AIEgen-Functionalized Mesoporous Silica Gated by Cyclodextrin

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Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

AIEgen-Functionalized Mesoporous Silica Gated by CyclodextrinModified CuS for Cell Imaging and Chemo-Photothermal Cancer Therapy Qing-Lan Li,† Duo Wang,† Yuanzheng Cui,† Zhiying Fan,† Li Ren,*,†,§ Dongdong Li,†,∥ and Jihong Yu*,†,‡ †

State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, ‡International Center of Future Science, and ∥Department of Materials Science, Jilin University, 2699 Qianjin Street, Changchun 130012, P. R. China § College of Food Science and Engineering, Jilin University, 5333 Xi’an Street, Changchun 130000, P. R. China S Supporting Information *

ABSTRACT: A novel multifunctional drug delivery system has been constructed by assembling per-6-thio-β-cyclodextrin-modified ultrasmall CuS nanoparticles (CD-CuS) onto fluorescent AIEgen-containing mesoporous silica nanoparticles (FMSN). The CD-CuS nanoparticles are anchored on the surface of benzimidazole-grafted FMSN, acting as a gatekeeper and photothermal agent. The prepared blue-emitting nanocomposite (FMSN@CuS) exhibits good biocompatibility and cell imaging capability. Anticancer drug doxorubicin hydrochloride (DOX) molecules are loaded into FMSN@CuS, and zero prerelease at physiological pH (7.4) and on-demand drug release at an acidic environment can be achieved due to the pH-responsive gate-opening of CD-CuS only at an acidic condition. The FMSN@CuS nanocomposite can generate obvious thermal effect after the exposure of 808 nm laser, which can also accelerate the DOX release. Meanwhile, the fluorescence intensity of DOX-loaded FMSN@CuS increases with the release of DOX, and the intracellular drug release process can be tracked according to the change of luminescence intensity. More importantly, DOX-loaded FMSN@CuS displays efficient anticancer effects in vitro upon 808 nm laser irradiation, demonstrating a good synergistic therapeutic effect via combining enhanced chemotherapy and photothermal therapy. KEYWORDS: aggregation-induced emission luminogen, mesoporous silica nanoparticles, CuS, cell imaging, drug release, chemo-photothermal therapy

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INTRODUCTION

alized MSN as an excellent platform for biomedical applications.16−19 As is well-known, MSN is an excellent nanocarrier for drug delivery and release. Construction of stimuli-responsive controlled drug delivery systems (CDDS) based on MSN not only improves the efficacy of conventional chemotherapy, but also reduces the side effects of drugs to healthy tissues.20,21 So far, different kinds of gatekeepers such as polymers,22 biomolecules,23,24 supramolecular macrocyclic receptors,25,26 and inorganic nanoparticles (Au, ZnO, Fe3O4, etc.),27−30 have been anchored on the surface of MSN to realize controlled drug release upon stimuli of pH, light, redox, competitive binding, ultrasound, etc. Notably, cyclodextrin (CD), as a wellknown supramolecular macrocyclic receptor, has been extensively studied for construction of CDDSs because of its regulated assembly behavior with guest molecules grafted on MSN.31 In particular, CD-based gatekeepers with pH stimuli-

Fluorescent nanoparticles for biomedical applications have attracted tremendous research interests over the past few decades.1−3 Fluorescent nanoparticles, such as quantum dots,4 upconversion nanoparticles,5,6 and dye-based silica nanoparticles,7,8 can be used to real-time visualize or monitor biological events in vivo through fluorescence imaging. Among them, dye-based silica nanoparticles have been studied systematically due to their high stability, easy surfacemodification, and good biocompatibility.9 However, conventional dyes often suffer from photobleaching and aggregation caused quenching (ACQ) after incorporation in silica nanoparticles at high concentrations, thus hindering their further applications in biological imaging. Thanks to the discovery of aggregation-induced emission luminogens (AIEgens) with high fluorescent efficiency in aggregated states,10−12 various AIEgens were embedded into silica nanoparticles via physical encapsulation or covalent conjugation for efficient bioimaging.13−15 Particularly, AIEgens were successfully introduced into mesoporous silica nanoparticles (MSN) via covalent bonding by our group to integrate bioimaging and drug delivery into one single system, promising AIEgen-function© XXXX American Chemical Society

Special Issue: AIE Materials Received: September 25, 2017 Accepted: December 8, 2017

