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Xijian Liu,a,b Qilong Ren,b Fanfan Fu,c Rujia Zou,b,d Qian Wang,b,e Guobing Xin, Zhiyin Xiao,b Xiaojuan Huang,b Qian Liub and Junqing Hu*,b 5
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x We report a facile and low-cost approach to design a difunctional nanoplatform (CuS@mSiO2-PEG) as a near-infrared (NIR) light responsive drug delivery system for efficient chemo-photothermal therapy. The nanoplatform demonstrated good biocompatibility and colloidal stability, as well as high loading capacity for anticancer drug (26.5% wt for doxorubicin (DOX)). The CuS nanocrystals (core) within these CuS@mSiO2-PEG core-shell nanoparticles can effectively absorb and convert NIR light to fatal heat under NIR light irradiation for photothermal therapy, and the release of DOX from the mesoporous silica (shell) can be triggered by pH and the NIR light for chemotherapy. When the CuS@mSiO2-PEG/DOX nanocomposites were irradiated by 980 nm light, both chemotherapy and photothermal therapy were simultaneously driven, resulting in a synergistic effect for killing cancer cells. Importantly, compared with chemotherapy or photothermal treatment alone, the combined therapy significantly improved the therapeutic efficacy.
1. Introduction 20
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Chemotherapy is one of the most commonly used cancer treatment strategies, but it shows many limitations, such as systemic side effects, low efficacy, and drug resistance1. In order to improve anticancer efficacy and optimize therapy, nanocarriers were used for drug delivery2, 3. Particularly, stimuli-responsive drug delivery system using “smart” nanocarriers has become a promising modality for enhancing effects of the chemotherapy due to the ability of drug release with control of the area, time, and dosage4-6. To date, various strategies, such as hydrophobicity, pH, temperature, biomolecular reactions, and light have been developed to control the drug delivery for carriers3, 7. Among them, near-infrared (NIR) light responsive release is a very important method due to the non-invasive nature, high spatial resolution, and minimal damage to normal tissues of NIR light4, 6. Importantly, the NIR light responsive system for drug delivery combines the chemotherapy and photothermal therapy, exhibiting synergistic effect for cancer therapy due to enhanced cytotoxicity of anticancer drug doxorubicin hydrochloride (DOX) at elevated temperatures and higher heat sensitivity for the cells exposed to the DOX8, 9. Copper chalcogenides are a new class of photothermal agent which has been considered as a prospective candidate for photothermal therapy10-15. Moreover, CuS nanocrystals can be biodegradable and have lower long-time toxicity than most used photothermal agent-gold nanorod16. Zhou and coworkers have prepared CuS nanocrystals and used them for photothermal therapy, which exhibited good cancer treatment efficacy10, This journal is © The Royal Society of Chemistry [year]
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however, the exorbitant power density of the laser (12 W/cm2) were used in photothermal therapy that probably harmed skins and normal tissues. To solve this problem, combination of photothermal therapy of CuS nanocrystals with chemotherapy can be adopted due to the fact that the combined therapy can effectively enhance cancer treatment efficacy and reduce the power density of laser in photothermal therapy. And numerous good results showed that the combination of photothermal therapy and chemotherapy had better effects on destroying cancer cells than each separate treatment17.For example, hollow CuS nanoparticles combined photothermal therapy and chemotherapy that achieved enhanced tumoricidal efficacy with a lower drug dose and mild irradiation conditions due to the synergistic effects18. However, the biocompatibility of CuS nanoparticles at high concentration is alarming. Mesoporous silica nanoparticles are widely used for drug controllable release in chemotherapy due to excellent stability, good biocompatibility, large surface and cavity volumes, tunable porosity and facile modification2, 19, 20 . So the combination of chemotherapy and photothermal therapy in a platform can be achieved by mesoporous silica coating CuS nanocrystals for enhancing therapy effect. In our previous works, core (photothermal agents)-shell (mesoporous silica) nanocomposites based on hydrophobic inorganic nanoparticles, such as Cu9S5@mSiO2-PEG21, Cu2-xSe@mSiO2PEG22 that combined photothermal therapy and chemotherapy in a platform, exhibited remarkable cancer-cell killing efficiency due to the synergistic effect. Unfortunately, the synthesis process of Cu9S5 and Cu2-xSe nanocrystal is not convenient due to synthesis in high temperature, anhydrous and anaerobic condition, and the synthesis cost is not cheap due to use of [journal], [year], [vol], 00–00 | 1
Dalton Transactions Accepted Manuscript
