSO to minimize the scattering effect of silica. The amount of surface amine was quantified by fluorescamine assay.

Reagents. Molybdenum (IV) sulfide (MoS 2 ), n-butyllithium solution 1.6 M in hexane (BuLi), cetyltrimethylammonium bromide (CTAB), sodium hydroxide (NaOH), tetraethyl orthosilicate (TEOS), (3-aminopropyl)triethoxysilane (APTES), 1,1'carbonyldiimidazole (CDI), fluorescein isothiocyanate (FITC), thiazolyl blue tetrazolium bromide (MTT) and all other solvents were purchased from Sigma Aldrich (St. Louis, MO). Aminefunctionalized methoxy PEG (PEG-NH 2 , MW = 5 kDa) was purchased from Sunbio (Korea). Doxorubicin hydrochloride (DOX) was purchased from Wako chemical (Japan). Lysotracker® Red was purchased from Invitrogen (MA). All reagents were used as received without further purification.

Preparation of PEGylated PSMS-NH 2 (PSMS-PEG) and FITClabelled PSMS-PEG (FITC-PSMS-PEG). The surface of PSMSNH 2 was PEGylated using carbodiimide coupling. PSMS-NH 2 (20 mg) was dispersed in 20 mL of DMSO and 25 mg of CDI was added. After 3 h, the suspension was centrifuged (13,000 rpm, 30 min) to remove the unreacted CDI. The pellet was re-dispersed in 20 mL of DMSO and 50 mg of PEG-NH 2 was added. After stirring overnight, the PSMS-PEG was obtained by centrifugation and washed with MeOH, PBS, DW, and MeOH, then dried in vacuo. The weight fraction of MoS 2 in PSMS-PEG was estimated by UV-vis absorption at 750 nm in DMSO. The amount of residual surface amine was quantified by fluorescamine assay.

Instrumental methods. High-resolution TEM imaging, EDS, and EELS elemental mapping were performing using a transmission electron microscope (JEM-2200FS with image CS-corrector, JEOL) as sample loaded on the grid (400 mesh, Cu) and analyzed by Gatan Digital Microscope. UV-vis spectra were measured using a UV-vis spectrometer (UV 2550, Shimadzu) and fluorescence spectra were measured using a spectrofluorophotometer (RF-5301 PC, Shimadzu). Fourier transform infrared (FT-IR) spectra were obtained with an FT-IR spectrophotometer (VERTEX70, Bruker) as KBr solid pellets. Raman spectra and image were measured using

In order to observe the distribution and movement of the carrier, PSMS-PEG was labeled with the green fluorescent dye FITC using residual amines on the surface. PSMS-PEG (4 mL of 1 mg/mL) in DW was mixed with 50 μL of 1 mM FITC solution in DMSO and stirred overnight. FITC-PSMS-PEG was obtained by centrifugation (13,000 rpm, 10 min) and resuspended in 4 mL of DW.


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Scheme 2. a) Synthesis of silica modified MoS 2 (PSMS-NH 2 ). b) Preparation of DOX loaded and PEGylated PSMS-NH 2 (DOX-PSMSPEG). Loading doxorubicin into PSMS-PEG (DOX-PSMS-PEG). In order to load Doxorubicin into the porous silica shell, PSMS-PEG was dispersed in 1 mg/mL DOX solution in DW at a final concentration of 1 mg/mL and stirred in the dark for 24 h. The mixture was centrifuged and rinsed 3 times with PBS to remove the adsorbed DOX. For quantification of loaded DOX, the final product was dispersed in MeOH and the fluorescence of extracted DOX (ex = 495 nm / em = 555 nm in MeOH) was measured. Photothermal effect of nanoparticles. Since single-layered MoS 2 shows a good photothermal effect, temperature change of nanoparticles under NIR irradiation was monitored. 1 mL of DW, 0.1 mg/mL mesoporous silica nanoparticle suspension, 0.05 mg/mL eMoS 2 solution, 0.05 mg/mL MoS 2 equivalent MSMS-NH 2 and MSMS-PEG solution were irradiated by NIR laser (808 nm diode laser, JENOPTIK unique-mode GmbH, Germany) at power density of 1 W/cm2. Temperature of the solution was measured by thermocouple at predetermined time interval. Photo-responsive release of DOX from DOX-MSMS-PEG. The photo-responsive release of DOX under NIR irradiation was evaluated by preparing a 0.1 mg/mL suspension of DOX-PSMS-PEG in buffer solution (DPBS, pH = 7.4; 0.1 M acetate buffer, pH = 5.0) and it was continuously irradiated by an NIR laser (3 W/cm2). At each desired time, a 20 μL aliquot of the suspension was collected, diluted into 200 μL and centrifuged. The fluorescence of DOX in the supernatant was measured. The suspension incubated in the dark was used as a control and sampled according to the same procedure.