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DOI: 10.1021/acsami.7b14566 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Synthesis of TPE-Containing Organosilica Precursor (TPESi). 1-[(4-Bromomethyl)phenyl]-1,2,2-triphenylethene (TPE-CH2Br) was prepared according to the reported literature.46 The anhydrous THF solution (10 mL) containing TPE-CH2Br (106 mg), K2CO3 (173 mg) and APTS (0.12 mL) was refluxed for 6 h in a N2 atmosphere. The white transparent solid denoted as TPE-Si was obtained via rapid silica-gel chromatography using DCM and methanol (v/v = 30:1) as eluent. HR-MS (MALDI-TOF): m/z 566.462 [(M +H)+, calcd 566.301]. Synthesis of TPE-Functionalized MSNs. The mixture solution of CTAB (0.1 g), H2O (48 mL), and 2 M NaOH (aq, 0.35 mL) was stirred at 80 °C for 30 min. TEOS (0.5 mL) and TPE-Si (12 mg) were then added simultaneously, and the solution was stirred vigorously at 80 °C for 3 h. The resulting white solid was obtained via centrifugation and freeze-drying. To remove the occluded surfactant, the assynthesized white material (0.24 g) was refluxed in the methanol (25 mL) solution of concentrated HCl (0.25 mL) for 6 h. After centrifugation and freeze-drying, TPE-functionalized mesoporous silica nanoparticles with strong blue fluorescence were obtained, denoted as FMSN. Functionalization of FMSN with Benzimidazole. One hundred milligrams of FMSN and 1 mmol of γ-chloropropyl triethoxysilane were added into distilled toluene (10 mL). The reaction was operated at 110 °C under N2 for 20 h. After filtration and drying, the obtained materials (FMSN-Cl) were dispersed in anhydrous dimethylformamide (DMF) solution dissolving tetrabutylammonium iodide (22 mg), benzimidazole (118 mg), and triethylamine (0.42 mL). The suspension was heated to 89 °C for 3 days at the atmosphere of N2. Finally, the benzimidazole-functionalized material denoted as FMSNBM was washed with DMF and MeOH, and dried in vacuum conditions at room temperature. Synthesis of Per-6-thio-β-cyclodextrin-Modified CuS NPs (CD-CuS). Ultrasmall CD-CuS particles were prepared via the method of ligand exchange. Oleylamine-capped CuS (OA-CuS) nanoparticles were first synthesized according to the literature.47 Ten milligrams of OA-CuS was then added to 5 mL of DMF containing100 mg of CD. The solution was shaken for 30 min with sonication and stirred for 24 h at room temperature. The CD-CuS nanoparticles were collected by centrifugation at 15 000 rpm and washed several times with chloroform and DMF. Finally, the product was dispersed in DMF (1 mg mL−1) for later use. DOX Loading, CD-CuS Capping, and Drug Release of FMSN. The FMSN-BM material (15 mg) was dispersed in an aqueous solution of DOX (2 mM, 6 mL) through ultrasound. After the suspension solution was stirred for 24 h, 5 mg of CD-CuS was added and the mixture was stirred for 24 h. The DOX-loaded FMSN@CuS was then centrifuged, washed 3 times by the mixed solution of DMF and deionized water, and finally dried in a freezer dryer. Meanwhile, the FMSN@CuS composite was prepared via the same method without adding DOX. DOX-loaded FMSN@CuS (1.5 mg) was dispersed in 1.5 mL of PBS (pH 7.4, 6.0, 5.0, and 3.5, respectively) at room temperature. The supernatant containing released DOX was obtained via centrifugation at predetermined time intervals. Then the precipitated DOX-loaded FMSN@CuS was dispersed in 1.5 mL of fresh PBS again. The collected supernatant was measured by an UV−vis spectrophotometer at a wavelength of 480 nm to monitor controlled release processes of DOX. Meanwhile, the drug release experiments at 55 °C were also carried out. In order to study the effects of NIR on DOX release, the dispersed solution of DOX-loaded FMSN@CuS (pH 7.4 and pH 5.0) was further irradiated with an 808 nm fiber laser device (2 W) for 10 min in the original time intervals at room temperature. Photothermal Performance Assessment of FMSN@CuS. Aqueous dispersions of the FMSN@CuS with different concentrations (50, 100, 200, 400, 800 μg mL−1) were put in quartz cells and irradiated with an 808 nm laser device for 7 min. And the temperature of the solutions was recorded per 10 s using a digital thermometer. Biocompatibility of FMSN@CuS Nanocomposite. The biocompatibility of FMSN@CuS nanocomposite was evaluated by the MTT assay. Human embryonic kidney cell line (HEK293 cells) and

responsive property can encapsulate anticancer drug in the mesopores of MSN at the physiological environment, and be triggered at an acidic environment of tumor to realize controlled drug release.32−35 On the other hand, near-infrared (NIR) light is a promising stimuli because of its noninvasive and deep tissue penetration.36 NIR-induced drug release and photothermal therapy can be achieved simultaneously by the combination of MSN and photothermal agents into a nanocomposite structure. Photothermal agents, including gold nanoparticles, carbon nanomaterials, and copper chalcogenides, can convert photoenergy from NIR light into thermal effects and further promote drug release, leading to synergistic therapeutic treatment for cancer cells.37−39 Among them, copper sulfide (CuS) nanoparticles have been widely applied for construction of multifunctional theranostic nanoplatforms because of their high photothermal conversion efficiency, easy preparation, and low cytotoxicity.19,40−43 Notably, researchers have anchored CuS nanoparticles onto MSN as gatekeepers and photothermal agent via two complementary oligonucleotide sequences to fabricate CDDS.44 Herein, per-6-thio-β-cyclodextrin molecules were modified on CuS via ligand exchange to form ultrasmall CD-CuS nanoparticles that act as both gatekeeper and photothermal agent. The CD-CuS nanoparticles were further anchored onto benzimidazole-grafted AIEgens-containing MSN to construct a pH stimuli-responsive CDDS. The CD-CuS not only promises a zero prerelease at physiological pH (7.4) and on-demand drug release at an acidic condition for DOX-loaded FMSN@ CuS, but also further accelerates the drug release upon NIR irradiation, promising a synergistic therapeutic effect via the combination of enhanced chemotherapy and photothermal therapy for cancer cells.

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EXPERIMENTAL SECTION

Materials. Chemicals and reagents used herein were of analytical grade. 4-Methylbenzophenone, diphenylmethane, cetyltrimethylammonium bromide (CTAB), and tetraethylorthosilicate (TEOS) were purchased from Sinopharm Chemical Reagent Co. (Beijing, China). Sodium hydroxide (NaOH), tetrahydrofuran (THF), and N,Ndimethylformamide (DMF) were purchased from Beijing Chemical Reagent Co., Ltd. Sulfur and triethylamine were obtained from Tianjin Fuchen Chemical Reagents Factory. DOX was from Adamas-beta; (3aminopropyl)triethoxysilane (APTS) and γ-chloropropyl triethoxysilane were from J&K Scientific Ltd.; and oleylamine, Cu (acac)2, tetrabutylammonium iodide, and benzimidazole were from Aladdin (Shanghai, China). Per-6-thio-β-cyclodextrin (CD) was synthesized according to the method reported in the literature.45 A series of phosphate buffers (PBS) was prepared in line with the Chinese Pharmacopeia (Second Part, 2010 Edition). Characterization. Molecular weight was measured on Bruker autoflex speed MALDI-TOF. Powder X-ray diffraction (XRD) patterns were determined by a Rigaku Ultima IV powder diffractometer. The size and morphology of prepared materials were observed via scanning electron microscopy (SEM, JSM-6700F JEOL) and transmission electron microscopy (TEM, Tecnai G2 S-Twin F20). Nitrogen adsorption and desorption measurements were operated on a Micromeritics Tristar 3000 surface area and porosity analyzer. FT-IR and UV−vis absorbance spectra were recorded by using IFS-66 V/S and UV-2450 spectrometers. Zeta potentials of materials were measured on Zetasizer Nano ZS. Thermogravimetric analysis (TGA) was carried out on a TGA Q500 instrument with a 10 K/min heating rate at the air atmosphere. Confocal laser scanning microscopy (CLSM) images were recorded by a confocal fluorescence microscope (LSM710, Zeiss) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed on a microplate reader (infinite F200 Pro, TECAN). B