CuS@mSiO2-PEG core-shell nanoparticles as a NIR light responsive drug delivery nanoplatform for efficient chemo-photothermal therapy
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oleylamine as solvent. In addition, it takes long time and complicated procedures to change Cu9S5 and Cu2-xSe nanocrystals from hydrophobicity to hydrophilcity before silica coating. Thus, it is a great need to develop a facile, low-cost and green synthetic approach to fabricate core (photothermal agents)shell (mesoporous silica) nanocomposites for chemophotothermal therapy. In this work, CuS@mSiO2-PEG core-shell nanoparticles were prepared by a facile, cheap and green way, and used as a NIR light responsive system for drug delivery. Compared with previous core-shell nanoparticles which combined photothermal therapy and chemotherapy, such as Au@SiO223, Cu9S5@mSiO2PEG21, Cu2-xSe@mSiO2-PEG22, the CuS@mSiO2-PEG coreshell nanoparticles had remarkable advantages. Not only the material cost for synthesis of the CuS nanocrystal is much cheaper than that of the Au nanorod, Cu9S5 and Cu2-xSe nanocrystal, but also the preparation of the CuS nanocrystal is more convenient than that of the Au nanorod, Cu9S5 and Cu2-xSe nanocrystal. The CuS nanocrystal can be synthesized in aqueous solution at low temperature only using cheap material (copper chloride, sodium sulphide, sodium citrate and water). Without any fussy pretreatment, mesoporous silica coating can be directly carried out after adding CTAB to CuS solution. Mesoporous silica shell as carrier for loading anticancer drug (DOX) and CuS nanocrystals core as photothermal agents, CuS@mSiO2-PEG core-shell nanoparticles can effectively convert the NIR light into fatal heat to kill cancer cells (photothermal therapy) under 980nm laser irradiation at a safe power density (0.72W/cm2)24, meanwhile the heat generated by the photothermal effect can lead to rapid release of the drug loaded inside the mesoporous SiO2 shells to kill cancer cells (chemotherapy). Importantly, the combination of photothermal therapy and chemotherapy significantly improved the therapeutic efficacy, and displayed better therapeutic effects for cancer treatment than individual therapies.
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2. Experimental Section
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2.1 Chemicals and reagents All reagents were used without further purification. Copper (II) chloride (CuCl2·2H2O), sodium citrate (C6H5Na3O7·2H2O), cetyltrimethylammonium bromide (CTAB), sodium hydroxide (NaOH) and anhydrous ethanol are analytically pure, which were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China), Tetraethylorthosilicate (TEOS, GR) and sodium sulfide (Na2S·9H2O) were obtained from Aladdin, and 2[Methoxy(polyethyleneoxy)propyl]-trimethoxysilane (PEG-silane, MW = 596-725 g/mol, 9-12 EO) was obtained from Gelest (Morrisville, PA) and doxorubicin hydrochloride (DOX) was got from Huafeng United Technology CO., Ltd. (Beijing, China) 2.2 Characterization Sizes, morphologies, and microstructures of the nanoparticles were measured by a transmission electron microscope (TEM; JEM-2100F). The surface area, pore size, and pore-size distribution of the products were determined by BrunauerEmmett-Teller (BET) nitrogen adsorption-desorption and BarettJoyner-Halenda (BJH) methods (Micromeritics, ASAP2020). Powder X-ray diffraction (XRD) was determined by a D/max2550 PCX-ray diffractometer (Rigaku, Japan). UV-visible absorption spectra were determined on an UV-Vis 1901
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Spectrophotometer (Phoenix) using quartz cuvettes with an optical path of 1 cm. Fourier transform infrared (FTIR) spectra were measured using attenuated total reflectance (ATR) methods by an IRPRESTIGE-21 spectrometer (Shimadzu). 29Si CP (crosspolarization)/MAS(magic-angle spinning) solid state NMR measurement was carried out on a Bruker Avance 400 spectrometer (9.4T), operating at Larmor frequencies 79.51 MHz (spinning at 5 kHz, 4 ms contact time and 2 s repetition time). 2.3 Synthesis of CuS nanocrystals The CuS nanocrystals was synthesized according to previous methods10. 0.2 mmol Copper (II) chloride and 0.136 mmol sodium citrate were dissolved in 180 mL deionized water under stirring, then 20 mL sodium sulfide solution (10 mmol/L) was dropwise added. The reaction mixture turned dark brown after stirring for 5 min at room temperature, then was heated to 90 oC and maintained for 15 min. Finally, a dark-green solution of citrate-coated CuS nanocrystals was obtained and cooled with ice-cold water, then stored at 4 oC for later use. 2.4 Synthesis of CuS@mSiO2-PEG core-shell nanoparticles 0.7 g of CTAB and 65 mL of CuS nanocrystals’ solution were added to a round flask, then heated to 40 oC and maintained 2 h under stirring. 