dium for 0, 3, 6, 9, 12 or 15 min and further incubated for 24 h. In the case of DOX toxicity, medium was replaced by fresh medium containing 0 to 20 μM DOX (HeLa and MCF-7) or 0 to 50 μM of DOX (HepG2 and HCT-8) and further incubated for 24 h. After incubation, cell viability was evaluated by MTT assay. MTT assay: medium was replaced by 180 μL of fresh medium and 20 μL of MTT solution (5 mg/mL) was added. After incubation in the dark for 4 h the medium was removed and the purple formazan was completely dissolved in 200 μL of DMSO. 100 μL of each sample was transferred into a new 96 well plate and UV-vis absorbance at 570 nm was measured by a microplate spectrofluorometer (VICTOR3 V multilabel counter). Non-treated cells i.e., those not receiving DOX were used to represent 100% of cell viability. Intracellular release of DOX observed by CLSM image. The intracellular release of DOX facilitated by NIR laser irradiation was analyzed by confocal microscopy. HeLa cells (40,000 cells/well) were seeded onto glass cover slips placed in a 12-well culture plate and incubated overnight. Medium was replaced by fresh serum-free medium containing 10 μg/mL DOX-PSMS -PEG. After 4 h of incubation, cells were washed carefully and irradiated by NIR laser (5 W/cm2) for 15 min under fresh medium. Cells were washed with PBS and fixed immediately with 10% neutrally buffered formalin (NBF) just after irradiation or after 4 h of further incubation. Cells on the coverslip were mounted by Vectashield anti-fade mounting medium including DAPI (Vector Labs). Intracellular fluorescence of DOX was observed by CLSM with excitation at 488 nm and false imaged as red.

Cell viability test. Prior to the evaluation of the carrier itself, the nonspecific cytotoxicity of the NIR laser and the dose-dependent cytotoxicity of free DOX were measured. Cells (HeLa for laser toxicity; HeLa, MCF-7, HepG2 and HCT-8 for DOX toxicity) were seeded onto a 96 well culture plate at a density of 8000 cells/well and incubated overnight. To determine the phototoxicity of the NIR laser, cells were irradiated (5 W/cm2) under fresh me-



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Figure 1. a) AFM image of PSMS-NH 2 . (scale bar = 300 nm) b) TEM image and c) corresponding elemental maps of PSMS-NH 2 . (scale bar = 100 nm)