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Scheme 1. Schematic Diagram of the Preparation Process of the DOX-Loaded FMSN@CuS and Its Intracellular Drug Release Process

Figure 1. (a) Fluorescence spectrum of FMSN (excitation wavelength: 336 nm); (b) SEM and (c) TEM images of FMSN; (d) N2 adsorption and desorption isotherm of FMSN and FMSN-BM; (e) FT-IR spectra and (f) TGA curves of FMSN, FMSN-Cl, and FMSN-BM. Intracellular Drug Delivery and Release Performance of DOX-Loaded FMSN@CuS. SGC-7901 cells were seeded and cultured in 6-well plates for attachment. Subsequently, DOX-loaded FMSN@CuS (50 μg mL−1) was added. After NIR irradiation for 7 min, cells were further cultured for 30 min, 4 and 24 h, respectively. The intracellular drug delivery performance of DOX-loaded FMSN@ CuS and the distribution of DOX in real time were observed by CLSM with excitation at 340 nm and emission at 465 nm for AIEgen (blue channel), and with excitation at 530 nm and emission at 617 nm for DOX (red channel). Chemo-Photothermal Therapy of DOX-Loaded FMSN@CuS. SGC-7901 cells were seeded and cultured in 96-well plates at a density of 5 × 103/well for 24 h. The complete media were then replaced by fresh media containing different concentrations of DOX, FMSN@CuS and DOX-loaded FMSN@CuS. After the cells were incubated for 4 h, wells of FMSN@CuS and DOX-loaded FMSN@CuS were irradiated by 808 nm laser for 7 min. In contrast, wells of DOX and DOX-loaded FMSN@CuS were without irradiation. Finally, all of them were kept at 37 °C and 5% CO2 conditions for another 20 h, and relative cell viabilities were measured by the MTT assay. Meanwhile, living SGC7901 cells after different treatments were stained with calcium-AM, the

gastric cancer cell line (SGC-7901 cells) were cultured in RPMI-1640 containing 10% fetal bovine serum and incubated at 37 °C and 5% CO2, respectively. They were then seeded and cultured in 96-well plates under 37 °C and 5% CO2 conditions for 24 h. When cells attached to the plates completely, they were incubated at the fresh medium containing different concentrations of FMSN@CuS (50, 25, 12.5, 6, and 3 μg mL−1) for another 24 h. After the dispose of MTT solution and DMSO, the absorbance intensity at 490 nm was measured by a microplate reader and relative cell viabilities were calculated. Fluorescence Imaging of FMSN@CuS. SGC-7901 cells were seeded in 6-well plates at a density of 6 × 103/well at 37 °C and 5% CO2 for 24 h. When cells attached to the plates completely, the fresh medium containing FMSN@CuS (50 μg mL−1) was added to replace the original. After different time intervals (4 and 24 h), cells were stained with 2 nM calcium-AM (beyotime biotechnology, china) according to the protocol of the manufacturer, washed with PBS to remove excess materials and dyes, and fixed with 4% paraformaldehyde for 20 min at 37 °C. After cells were placed onto microscope slides, FMSN@CuS nanoparticles taken up by cells were examined by using CLSM and a fluorescence microscope. C


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Figure 2. TEM images of (a) OA-CuS NPs and (b) FMSN@CuS; (c) EDS of FMSN@CuS; (d) UV−vis−NIR absorption spectra of an aqueous dispersion of CD-CuS, FMSN@CuS and FMSN-BM; (e) temperature changes of FMSN@CuS solution at different concentrations after exposure to NIR light (808 nm). survival states of which were observed directly by the fluorescence microscopy.

functionalization is detected by FT-IR (Figure 1e). The peaks at 1400 and 1455 cm−1 of FMSN belong to skeletal vibrations of benzene ring of TPE. After grafting γ-chloropropyl triethoxysilane on FMSN, the absorption signal at around 2979 cm−1 assigned to stretching vibration of −CH2 becomes stronger. Furthermore, the sharp stretching vibration band of CN at 1656 cm −1 appears, proving the successful modification of benzimidazole on FMSN-BM.48 In addition, the average surface potentials vary with the modification of FMSN (Figure S5). The zeta potential of FMSN is 11.2 mV, due to the existence of secondary amine in TPE-Si. After modification with benzimidazole, the zeta potential decreases from 19.7 mV to −16.9 mV. TGA curves of FMSN, FMSN-Cl and FMSN-BM are shown in Figure 1f, and the weight losses of materials below 100 °C belong to the physical loss of water. By calculating the difference in weight loss of FMSN, FMSN-Cl and FMSN-BM, about 1.13 mmol g−1 of γ-chloropropyl triethoxysilane and 0.32 mmol g−1 of benzimidazole are decorated on FMSN, which are consistent with the elemental analysis results (1.35 mmol g−1 of γ-chloropropyl triethoxysilane and 0.30 mmol g−1 of benzimidazole) (Table S1), note that the weight loss of water has been subtracted. The diameter of as-prepared ultrasmall OA-CuS nanoparticles is about 4.2 nm (Figure 2a and Figure S3). OA can be exchanged by CD forming CD-CuS via Cu−S bond, which is confirmed by FT-IR spectroscopy (Figure S6). From the FTIR spectrum of CD-CuS, a typical absorption peak at 1153 cm−1 assigned to the coupled C−O−C stretching/O−H bending vibrations of CD is displayed and the stretching vibration band of S−H at 2567 cm−1 of CD disappears, demonstrating CD molecules are modified on CuS nanoparticles. As shown in Figure 2b, CD-CuS nanoparticles are successfully assembled on the surface of FMSN-BM via host− guest interaction between CD and benzimidazole.33 And the existence of Si, O and Cu in FMSN@CuS is confirmed by the energy dispersive spectroscopy (EDS) (Figure 2c). After FMSN@CuS was dispersed in simulated body fluids (Hank’s balanced salt solution, HBSS) for different time (0 h, 36 and 84 h), the nanocomposites keep spherical in shape and CD-CuS