3 mL of ethanol and 100 µL of NaOH solution (30 mg/mL) were added, subsequently 100 µL of TEOS was dropped into this mixture. The mixture maintained 40 oC under continuously stirring for 1.5 h, then 100 µL of PEG-silane was added and the mixture maintained at 40 oC for another 6.5 h upon stirring. Finally, CuS@mSiO2-PEG core-shell nanoparticles were collected by centrifugation (12000 rpm, 10 min), and washed three times with ethanol. Then the products were dispersed in the ethanol solution of NH4NO3 (50 mL, 10 mg/mL), and the mixture stirred at 50 °C for 2 h to remove the template of CTAB by ion exchange. CuS@mSiO2-PEG core-shell nanoparticles were further washed three times with ethanol, then dispersed in deionized water and stored at 4 °C for further use. 2.5 Measurement of photothermal performance Photothermal performance was measured according to our previous method25. Aqueous dispersions of the CuS@mSiO2PEG core-shell nanoparticles with different concentrations (10, 25, 50, 100, 200 mg/mL) were illuminated by 980 nm laser (power density ~ 0.72 W/cm2). The temperature of the dispersions was measured by a digital thermometer (with an accuracy of 0.1 oC) using a thermocouple probe every 5 s. 2.6 DOX loading and in vitro release 3 mg of CuS@mSiO2-PEG nanoparticles was dispersed in 5 mL of PBS solution (pH = 7.4) containing DOX (0.24 mg/mL), and stirred for 48 h in darkness at room temperature to reach the equilibrium state. The DOX-loaded CuS@mSiO2-PEG nanocomposites (CuS@mSiO2-PEG/DOX) were collected by centrifugation and were washed three times with PBS solution to remove the unbound DOX. All the supernatant solution was collected together and measured by a UV-Vis spectrophotometer at 482 nm to calculate the amount of DOX payload in the CuS@mSiO2-PEG nanoparticles. The encapsulation efficiency of the CuS@mSiO2-PEG nanoparticles = (weight of DOX loaded into the CuS@mSiO2-PEG)/(initial weight of DOX). The loading content of the CuS@mSiO2-PEG nanoparticles = (weight of DOX loaded into the CuS@mSiO2-PEG)/(weight of the CuS@mSiO2-PEG + DOX loaded into the CuS@mSiO2-PEG). This journal is © The Royal Society of Chemistry [year]
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The release of DOX from the CuS@mSiO2-PEG/DOX nanocomposites with or without the 980 nm NIR laser irradiation was studied. The CuS@mSiO2-PEG/DOX nanocomposites were dispersing in PBS solution at pH 7.4 and pH 5.0 with ultrasonic, respectively. The laser-triggered drug release experiments were performed by irradiating (980 nm, 0.72W/cm2) the dispersion liquid for 5 min under stirring. Then the dispersion liquid was centrifuged and supernatant was collected, and bottom CuS@mSiO2-PEG/DOX nanocomposites were dispersed in fresh PBS solution again and the dispersion liquid was placed in room temperature with stirring. As the controls, dispersion liquid of the CuS@mSiO2-PEG/DOX under stirring without laser irradiation was placed in room temperature with stirring and was centrifuged every hour or two hour intervals to collect supernatant. The amount of released DOX in the supernatant was determined by UV-Vis-NIR spectrophotometer. At a certain time intervals, above operation was repeated. 2.7 CLSM Imaging. The intracellular DOX uptake was qualitatively confirmed by CLSM(Confocal Laser Scanning Microscopy). HeLa cells were seeded in a 12-well plate and cultured for 24 h to allow the HeLa cells to attach onto the coverslips. Before CLSM imaging, the HeLa cells attached onto the coverslips were incubated with 1 mL of fresh cell medium containing the CuS@mSiO2-PEG/DOX nanocomposites (DOX concentration 5 µg/mL) for 4 h and 8 h, respectively. Then the cells were washed with PBS several times. Then the cells were fixed with glutaraldehyde for 15 min at 4 °C and counterstained with Hoechst 33342 for 15 min at 37 °C using a standard
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procedure. Finally, samples were imaged by CLSM (Leica TCS SP5, Germany). 2.8 Cytotoxicity of the CuS@mSiO2-PEG nanoparticles The in vitro cytotoxicity was determined by the MTT assay in human cervical carcinoma cell line (HeLa)13. HeLa cells were plated into a 96-well plate in a complete medium at 37 oC and 5% CO2 for 24 h before the experiments. The culture medium was replaced and cells were incubated with complete medium containing the CuS@mSiO2-PEG nanoparticles at a series of concentrations at 37 oC with 5% CO2 for further 24 h. Relative cell viabilities were determined by the standard MTT assay. 2.9 Chemo-photothermal therapy Chemo-photothermal therapy experiments were referenced by our previous method22. HeLa cells were plated into a 96-well plate in a complete medium for 24 h before the experiments. The culture medium was replaced with fresh medium or complete medium containing the DOX, CuS@mSiO2-PEG and CuS@mSiO2PEG/DOX nanocomposites at a series of concentrations. Especially, at each equivalent DOX point, CuS@mSiO2-PEG has the same Cu concentration as the CuS@mSiO2-PEG/DOX. After 4 h of incubation, the cells were washed with PBS and replaced with fresh medium. The cells were treated with or without irradiation of 980 nm light (0.72 W/cm2) for 5 min, respectively, and then incubated for further 24 h. The standard MTT assay was carried out to quantify the cell viabilities.