NIR-triggered cytotoxicity in vitro. The cytotoxicity of nanoparticles with and without irradiation was evaluated using various concentrations. Cells (8000 cells/well; HeLa, MCF-7, HepG2 and HCT-8) were seeded on 96 well culture plates and incubated overnight. DOX-PSMS-PEG or PSMS-PEG at final concentration of 0 to 20 μg/mL was applied in serum-free medium and incubated for 4 h. Cells were then washed with PBS and irradiated by laser with power density of 5 W/cm2 for 15 min or incubated in the dark under fresh medium for 24 h, followed by MTT assay. Photothermally triggered endosomal escape monitored by confocal laser scanning microscopy (CLSM) image. In order to prove photothermally triggered endosomal escape, the localization of the FITC-labeled carrier (FITC-PSMS-PEG) and the endolysosome were observed by confocal microscope. Briefly, HeLa cells (20,000 cells/well) were seeded onto glass cover slips placed in a 12 well culture plate and incubated overnight. The medium was then replaced by fresh serum-free medium containing 10 μg/mL FITCPSMS-PEG and incubated for 4 h. Cells were irradiated by NIR laser (5 W/cm2) for 15 min and lysotracker was added immediately at a final concentration of 4 μM. After 5 min of incubation, cellular uptake was blocked by adding cold DPBS and the cells were washed thoroughly and finally fixed with NBF overnight at 4 oC. Cells on the coverslip were mounted in Vectashield anti-fade mounting medium including DAPI (Vector Labs) and observed with CLSM. RESULTS AND DISCUSSION Preparation and characterization of amine-functionalized silicacoated MoS 2 (PSMS-NH 2 ). Single-layered MoS 2 (eMoS 2 ) was prepared by chemical exfoliation from bulk MoS 2 using the lithium ion intercalation method (Scheme 2a).37 Newly prepared eMoS 2 was covered by a porous silica structure in order to enlarge the surface area for effective loading of the drug and to maintain the aqueous stability of single layered MoS 2 . The silica layer was formed on the surface of eMoS 2 by sol-gel condensation of a silica precursor with cetyltrimethylammonium bromide (CTAB) under

basic conditions.42 Amine functional groups were introduced during the silica coating by addition of amine-functionalized silica precursor for further PEGylation. The morphology and composition of the synthesized porous silica-coated MoS 2 nanoplate (PSMS-NH 2 ) was confirmed by atomic force microscopy (AFM) and elemental mapping by electron energy loss spectroscopy (EELS) with a high-resolution transmission electron microscope (HR-TEM) (Figure 1). The thickness of the nanoplates increased from ~ 0.7 nm, the dimension of a single MoS 2 , layer to ~ 60 nm, representing the addition of a silica layer on the surface (Figure 1a, S1a).43 The elemental map shows the overlapping images of Mo, S, Si, O and N, indicating that PSMS-NH 2 consists of MoS 2 (Mo, S) and amine-functionalized silica (Si, O, N) as designed (Figure 1b). The surface area of the carrier was determined to be 421 m2/g by N 2 adsorption isotherm and the average pore size was around 5 nm (Figure S2). In addition, it was confirmed by high-resolution TEM and corresponding fast Fourier transform (FFT) image that the original lattice distance of MoS 2 (~0.32 nm) was maintained even after functionalization with silica (Figure S3a-b).44-46 PEGylation and drug loading (DOX-PSMS-PEG). In order to enhance the colloidal stability of the carrier still further, PEG was attached to the surface of the silica layer (Scheme 2b). Aminemodified PEG (PEG-NH 2 ) was conjugated to the amine groups of the silica surface using carbodiimide (CDI). After PEGylation, the thickness of the nanoplate increased to approximately 80 nm, as measured by AFM (Figure S4). The average hydrodynamic diameter of the final material was about 450 nm as determined by dynamic light scattering (DLS) and the surface charge altered from negative (eMoS 2 ) to positive (PSMS-NH 2 ) and then to negative (PSMS-PEG) through the process of successive surface modifications (Figure S5). As shown in Figure S6a, the absorbance in the NIR range was preserved after the modification steps. In addition, interestingly, the A-B extinction at 630-650 nm was observed even after silica coating, which confirms the single layered nature of MoS 2 in the final product.47, 48 The weight percentage of eMoS 2 was found to be 151 mg eMoS 2 per g of PSMS-PEG calculated from the absorbance at 750 nm. In addition, the presence of silica and PEG


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was confirmed by IR spectroscopy (Figure S6b). The characteristic Raman spectrum of MoS 2 at 350-450 cm-1 was observed even after PEGylation and the localization of MoS 2 (350-450 cm-1) and amorphous PEG (1250-1750 cm-1) in the Raman microscopic image also proved the successful addition of PEG to the surface of the PSMS-NH 2 nanoplates (Figure S7).49 The colloidal stability of PSMS-PEG was maintained for more than a month in aqueous solution whereas severe aggregation of eMoS 2 solution was observed during that period (Figure S8). In the following step, a representative anticancer drug, doxorubicin (DOX) was loaded into the porous silica layer. DOX was physically adsorbed onto the silica surface via charge interactions and hydrogen bonding. The amount of loaded drug was quantified by fluorescence spectroscopy and determined to be up to 230 mg/g of nanoplate depending on loading condition, corresponding to around 190% (w/w) per eMoS 2 sheet due to the added weight fraction of silica and PEG.