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RESULTS AND DISCUSSION Preparation and Characterization of Functionalized FMSN. TPE-containing organosilica precursor (TPE-Si) was synthesized via the nucleophilic substitution reaction of APTS and TPE-CH2Br. The presence of the TPE-Si was proved by the mass spectrum (Figure S1). As illustrated in Scheme 1, AIE luminogen was introduced into MSN by coprecipitation of TPE-Si and TEOS at conditions of NaOH as catalyst and CTAB as template. The as-prepared material (FMSN) was then functionalized with γ-chloropropyl triethoxysilane and benzimidazole to generate FMSN-BM, which was further assembled with CD-CuS to construct a pH stimuli-responsive and photothermoenhanced drug delivery system combining cell imaging, chemotherapy, and photothermal therapy multifunctionalities. The amount of the TPE unit embedded into the MSN is approximately 0.14 mmol g−1, calculated by elemental analysis (Table S1). FMSN emits typical blue fluorescence at 465 nm (Figure 1a), and the solid fluorescence quantum yield of FMSN under excitation at 336 nm is 19%. The XRD pattern of FMSN (Figure S2) shows characteristic diffraction peaks (100, 110, 200, and 210) of MCM-41 with highly ordered 2D hexagonal pore channel array. As shown in Figure 1b and Figure S3, FMSN has a spherical morphology with average diameter of 100 nm. Ordered mesoporous structure of FMSN can be observed in the TEM image (Figure 1c). The N2 adsorption− desorption isotherms of FMSNs and FMSN-BM belong to the typical IV isotherm, indicating the presence of well-defined mesoporous channels (Figure 1d). The Brunauer− Emmett− Teller (BET) surface area and total pore volume of FMSNs are 917 m2 g−1 and 1.12 cm3 g−1, dropping to 687 m2 g−1 and 0.62 cm3 g−1 for FMSN-BM, attributed to the modification of γchloropropyl triethoxysilane and benzimidazole on FMSN. The pore-size distributions of FMSNs and FMSN-BM are concentrated at 2.9 and 2.8 nm, calculated by the Barrett− Joyner−Halenda (BJH) method (Figure S4). FMSN surface D


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Figure 3. CLSM images of SGC-7901 cells incubated with FMSN@CuS (50 μg mL−1) for 4 h and 24 h. From left to right, bright-field, blue fluorescence images of FMSN@CuS, green fluorescence images of calcein-AM, and their overlapping images. Scale bar: 50 μm.

Figure 4. Drug release curves of DOX-loaded FMSN@CuS (a) in different pH valued PBS solutions at room temperature (20 °C); (b) upon NIR laser irradiation in pH 5.0 (black) and pH 7.4 (red) solutions; (c) fluorescence spectra of DOX-loaded FMSN@CuS with the drug release in pH 5.0 solution upon NIR laser irradiation.

studied via monitoring the temperature change of the solution of FMSN@CuS nanocomposites upon periodic NIR laser ON/ OFF irradiation for five repeat cycles. As shown in Figure S8, there is no noticeable difference in the thermal conversion efficiency, demonstrating high photothermal stability of FMSN@CuS. Biocompatibility and Fluorescence Imaging Capability of FMSN@CuS. The biocompatibility of FMSN@CuS was evaluated by the standard MTT assay for both HEK293 cells and SGC-7901 cells. As shown in Figure S9, the viability of HEK293 cells remained above 84% after incubation with FMSN@CuS for 24 h at concentrations from 3 to 50 μg/mL, indicating that FMSN@CuS has slight cytotoxicity toward normal cells. SGC-7901 cells treated with FMSN@CuS also showed high cell viability up to 88% at a concentration of 50 μg/mL. Then SGC-7901 cells were incubated with FMSN@CuS (50 μg/mL) for different time (4 and 24 h) and calcein-AM was added to intuitively assess the biocompatibility of FMSN@CuS via fluorescence imaging. As shown in Figure 3 and Figure S10, the FMSN@CuS nanocomposite can be efficiently taken up by cells and emit bright blue fluorescence in cytoplasm. After 24 h of incubation, cells still kept normal morphologies and can be further stained by calcein-AM with green color, suggesting that FMSN@CuS has negligible cytotoxicity to cells. Therefore, good biocompatibility and fluorescence imaging performance

nanoparticles are still anchored on the surface of FMSN-BM, demonstrating good stability of FMSN@CuS at physiological conditions (Figure S7). As shown in Figure 2d, CD-CuS has a characteristic absorption peak in the range of 700−1100 nm, whereas FMSN-BM shows no absorption in this range. After attachment with CD-CuS, FMSN@CuS also shows absorbance in the NIR range, promising that FMSN@CuS can convert the absorbed light into heat upon NIR laser irradiation. The absorbance intensity of FMSN@CuS is smaller than that of CD-CuS, indicating that SiO2 may block the light and thus affect the absorption of CD-CuS nanoparticles.44 Because of lower tissue invasion and deeper tissue penetration of 808 nm laser, the photothermal effect of FMSN@CuS was investigated under the 808 nm laser irradiation. Various concentrations of FMSN@ CuS nanocomposites were dispersed in distilled water. The temperature of dispersed solutions increased markedly with the increase of concentrations of FMSN@CuS under NIR irradiation. After irradiation for 7 min, the elevated temperature of the dispersed solution in the concentration range of 50−800 μg mL−1 is 5−28 °C (Figure 2e). Therefore, mild hyperthermia (39−42 °C) can be reached toward body tissue via irradiating FMSN@CuS at a concentration of 50 μg mL−1 with an NIR laser for 7 min, which can efficiently kill cancer cells through the combination with other therapies, such as chemotherapy.37 The photothermal stability of FMSN@CuS was then further E