3. Results and Discussions
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Fig. 1 Schematic illustration of the synthesis of CuS@mSiO2-PEG core-shell nanoparticles The typical procedure of fabricating CuS@mSiO2-PEG core-shell potential of CuS nanocrystals is -42.8 mV due to the citric acid nanoparticles was illustrated in Fig. 1. As-prepared CuS 45 ligands. After adding CTAB, the Zeta potential of the CuS/CTAB nanocrystals were capped by hydrophilic citric acid ligands in nanoparticles increases to 31.2 mV, indicating a change from aqueous solution, thus the nanoparticles were electronegative due negatively charge to positively charge. After removal of CTAB template, the Zeta potential decreases to -20.0 mV, attributing to to citric acid ligands. Mesoporous silica shell can’t coat directly on such electronegative nanoparticles because of the hydrolysate the OH groups on the surface. of TEOS with negative charge. So the surfactant-CTAB was used to change the chemistry of the surfactant head groups on the surface of the CuS nanocrystals. The bilayer of cationic CTAB coats around the CuS nanocrystals by electrostatic interaction and hydrophobic interaction, and changes the nanocrystals from negatively charge to positively charge26. The micelles formed by excess CTAB and bilayer of CTAB around the nanocrystals were used as a soft template, and the TEOS hydrolyzed and condensed under basic conditions and SiO2 grew around the template due to electrostatic interaction (between electropositive CTAB and anionic silicate species)27, forming CuS@SiO2 core-shell nanoparticles. In order to further improve the biocompatibility of the nanoparticles, the PEG was grafted onto the surface of SiO2 shell. The template CTAB was removed by treatment with NH4NO3 ethanol solution, and CuS@mSiO2-PEG core-shell nanoparticles with mesoporous structure were obtained. CuS nanocrystals capped with citric acid were first prepared according to the literature10. The transmission electron microscope (TEM) images of the obtained CuS nanocrystals showed an average diameter of ~12 nm and high crystallinity (Fig S1). The interplanar spacing is ~0.305 nm, corresponding to d spacing for the (102) crystal planes of standard hexagonal structured CuS crystal.
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Fig. 2 Low magnified TEM image (a), medium magnified TEM image (b), HRTEM image (c) and magnified HRTEM image (d) of CuS@mSiO2-PEG core-shell nanoparticles. In the silica-coating process, CTAB acted as an organic template for preparing CuS@SiO2 core-shell nanoparticles. The CuS@SiO2-PEG core-shell nanoparticles were obtained by grafting with PEG onto the SiO2 shell. After removal of CTAB template by ion exchange of NH4NO3, the CuS@mSiO2-PEG core-shell nanoparticles were obtained. The Zeta potentials of CuS nanocrystals, CuS/CTAB and CuS@mSiO2-PEG nanoparticles displayed the surface charge change of nanoparticles before and after silica coating (Fig S3). The Zeta 4 | Journal Name, [year], [vol], 00–00
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Fig. 3 (a) XRD patterns of the standard CuS (lower), as-prepared CuS (middle) and CuS@mSiO2-PEG nanoparticles (upper). (b) The FTIR spectra of as-prepared CuS@mSiO2 and CuS@mSiO2PEG nanoparticles (Inset: photo of as as-prepared dispersion of CuS@mSiO2-PEG nanoparticles). As shown in Fig. 2a-b, the CuS nanocrystals are completely encapsulated into mesoporous SiO2 shell with an estimated thickness of ~13 nm, and CuS@mSiO2-PEG nanoparticles have a diameter of ~37 nm. The diameter of the CuS@mSiO2-PEG coreshell nanoparticles in the water was also measured by dynamic light scattering (Zetasizer Nano Z). As shown in Fig. S4, there is a peak around 90 nm, and the average diameter is ~ 179.7 nm, suggesting that the CuS@mSiO2-PEG core-shell nanoparticles have slight aggregation in the water. As shown in the HRTEM images of CuS@mSiO2-PEG core-shell nanoparticles, the interplanar spacing of the core is ~0.189 nm, corresponding to the d spacing for the (110) crystal planes of standard hexagonal structured CuS crystal, which suggests that the phase structure of CuS nanocrystals well retained after silica coating. The phase structures of as-obtained CuS nanocrystals and CuS@mSiO2-PEG core-shell nanoparticles were examined by This journal is © The Royal Society of Chemistry [year]