was measured using the intrinsic fluorescence of DOX (Figure 2b). As expected, around 30% of the loaded drug was release at pH 5.0 with NIR irradiation whereas less than 5% leaked out at pH 7.4 without irradiation. The amine functional group of DOX is protonated at acidic pH, and thus its hydrophobicity is reduced and the elevated local temperature caused by the photothermal effect of the core MoS 2 accelerates the diffusion rate of loaded DOX into the aqueous environment.52, 53 Since the pH is reduced during maturation of an endosome, facilitated intracellular DOX release under NIR irradiation is predicted from this result.54 Although the release of loaded DOX was facilitated by external NIR irradiation in vitro, a true test at the cellular level would be to show successful NIR-responsive cytotoxicity. When DOX is loaded into PSMS-PEG drug carrier particles, it exhibits relatively lower fluorescence level due to intermolecular π-π interactions and the strong quenching effect of MoS 2 . This signal was compared to the elevated fluorescence signal produced on release of DOX from DOX-PSMS-PEG (Figure S9).55, 56 Intracellular drug release under external stimulus was observed by confocal fluorescence microscopy. Prior to irradiation, the DOX signal was relatively low and mainly confined to the perinuclear region, signifying the endosomal uptake of nanoplates (Figure 2c). In contrast, the successful intracellular release of DOX after NIR irradiation was confirmed by the large increase in intensity and the wider distribution of DOX fluorescence. These results support the potential of DOX-PSMS-PEG as a NIR-responsive drug delivery carrier.

Figure 2. a) NIR-responsive temperature elevation of nanoplates. b) Photo-responsive drug release of DOX from DOX-PSMS-PEG in vitro. c) Confocal microscopic images of DOX-PSMS-PEG treated HeLa cells which was further irradiated or not irradiated by 808 nm laser (5 W/cm2). Nucleus was stained by DAPI (blue) and DOX was falseimaged as red. (scale bar = 50 μm)

NIR-responsive properties. The nanoplate consists of a porous silica shell and eMoS 2 core, which is known to show a strong photothermal effect.37 In order to demonstrate the photothermal effect of eMoS 2 in the core, the temperature change under NIR irradiation was monitored. As shown in Figure 2a, the temperature of the medium containing 0.05 mg/mL eMoS 2 increased by over 50 °C in 5 min when irradiated with an 808 nm laser with a power density of 1 W/cm2. The PSMS-NH 2 and PSMS-PEG at a similar concentration of 0.05 mg/mL of eMoS 2 also showed a similar pattern, confirming preservation of the photothermal property of eMoS 2 , even after surface modification with silica and PEGylation. The temperature increase observed is high enough to induce thermal effects in the cancer cell and to facilitate both endosomal escape and intracellular drug release.50 One of the most important criteria for a successful, stimulusresponsive, drug releasing platform is that the loaded drug should be released only under the desired condition with minimal nonspecific leakage.51 In order to evaluate the stimulus sensitivity of the nanoplate, release of loaded drug, with and without NIR irradiation,

Figure 3. NIR-induced cytotoxicity of DOX-PSMS-PEG or PSMSPEG against a) HeLa, b) MCF-7, c) HepG2 and d) HCT-8 cell line. (** p < 0.01, *** p < 0.001)

NIR-responsive cytotoxicity and photothermally triggered endosomal escape. As previously discussed, the drug loading of DOXPSMS-PEG is released under NIR irradiation, especially at endosomal acidic pH. In addition, the photothermal effect of the eMoS 2 particle core may induce thermal damage in cancer cells. In order to evaluate of DOX-PSMS-PEG as a stimuli-responsive drug delivery system, the NIR-induced cytotoxicity of the DOX-loaded carrier (DOX-PSMS-PEG) was compared to that of the free carrier (PSMS-PEG) by MTT assay (Figure 3). In the absence of NIR, both carriers were biocompatible regardless of the presence of loaded drug. Irradiation of a naïve cell with NIR also did not show any significant cytotoxicity (Figure S10). However, significant cytotoxicity against nanoplate pre-treated cells was observed when irradiated. As a result of photothermal effect of the core MoS 2 , the