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Figure 5. CLSM images of SGC-7901 cells after treatment with DOX-loaded FMSN@CuS (50 μg mL−1) for 30 min, 4 and 24 h. The blue fluorescence represents FMSNs, and the red fluorescence represents DOX. Scale bar: 50 μm.

promise that FMSN@CuS can serve as a fluorescent biomarker for intracellular imaging. In Vitro Drug Release. To study the stimuli-responsive release performance of FMSN@CuS, we chose anticancer drug DOX as a model drug to be loaded in the nanocomposite. The drug loading capacity is about 19.7 wt % calculated by loading experiments. Because CD-CuS is assembled on the surface of FMSN-BM via hydrogen-bonding interactions between CD and benzimidazole, such interaction would weaken with the protonation of benzimidazole in acidic environment, leading to gateopening of CD-CuS and controlled drug release. In vitro drug release experiments were carried out in different PBS solutions (pH 7.4, 6.1, 5.0, and 3.5, respectively). As shown in Figure 4a, zero premature release of DOX at pH 7.4 is realized due to the gatekeeper effect of CD-CuS. When reducing the pH value from 6.1 to 3.5, the release rate and total release amount of DOX increase, which would promise the on-demand drug release in acidic cancer cells. Meanwhile, the temperature influence on drug release was also studied (Figure S11). At elevated temperature (55 °C), the drug release was raised to 81.6% at pH 5.0. The heat could dissociate the strong electrostatic interaction between DOX and silica and thus more DOX are released from FMSN upon heating.49 Notably, there was no drug release at pH of 7.4 because of the capping of CD-CuS on FMSN. The drug release behavior of DOX-loaded FMSN@CuS was further studied under NIR irradiation. As shown in Figure 4b, no DOX escapes from FMSN at pH 7.4 even under NIR irradiation, which is consistent with the release experiments

treated with external heating. However, an approximate “ladder” release curve was obtained when DOX-loaded FMSN@CuS in PBS solution at pH 5.0 was irradiated with the NIR laser for 10 min every hour. The rapid drug release and the slow release appeared alternately upon periodic NIR laser ON/OFF irradiation. Significantly, after 8 h, the total DOX release reached to 86.5% upon NIR irradiation, which is about 1.5 times of that without NIR irradiation at pH 5.0. Therefore, the drug release of DOX-loaded FMSN@CuS can be triggered by acidic pH, and the DOX release rate and the total amount can be enhanced by external heating and NIR light. On the other hand, the fluorescence emission intensity of the DOXloaded FMSN@CuS at 465 nm gradually increases with the release of DOX, shown in Figure 4c. In fact, the emission intensity of FMSN@CuS decreases after loading with DOX because of the fluorescence self-absorption of DOX, but it would recover when DOX is released from the nanocomposite. Therefore, the change in fluorescence intensity can be utilized to track the drug release process of DOX-loaded FMSN@CuS. Intracellular Drug Delivery Performance of DOXLoaded FMSN@CuS. As an anticancer drug, DOX kills cancer cells via destroying DNA in the nucleus,50 so the intracellular distribution of released DOX was investigated. CLSM was employed to monitor the intracellular drug delivery performance of DOX-loaded FMSN@CuS and the distribution of DOX in real time. As seen in Figure 5, the nanoparticles with blue fluorescence are taken up by SGC-7901 cells and are localized in cytoplasm all the time in line with the aforementioned cell imaging results. After 30 min incubation, F


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Figure 6. (a) In vitro viabilities of SGC-7901 cells after incubation with different concentrations of free DOX, FMSN@CuS and DOX-loaded FMSN@CuS with or without 808 nm NIR laser irradiation. * denotes p ≤ 0.05 versus DOX-loaded FMSN@CuS+NIR treatment. Error bars represent standard deviation. (b) Fluorescence microscope images of SGC-7901 cells with various treatments stained with calcium-AM. Scale bar: 400 μm.

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the red fluorescence of DOX is overlapped with the blue of the nanocomposite, indicating that most of DOX is still loaded in the FMSN@CuS at this stage. With the increase in incubation time, DOX accumulation gradually shifts from cytoplasm to nucleus and the blue fluorescence intensity of DOX-loaded FMSN@CuS becomes stronger (Figure S12), suggesting that the DOX molecules can be efficiently released from the FMSN@CuS in the acidic environment of SGC-7901 cells and enter nucleus to perform chemotherapy. Synergistic Therapeutic Effects of DOX-Loaded FMSN@CuS. The synergistic therapeutic effect combining chemotherapy and photothermal therapy of DOX-loaded FMSN@CuS toward SGC-7901 cells was investigated by MTT assays. As shown in Figure 6a, the inhibition ratio of materials against SGC-7901 cells increases with the increasing concentrations of materials. FMSN@CuS exhibits a certain cellkilling efficacy under NIR irradiation due to the generated thermal effect, which is gradually enhanced with the increase in laser power (Figure S13). The viability of cells treated with DOX-loaded FMSN@CuS obviously decreases attributed to the released DOX for chemotherapy. The inhibition ratio of DOX against SGC-7901 cells is higher than that of DOXloaded FMSN@CuS, mostly because part of DOX is still loaded in FMSN after 24 h, which cannot enter the nucleus. Notably, under the NIR laser irradiation, the photothermal effect of nanocomposites is gradually enhanced with the increasing concentrations of materials, leading to more effective photothermal therapy and more DOX molecule released. Therefore, DOX-loaded FMSN@CuS at high concentrations (above 12.5 μg/mL) exhibits the highest cell-killing ability under the NIR laser radiation, demonstrating a good synergistic therapeutic effect via the combination of enhanced chemotherapy and photothermal therapy. To visually evaluate the therapeutic effects in cellular level, we stained living cells after treatment with DOX, FMSN@CuS, and DOX-loaded FMSN@ CuS with or without NIR irradiation by calcein-AM with green color. The cell-killing capacity for FMSN@CuS, DOX-loaded FMSN@CuS, and DOX-loaded FMSN@CuS with NIR irradiation increases in turn, proving a better therapeutic effect for cancer cells than the individual chemotherapy or photothermal therapy, which is consistent with the results of MTT assays (Figure 6b).