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Fig. 4 (a) UV-Vis-NIR absorbance spectra of as-prepared CuS@mSiO2-PEG nanoparticles. (b) Plots of linear fitting absorbance at 980 nm versus the concentration of CuS@mSiO2PEG nanoparticles. PEG modification of the silica shells can improve colloidal stability, decrease immunogenicity and enhance biocompatibility12, 28, thus the CuS@mSiO2 nanoparticles were further grafted with PEG on the outside surface of silica shell. The Fourier transform infrared (FTIR) spectra of the CuS@mSiO2 and CuS@mSiO2-PEG core-shell nanoparticles are shown in Fig 3b. The CuS@mSiO2 and CuS@mSiO2-PEG coreshell nanoparticles both show typical stretching vibration peaks of Si-O-Si at 1074 cm-1 and 962 cm-1, and a vibration peak of H2O molecule adsorbed by mesoporous silica at 1634 cm-1 29, 30. Compared with the CuS@mSiO2 nanoparticles, the additional adsorption peak of the CuS@mSiO2-PEG at 2928 cm-1 is assigned to stretching vibration of methylene (CH2) in the long alkyl chain of the backbone of PEG, and at 1350 cm-1 is corresponded to deformation vibration of backbone of PEG31. These results indicate that the PEG was successfully grafted on the mesoporous silica shell. The 29Si NMR spectrum of CuS@mSiO2-PEG nanoparticles is shown in Fig. S5. The signal This journal is © The Royal Society of Chemistry [year]
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at -64.2 ppm can be assigned to Si of C-Si(OSi)3 (T3 sites)32-34, which further proves the PEG was successfully grafted on the mesoporous silica shell. The CuS@mSiO2-PEG core-shell nanoparticles show excellent colloidal stability due to the PEG modification. After aqueous dispersion of the CuS@mSiO2-PEG core-shell nanoparticles standing for a week, no aggregation was discovered (Fig 3b, inset).
Fig.5 (a) Temperature change of the aqueous solution containing the CuS@mSiO2-PEG nanoparticles with different concentrations under irradiation of the 980 nm laser (0.72 W/ cm2). (b) Plot of temperature elevation over a period of 360 s versus the concentration of the CuS@mSiO2-PEG nanoparticles. As shown in Fig. S2a, as-synthesized CuS@mSiO2-PEG coreshell nanoparticles after removal of CTAB have a BrunauerEmmett-Teller (BET) surface area as high as 802 m2/g and exhibit the type IV isotherm. The pore size distribution exhibits a sharp peak centered at a mean value of 3.6 nm (Fig. S2b), and the Barrett-Joyner-Halenda (BJH) pore volume of the CuS@mSiO2PEG core-shell nanoparticles is calculated to be 0.829 m3/g. Thus, a large surface area, great pore volume and appropriate pore size insure high drug loading capacity of the CuS@mSiO2-PEG coreshell nanoparticles. Ideal photothermal agents should have strong adsorption in NIR region, so the optical property of the aqueous dispersion of the CuS@mSiO2-PEG nanoparticles was examined by UV-VisNIR spectroscopy. As shown in Fig. 4a, the CuS@mSiO2-PEG aqueous dispersion exhibits a strong absorption in the NIR region due to the localized surface plasma resonances (SPR) of valenceband free carriers (positive holes) of CuS core10. Moreover, the Journal Name, [year], [vol], 00–00 | 5
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XRD pattern (Fig. 3a). The XRD patterns of both samples match well with the standard hexagonal phase of CuS (JCPDS card no: 79-2321). In addition, CuS@mSiO2-PEG nanoparticles have a wide peak at ~ 23°, which is clearly due to the amorphous silica coating. These results further prove that the phase structures of CuS nanocrystals were well retained after silica coating.
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absorptions at 980 nm were lineally enhanced with the increase of the concentration (Fig. 4b). Particularly, the absorbance peak locates at 975 nm, and is very close to the wavelength (980 nm) of excitation laser, which ensures that the CuS@mSiO2-PEG aqueous dispersion can convert the absorbed light into heat more effectively and nearly gain the highest temperature under irradiation of 980 nm laser35.