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viability of PSMS-PEG treated cells was approximately 70% after irradiation for all four cell lines at a dose of 20 μg/mL carrier. The anticancer effect of DOX-PSMS-PEG was significantly greater than PSMS-PEG alone, driven by intracellular release of DOX. The half maximal inhibitory concentration (IC 50 ) is a suitable index to quantitatively compare the efficacy of drug delivery systems. Thus, the IC 50 of DOX-PSMS-PEG against MCF-7 cell line with external stimulus was measured at 1.81 μM of DOX equivalent concentration, whereas that of free DOX was 15.5 μM (Figure S11). The corresponding IC 50 values of the loaded carrier against HeLa, HepG2, and HCT-8 cell lines indicated 1.4-, 36-, and 12-fold greater effectiveness than that of free DOX, respectively.

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and PEG. Doxorubicin, an anticancer drug, was loaded into the nanoplate and the release was facilitated by external NIR irradiation at acidic pH. Intracellular release of DOX was further accelerated by exposure to NIR. The system exhibited an outstanding anticancer effect, more than 30 times more effective against hepatocarcinoma than free DOX. The significantly enhanced anticancer effect of DOX-PSMS-PEG was achieved by the combination of 1) thermal damage through heating of the carrier, 2) enhanced release of DOX and 3) successful endosomal escape. Taken together, the results in this study suggest the potential of PSMS-PEG as an ondemand delivery system for anticancer drugs and a further application in the field of photothermo-responsive drug delivery systems.

ASSOCIATED CONTENT Supporting Information. AFM, N 2 isotherm, high resolution TEM, DLS, Zeta potential, UV-Vis, IR and Raman spectra, stability and basic cell viability are included in Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author [email protected]

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes Figure 4. a) Schematic illustration of photothermally-assisted endosomal escape for effective drug release. b) Confocal microscopic images of FITC-PSMS-NH 2 treated HeLa cells which was further irradiated or not irradiated by 808 nm laser (5 W/cm2). Nucleus was stained by DAPI (blue), lysosome was stained by lysotracker (red) and the carrier was stained by FITC (green). (scale bar = 50 μm)

It is well known that most nanoparticulate drug delivery carriers enter the cell through endocytosis. Endosomal maturation involves acidification and an elevation of enzymatic activity and therefore it is critical to design the carrier to escape the endosome as quickly as possible for effective delivery of drug. Recently, it has been reported that the photothermal effect promotes the escape of nanoparticles through heat mediated disruption of the endosomal membrane (Figure 4a).15-17 The endosomal localization and escape of PSMSPEG was monitored following triggering by NIR irradiation (Figure 4b). The green fluorescence signal from FITC-labeled PSMSPEG was co-localized with endosome positions stained by lysotracker, confirming the endocytic uptake of PSMS-PEG. After irradiation, the carrier was shown to have escaped from the endosomes, as inferred from the non-overlapping signals of carrier (FITC) and endosomes (lysotracker). CONCLUSION In this study, the use of a NIR-responsive MoS 2 -based nanoplate as a drug delivery system was demonstrated. The colloidal stability of single layered MoS 2 in an aqueous suspension was maintained for over a month by covering the surface with a porous silica shell

The authors declare no competing financial interests.

ACKNOWLEDGMENT This research was supported by the Institute for Basic Science (IBS) in Korea (IBS-R007-D1). The authors would like to thank Professor Hee Cheul Choi and his student Intek Song for valuable help regarding Raman spectroscopy. Also the authors appreciate to Professor Kimoon Kim and Dr. Yonghwi Kim for valuable help for measuring N 2 sorption isotherm.

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Photothermally Controllable Cytosolic Drug Delivery Based On Core

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