CONCLUSION In conclusion, a multifunctional drug delivery system based on CD-CuS and AIEgen-containing MSN (FMSN) has been constructed for cell imaging and synergistic chemo-photothermal cancer therapy. The obtained FMSN@CuS nanocomposite exhibits good biocompatibility and excellent fluorescence imaging capability, indicating that it can function as an efficient fluorescent biomarker for cell imaging. CD-CuS nanoparticles assembled on FMSN act as both pH stimuliresponsive gatekeeper and photothermal agent. The capping of CD-CuS on FMSN results in zero prerelease of DOX at physiological pH (7.4) and stimuli-responsive drug release at an acidic condition for DOX-loaded FMSN@CuS. Moreover, NIR irradiation can not only induce thermal effect, but also accelerate drug release of DOX-loaded FMSN@CuS, leading to a good synergistic therapeutic effect toward SGC-7901 cells. This multifunctional drug delivery system combining luminescence, pH stimuli-responsive drug release, and photothermal effect shows promising potentials for cell imaging and cancer therapy.

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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/acsami.7b14566. MS spectrum of TPE-Si; XRD patterns, particle size distributions, pore diameter distributions, average zeta potentials, elemental analysis, and FT-IR spectra of materials; photothermal stability of FMSN@CuS; viability of HEK293 cells and SGC-7901 cells after treatment with FMSN@CuS; fluorescence microscope images of SGC-7901 cells incubated with FMSN@CuS; drug release curves of DOX-loaded FMSN@CuS at different temperature (PDF)

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AUTHOR INFORMATION

Corresponding Authors

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

Qing-Lan Li: 0000-0002-6090-6408 Li Ren: 0000-0002-0564-4265 Jihong Yu: 0000-0003-1615-5034 G


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ACS Applied Materials & Interfaces Notes

(16) Li, D.; Yu, J.; Xu, R. Mesoporous Silica Functionalized with an AIE Luminogen for Drug Delivery. Chem. Commun. 2011, 47, 11077− 11079. (17) Li, D.; Yu, J. AIEgens-Functionalized Inorganic-Organic Hybrid Materials: Fabrications and Applications. Small 2016, 12, 6478−6494. (18) Wang, D.; Chen, J.; Ren, L.; Li, Q.; Li, D.; Yu, J. AIEgenFunctionalised Mesoporous Silica Nanoparticles as a FRET Donor for Monitoring Drug Delivery. Inorg. Chem. Front. 2017, 4, 468−472. (19) Fan, Z.; Ren, L.; Zhang, W.; Li, D.; Zhao, G.; Yu, J. AIE Luminogen-Functionalised Mesoporous Silica Nanoparticles as Nanotheranostic Agents for Imaging Guided Synergetic Chemo-/Photothermal Therapy. Inorg. Chem. Front. 2017, 4, 833−839. (20) Yang, P.; Gai, S.; Lin, J. Functionalized Mesoporous Silica Materials for Controlled Drug Delivery. Chem. Soc. Rev. 2012, 41, 3679−3698. (21) Chen, Y.; Chen, H.; Shi, J. In Vivo Bio-Safety Evaluations and Diagnostic/Therapeutic Applications of Chemically Designed Mesoporous Silica Nanoparticles. Adv. Mater. 2013, 25, 3144−3176. (22) Paris, J. L.; Cabañas, M. V.; Manzano, M.; Vallet-Regí, M. Polymer-Grafted Mesoporous Silica Nanoparticles as UltrasoundResponsive Drug Carriers. ACS Nano 2015, 9, 11023−11033. (23) Oroval, M.; Díez, P.; Aznar, E.; Coll, C.; Marcos, M. D.; Sancenón, F.; Villalonga, R.; Martı ́nez-Máñez, R. Self-Regulated Glucose-Sensitive Neoglycoenzyme-Capped Mesoporous Silica Nanoparticles for Insulin Delivery. Chem. - Eur. J. 2017, 23, 1353−1360. (24) Ruehle, B.; Clemens, D. L.; Lee, B.-Y.; Horwitz, M. A.; Zink, J. I. A Pathogen-Specific Cargo Delivery Platform Based on Mesoporous Silica Nanoparticles. J. Am. Chem. Soc. 2017, 139, 6663−6668. (25) Song, N.; Yang, Y.-W. Molecular and Supramolecular Switches on Mesoporous Silica Nanoparticles. Chem. Soc. Rev. 2015, 44, 3474− 3504. (26) Huang, X.; Wu, S.; Ke, X.; Li, X.; Du, X. Phosphonated Pillar[5]arene-Valved Mesoporous Silica Drug Delivery Systems. ACS Appl. Mater. Interfaces 2017, 9, 19638−19645. (27) Wang, X.; Tan, L.-L.; Li, X.; Song, N.; Li, Z.; Hu, J.-N.; Cheng, Y.-M.; Wang, Y.; Yang, Y.-W. Smart Mesoporous Silica Nanoparticles Gated by Pillararene-Modified Gold Nanoparticles for on-Demand Cargo Release. Chem. Commun. 2016, 52, 13775−13778. (28) Zhang, J.; Wu, D.; Li, M.-F.; Feng, J. Multifunctional Mesoporous Silica Nanoparticles Based on Charge-Reversal PlugGate Nanovalves and Acid-Decomposable ZnO Quantum Dots for Intracellular Drug Delivery. ACS Appl. Mater. Interfaces 2015, 7, 26666−26673. (29) Chen, Y.; Ai, K.; Liu, J.; Sun, G.; Yin, Q.; Lu, L. Multifunctional Envelope-Type Mesoporous Silica Nanoparticles for pH-Responsive Drug Delivery and Magnetic Resonance Imaging. Biomaterials 2015, 60, 111−120. (30) Wang, Y.; Shi, W.; Wang, S.; Li, C.; Qian, M.; Chen, J.; Huang, R. Facile Incorporation of Dispersed Fluorescent Carbon Nanodots into Mesoporous Silica Nanosphere for pH-Triggered Drug Delivery and Imaging. Carbon 2016, 108, 146−153. (31) Hu, Q.-D.; Tang, G.-P.; Chu, P. K. Cyclodextrin-Based HostGuest Supramolecular Nanoparticles for Delivery: from Design to Applications. Acc. Chem. Res. 2014, 47, 2017−2025. (32) Du, L.; Liao, S.; Khatib, H. A.; Stoddart, J. F.; Zink, J. I. Controlled-Access Hollow Mechanized Silica Nanocontainers. J. Am. Chem. Soc. 2009, 131, 15136−15142. (33) Meng, H.; Xue, M.; Xia, T.; Zhao, Y.-L.; Tamanoi, F.; Stoddart, J. F.; Zink, J. I.; Nel, A. E. Autonomous in Vitro Anticancer Drug Release from Mesoporous Silica Nanoparticles by pH-Sensitive Nanovalves. J. Am. Chem. Soc. 2010, 132, 12690−12697. (34) Liu, J.; Luo, Z.; Zhang, J.; Luo, T.; Zhou, J.; Zhao, X.; Cai, K. Hollow Mesoporous Silica Nanoparticles Facilitated Drug Delivery via Cascade pH Stimuli in Tumor Microenvironment for Tumor Therapy. Biomaterials 2016, 83, 51−65. (35) Vaupel, P.; Kallinowski, F.; Okunieff, P. Blood Flow, Oxygen and Nutrient Supply, and Metabolic Microenvironment of Human Tumors: A Review. Cancer Res. 1989, 49, 6449−6465.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the State Basic Research Project of China (Grant 2014CB931802), the National Natural Science Foundation of China (Grants 21320102001 and 21621001), and the 111 Project (B17020).