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Fig. 6 (a) Absorption spectra of the DOX and the CuS@mSiO2PEG/DOX dispersion (Inset: photo of as as-prepared CuS@mSiO2-PEG/DOX nanocomposites’ dispersion). (b) The cumulative release kinetics of the DOX from the CuS@mSiO2PEG/DOX nanocomposites in PBS (pH 7.4) and acetate buffer (pH 5.0) at room temperature with or without the NIR laser irradiation at a safe power density (0.72 W/cm2). To investigate the photothermal effect generated by the NIR laser irradiation, the temperatures of the CuS@mSiO2-PEG aqueous dispersion were measured under irradiation of 980 nm light with a safe power density (0.72 W/cm2). As shown in Fig. 5b, only a negligible temperature increase is observed from the control experiment of pure water (0 µg/mL) under the NIR irradiation. In contrast, the temperatures of the CuS@mSiO2-PEG aqueous dispersion were raised rapidly during the irradiation process under the same condition. For example, the temperature of the aqueous dispersion at the concentration of 200 µg/mL raised from 20.0 ℃ to 40.4 ℃ after 360 s of NIR irradiation. If the normal body temperature is 37 ℃, so the final temperature can reach 57.4 ℃ that is high enough to kill the cancer cells. Moreover, the temperature elevation increased as the concentration of the CuS@mSiO2-PEG nanoparticles increased 6 | Journal Name, [year], [vol], 00–00
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(Fig.5b). The photothermal conversion efficiency of CuS@mSiO2-PEG core-shell nanoparticles was also measured according to the references (see ESI for details) 13, 36, 37. As shown in Fig.S6, the photothermal conversion efficiency of CuS@mSiO2-PEG core-shell nanoparticles under irradiation of 980 laser (0.72W/cm2) can be calculated to be 29.5%, which is higher than those of previously reported Au rods (~ 21.0%, 800 nm laser), Cu2-xSe nanocrystals (~ 22%, 800 nm laser) and Cu9S5 nanocrystals (~ 25.7%,980 nm laser)37. These data illustrate that the CuS@mSiO2-PEG nanoparticles can effectively absorb and convert the NIR light into fatal heat due to their strong SPR absorption peak approximately at the wavelength (980 nm) of illumination laser, so the CuS@mSiO2-PEG core-shell nanoparticle is an excellent photothermal agent. These CuS@mSiO2-PEG core-shell nanoparticles are suitable for drug delivery due to the mesoporous silica shell. The anticancer drug DOX could be loaded into the CuS@mSiO2-PEG core-shell nanoparticles by simply mixing phosphate-buffer saline (PBS) solutions containing DOX with the nanoparticles. As shown in Fig. 6a, the CuS@mSiO2-PEG/DOX nanocomposites not only exhibit strong NIR absorbance from the CuS nanocrystals, but also show typical absorption peak near 480 nm region due to DOX, which indicates that the DOX has been successfully incorporated into the CuS@mSiO2-PEG nanopaticles. The color the CuS@mSiO2-PEG/DOX dispersion turned red brown (from dark yellow-green) due to loading DOX (Fig. 6a, inset). Because of large surface area and pore volume, appropriate pore size, the encapsulation efficiency and loading content of the DOX into the CuS@mSiO2-PEG core-shell nanoparticles are determined to be as high as 90.1％ and 26.5％ (by weight), respectively. Release of the DOX from the CuS@mSiO2-PEG core-shell nanoparticles against buffer solution at pH 7.4 and 5.0 was studied to simulate normal physiological environment and cellular lysosome environment, respectively (Fig. 6b). The cumulative release amount of the DOX at pH 7.4 was quite small within 9 h due to strong electrostatic interactions between the DOX and dissociated silanols23, indicating that the CuS@mSiO2PEG/DOX nanocomposites released very little DOX molecules in normal physiological environment. However, the drug release rate became much faster and the cumulative release of drug reached up to 37.9 % at pH 5.0 due to the enhancement of the solubility of the DOX for protonated daunosamine group under acidic conditions38. This pH-dependent release behavior with more the DOX release at lower pH value for the CuS@mSiO2PEG/DOX nanocomposites is beneficial for the cancer therapy since the microenvironments of extracellular tissues of tumors and intracellular lysosomes and endosomes are acidic28, 39. Whether the photothermal effect originated from CuS core under the irradiation (0.72 W/cm2, 980 nm) for 5 min can trigger the DOX release from the CuS@mSiO2-PEG/DOX nanocomposites at pH 5.0 and 7.4 was studied. The DOX released rapid upon the NIR irradiation, while it released slowly when the laser was shut off. At pH 5.0, the cumulative release of the DOX under irradiation increased from 37.9% to 67.2% within 9 h. At pH 7.4, the cumulative release of the DOX was also enhanced by laser irradiation. These results demonstrate that the release of the DOX from the CuS@mSiO2-PEG/DOX This journal is © The Royal Society of Chemistry [year]
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nanocomposites could be triggered by the NIR irradiation, which is due to the heat generated by the photothermal effect of the CuS@mSiO2-PEG dissociated the strong interactions between DOX and SiO228, and thus more DOX molecules released. Since the light can be manipulated precisely, the release of the DOX from the CuS@mSiO2-PEG nanocomposites can be conveniently and accurately controlled by the NIR irradiation. Thus, CuS@mSiO2-PEG core-shell nanoparticles have successfully achieved the drug release with control of the area, time, and dosage by pH and NIR light.
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Fig. 7 (a) Viabilities of HeLa cells incubated for 24 h with different concentrations of the CuS@mSiO2-PEG nanoparticles. (b) Viabilities of HeLa cells after incubation with different concentrations of free DOX, CuS@mSiO2-PEG and CuS@mSiO2-PEG/DOX with or without 5 min of NIR irradiation (0.72 W/cm2, 980 nm), and HeLa cells (incubated with only medium) with 5 min of NIR irradiation. At the each equivalent DOX point, CuS@mSiO2-PEG has a same Cu concentration as the CuS@mSiO2-PEG/DOX. To further investigate the intracellular drug delivery by CuS@mSiO2-PEG/DOX nanoparticles, DOX fluorescence in HeLa cell was examined using CLSM. As shown in Fig.S7, red fluorescence (DOX) of the cells was relatively weak after 4 h of incubation, but the intensity of red fluorescence was significantly enhanced after 8 h of incubation. These results indicated that increasing amounts of DOX delivered by the CuS@mSiO2PEG/DOX gradually passed through the cytomembrane, entered
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into cytoplasm, passed through the nucleus membrane and eventually assembled in nucleus with incubation time increasing. The undamaged nucleuses shown in Fig.S7d-f are much fewer than that of Fig.7Sa-c, indicating as the incubation time was prolonged the nucleus gradually collapsed and cells died due to DOX in nucleuses. Thus, the effective uptake of DOX indicates the effective delivery of DOX by the CuS@mSiO2- PEG/DOX nanoparticles to kill cancer cells. Although mesoporous silica has been widely reported to be suitable for drug delivery, we still investigated the cytotoxicity of CuS@mSiO2-PEG core-shell nanoparticles before biological applications to confirm such an argument. Thus, the standard MTT assay was carried out to determine the relative viabilities of HeLa cells after they were incubated with the CuS@mSiO2-PEG nanocomposites at various concentrations for 24 h. The results showed that the CuS@mSiO2-PEG core-shell nanoparticles exhibited no appreciable negative effect on the viability of cells, even at a high concentration up to 500 µg/mL (Fig. 7a), indicating the good biocompatibility of the CuS@mSiO2-PEG core-shell nanoparticles. The therapy effects of the photothermal therapy, chemotherapy, and their combination were studied in vitro. Free DOX and CuS@mSiO2-PEG/DOX nanocomposites have obvious cytotoxicity to Hela cells, which show an increasing inhibition ratio against HeLa cells with the increasing concentration (Fig. 7b). Moreover, the CuS@mSiO2-PEG/DOX nanoparticles demonstrated slightly higher cytotoxicity than free DOX at the all tested concentrations since DOX-loaded nanoparticles can enter cancer cells more easily than free DOX6, 40. Whether the NIR irradiation can improve cancer cell-killing efficacy was studied. There was hardly any negative effect to the cell viability when the cells were irradiated with NIR laser alone. However, NIR irradiation showed significant toxicity in the presence of the CuS@mSiO2-PEG nanoparticles due to their photothermal effect. The CuS@mSiO2-PEG/DOX nanocomposites under the NIR irradiation demonstrated a higher cell toxicity at all tested concentrations than the chemotherapy (without NIR irradiation) or the photothermal therapy (without DOX) alone (Fig.7b). For example, when the DOX concentration is 15 µg/mL, 63.0 % of the cells were inhibited by the CuS@mSiO2-PEG/DOX nanocomposites under the NIR irradiation, while 51.3% of the cells were killed by the CuS@mSiO2-PEG/DOX nanocomposites at the same concentration without the NIR illumination and only 30.9% of the cells were inhibited by the CuS@mSiO2-PEG nanocomposites (Cu concentration is identical as the CuS@mSiO2-PEG/DOX nanocomposites) in absence of DOX. These results demonstrated that the combination of chemotherapy and photothermal therapy by the CuS@mSiO2-PEG/DOX nanocomposites showed better therapy effect for cancer cells than individual therapy due to enhanced toxicity of DOX and hyperthermia8, 9. Therefore, as-prepared CuS@mSiO2-PEG nanoparticles can act as a difunctional drug delivery nanoplatform for combined chemo-photothermal therapy when driven by NIR irradiation.