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REFERENCES

(1) Michalet, X.; Pinaud, F. F.; Bentolila, L. A.; Tsay, J. M.; Doose, S.; Li, J. J.; Sundaresan, G.; Wu, A. M.; Gambhir, S. S.; Weiss, S. Quantum Dots for Live Cells, in Vivo Imaging, and Diagnostics. Science 2005, 307, 538−544. (2) Wang, K.; He, X.; Yang, X.; Shi, H. Functionalized Silica Nanoparticles: A Platform for Fluorescence Imaging at the Cell and Small Animal Levels. Acc. Chem. Res. 2013, 46, 1367−1376. (3) Zhou, J.; Liu, Z.; Li, F. Upconversion Nanophosphors for Smallanimal Imaging. Chem. Soc. Rev. 2012, 41, 1323−1349. (4) Lv, G.; Guo, W.; Zhang, W.; Zhang, T.; Li, S.; Chen, S.; Eltahan, A. S.; Wang, D.; Wang, Y.; Zhang, J.; Wang, P. C.; Chang, J.; Liang, X.J. Near-Infrared Emission CuInS/ZnS Quantum Dots: All-in-One Theranostic Nanomedicines with Intrinsic Fluorescence/Photoacoustic Imaging for Tumor Phototherapy. ACS Nano 2016, 10, 9637− 9645. (5) Liu, B.; Chen, Y.; Li, C.; He, F.; Hou, Z.; Huang, S.; Zhu, H.; Chen, X.; Lin, J. Poly (Acrylic Acid) Modification of Nd3+-Sensitized Upconversion Nanophosphors for Highly Efficient UCL Imaging and pH-Responsive Drug Delivery. Adv. Funct. Mater. 2015, 25, 4717− 4729. (6) Zhou, L.; Wang, R.; Yao, C.; Li, X.; Wang, C.; Zhang, X.; Xu, C.; Zeng, A.; Zhao, D.; Zhang, F. Single-Band Upconversion Nanoprobes for Multiplexed Simultaneous in Situ Molecular Mapping of Cancer Biomarkers. Nat. Commun. 2015, 6, 7350−7359. (7) Cho, E.-B.; Volkov, D. O.; Sokolov, I. Ultrabright Fluorescent Silica Mesoporous Silica Nanoparticles: Control of Particle Size and Dye Loading. Adv. Funct. Mater. 2011, 21, 3129−3135. (8) Yeh, C.-S.; Su, C.-H.; Ho, W.-Y.; Huang, C.-C.; Chang, J.-C.; Chien, Y.-H.; Hung, S.-T.; Liau, M.-C.; Ho, H.-Y. Tumor Targeting and MR Imaging with Lipophilic Cyanine-Mediated Near-Infrared Responsive Porous Gd Silicate Nanoparticles. Biomaterials 2013, 34, 5677−5688. (9) Montalti, M.; Prodi, L.; Rampazzo, E.; Zaccheroni, N. DyeDoped Silica Nanoparticles as Luminescent Organized Systems for Nanomedicine. Chem. Soc. Rev. 2014, 43, 4243−4268. (10) Luo, J.; Xie, Z.; Lam, J. W. Y.; Cheng, L.; Chen, H.; Qiu, C.; Kwok, H. S.; Zhan, X.; Liu, Y.; Zhu, D.; Tang, B. Z. AggregationInduced Emission of 1-Methyl-1,2,3,4,5-pentaphenylsilole. Chem. Commun. 2001, 1740−1741. (11) Ding, D.; Li, K.; Liu, B.; Tang, B. Z. Bioprobes Based on AIE Fluorogens. Acc. Chem. Res. 2013, 46, 2441−2453. (12) Hong, Y.; Lam, J. W. Y.; Tang, B. Z. Aggregation-Induced Emission: Phenomenon, Mechanism and Applications. Chem. Commun. 2009, 4332−4353. (13) Mahtab, F.; Lam, J. W. Y.; Yu, Y.; Liu, J.; Yuan, W.; Lu, P.; Tang, B. Z. Covalent Immobilization of Aggregation-Induced Emission Luminogens in Silica Nanoparticles Through Click Reaction. Small 2011, 7, 1448−1455. (14) Zhang, X.; Zhang, X.; Wang, S.; Liu, M.; Zhang, Y.; Tao, L.; Wei, Y. Facile Incorporation of Aggregation-Induced Emission Materials into Mesoporous Silica Nanoparticles for Intracellular Imaging and Cancer Therapy. ACS Appl. Mater. Interfaces 2013, 5, 1943−1947. (15) Li, Y.; Shao, A.; Wang, Y.; Mei, J.; Niu, D.; Gu, J.; Shi, P.; Zhu, W.; Tian, H.; Shi, J. Morphology-Tailoring of a Red AIEgen from Microsized Rods to Nanospheres for Tumor-Targeted Bioimaging. Adv. Mater. 2016, 28, 3187−3193. H


Forum Article

ACS Applied Materials & Interfaces (36) Weissleder, R. A Clearer Vision for in Vivo Imaging. Nat. Biotechnol. 2001, 19, 316−317. (37) Zhang, Z.; Wang, J.; Chen, C. Near-Infrared Light-Mediated Nanoplatforms for Cancer Thermo-Chemotherapy and Optical Imaging. Adv. Mater. 2013, 25, 3869−3880. (38) Wang, Y.; Wang, K.; Zhang, R.; Liu, X.; Yan, X.; Wang, J.; Wagner, E.; Huang, R. Synthesis of Core−Shell Graphitic Carbon@ Silica Nanospheres with Dual-Ordered Mesopores for CancerTargeted Photothermochemotherapy. ACS Nano 2014, 8, 7870−7879. (39) Wang, Y.; Wang, K.; Zhao, J.; Liu, X.; Bu, J.; Yan, X.; Huang, R. Multifunctional Mesoporous Silica-Coated Graphene Nanosheet Used for Chemo-Photothermal Synergistic Targeted Therapy of Glioma. J. Am. Chem. Soc. 2013, 135, 4799−4804. (40) Goel, S.; Chen, F.; Cai, W. Synthesis and Biomedical Applications of Copper Sulfide Nanoparticles: from Sensors to Theranostics. Small 2014, 10, 631−645. (41) Zhou, M.; Zhang, R.; Huang, M.; Lu, W.; Song, S.; Melancon, M. P.; Tian, M.; Liang, D.; Li, C. A Chelator-Free Multifunctional [64Cu] CuS Nanoparticle Platform for Simultaneous Micro-PET/CT Imaging and Photothermal Ablation Therapy. J. Am. Chem. Soc. 2010, 132, 15351−15358. (42) Xiao, Q.; Zheng, X.; Bu, W.; Ge, W.; Zhang, S.; Chen, F.; Xing, H.; Ren, Q.; Fan, W.; Zhao, K.; Hua, Y.; Shi, J. A Core/Satellite Multifunctional Nanotheranostic for in Vivo Imaging and Tumor Eradication by Radiation/Photothermal Synergistic Therapy. J. Am. Chem. Soc. 2013, 135, 13041−13048. (43) Song, G.; Wang, Q.; Wang, Y.; Lv, G.; Li, C.; Zou, R.; Chen, Z.; Qin, Z.; Huo, K.; Hu, R.; Hu, J. A Low-Toxic Multifunctional Nanoplatform Based on Cu9S5@mSiO2 Core-Shell Nanocomposites: Combining Photothermal- and Chemotherapies with Infrared Thermal Imaging for Cancer Treatment. Adv. Funct. Mater. 2013, 23, 4281− 4292. (44) Zhang, L.; Li, Y.; Jin, Z.; Yu, J. C.; Chan, K. M. An NIRTriggered and Thermally Responsive Drug Delivery Platform through DNA/Copper Sulfide Gates. Nanoscale 2015, 7, 12614−12624. (45) Rojas, M. T.; Königer, R.; Stoddart, J. F.; Kaifer, A. E. Supported Monolayers Containing Preformed Binding Sites. Synthesis and Interfacial Binding Properties of a Thiolated β-Cyclodextrin Derivative. J. Am. Chem. Soc. 1995, 117, 336−343. (46) Shi, H.; Kwok, R. T. K.; Liu, J.; Xing, B.; Tang, B. Z.; Liu, B. Real-Time Monitoring of Cell Apoptosis and Drug Screening Using Fluorescent Light-up Probe with Aggregation-Induced Emission Characteristics. J. Am. Chem. Soc. 2012, 134, 17972−17981. (47) Mou, J.; Li, P.; Liu, C.; Xu, H.; Song, L.; Wang, J.; Zhang, K.; Chen, Y.; Shi, J.; Chen, H. Ultrasmall Cu2‑xS Nanodots for Highly Efficient Photoacoustic Imaging-Guided Photothermal Therapy. Small 2015, 11, 2275−2283. (48) Bénard, S.; Neuville, L.; Zhu, J. Copper-Mediated NCyclopropylation of Azoles, Amides, and Sulfonamides by Cyclopropylboronic Acid. J. Org. Chem. 2008, 73, 6441−6444. (49) Zhang, Z.; Wang, L.; Wang, J.; Jiang, X.; Li, X.; Hu, Z.; Ji, Y.; Wu, X.; Chen, C. Mesoporous Silica-Coated Gold Nanorods as A Light-Mediated Multifunctional Theranostic Platform for Cancer Treatment. Adv. Mater. 2012, 24, 1418−1423. (50) Goto, S.; Ihara, Y.; Urata, Y.; Izumi, S.; Abe, K.; Koji, T.; Kondo, T. Doxorubicin-Induced DNA Intercalation And Scavenging by Nuclear Glutathione S-Transferase π. FASEB J. 2001, 15, 2702−2714.

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AIEgen-Functionalized Mesoporous Silica Gated by Cyclodextrin

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