In conclusion, we have developed a NIR light responsive drug delivery nanoplatform (CuS@mSiO2-PEG) for efficient chemoJournal Name, [year], [vol], 00–00 | 7
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photothermal therapy, which was fabricated by a facile, cheap and green way. CuS@mSiO2-PEG core-shell nanoparticles have good biocompatibility and colloidal stability, as well as a very high DOX loading content (26.5% wt). The CuS nanocrystals (core) can effectively absorb and convert NIR light to fatal heat under NIR light irradiation, and the release of DOX from the mesoporous silica (shell) can be triggered by pH and the NIR light. When the CuS@mSiO2-PEG/DOX nanocomposites were illuminated by NIR light, both chemotherapy and photothermal therapy were simultaneously driven, resulting in a synergistic effect for killing cancer cells. More importantly, the combination of therapies demonstrated significant enhancement of therapy effects for cancer cells than chemotherapy or photothermal therapy alone. Therefore, the NIR light responsive drug delivery nanoplatform has great potential in cancer chemo-photothermal therapy.
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Notes and references a College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai, 201620, China b State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University, Shanghai 201620, China; E-mail: [email protected] c College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China d Center of Super-Diamond and Advanced Films (COSDAF), Department of Physics and Materials Science, City University of Hong Kong, Hong Kong e Department of Orthopaedics, Shanghai First People’s Hospital, Shanghai Jiaotong University, 100 Haining Road, Hongkou District, Shanghai 200080, China
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Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grant no. 21171035, 51472049 and 51302035), the Key Grant Project of Chinese Ministry of Education (Grant no. 313015), the PhD Programs Foundation of the Ministry of Education of China (Grant no. 20110075110008 and 20130075120001), the National 863 Program of China (Grant no. 2013AA031903), the Science and Technology Commission of Shanghai Municipality (Grant no. 13ZR1451200), the Fundamental Research Funds for the Central Universities, the Program Innovative Research Team in University (IRT1221), the Shanghai Leading Academic Discipline Project (Grant no. B603) and the Program of Introducing Talents of Discipline to Universities (no. 111-2-04).
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36. 120
1. 50
2. 3. 4. 5.
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C. Xu, D. R. Yang, L. Mei, Q. H. Li, H. Z. Zhu and T. H. Wang, Acs Appl. Mater. Interfaces, 2013, 5, 12911-12920. F. Tang, L. Li and D. Chen, Adv. Mater., 2012, 24, 1504-1534. T. L. Doane and C. Burda, Chem. Soc. Rev., 2012, 41, 2885-2911. W. Yin, L. Yan, J. Yu, G. Tian, L. Zhou, X. Zheng, X. Zhang, Y. Yong, J. Li, Z. Gu and Y. Zhao, Acs Nano, 2014, 8, 6922-6933. J. H. Park, G. von Maltzahn, L. L. Ong, A. Centrone, T. A. Hatton, E. Ruoslahti, S. N. Bhatia and M. J. Sailor, Adv. Mater., 2010, 22, 880-885. W. Fang, J. Yang, J. Gong and N. Zheng, Adv. Funct. Mater., 2012, 22, 842-848. W.D. Jang, D. Yim and I.H. Hwang, J. Mater. Chem. B, 2014, 2, 2202-2211.
8 | Journal Name, [year], [vol], 00–00
37. 38. 125
39. 40.
H. Park, J. Yang, J. Lee, S. Haam, I. H. Choi and K. H. Yoo, Acs Nano, 2009, 3, 2919-2926. S. M. Lee, H. Park and K. H. Yoo, Adv. Mater., 2010, 22, 40494053. M. Zhou, R. Zhang, M. Huang, W. Lu, S. Song, M. P. Melancon, M. Tian, D. Liang and C. Li, J. Am. Chem. Soc., 2010, 132, 1535115358. Q. Tian, M. Tang, Y. Sun, R. Zou, Z. Chen, M. Zhu, S. Yang, J. Wang, J. Wang and J. Hu, Adv. Mater., 2011, 23, 3542-3547. Q. Xiao, X. Zheng, W. Bu, W. Ge, S. Zhang, F. Chen, H. Xing, Q. Ren, W. Fan, K. Zhao, Y. Hua and J. Shi, J. Am. Chem. Soc., 2013, 135, 13041-13048. X. Liu, B. Li, F. Fu, K. Xu, R. Zou, Q. Wang, B. Zhang, Z. Chen and J. Hu, Dalton Trans., 2014, 43, 11709-11715. Y. Li, W. Lu, Q. Huang, M. Huang, C. Li and W. Chen, Nanomedicine, 2010, 5, 1161-1171. J. Mou , P. Li , C. Liu , H. Xu , L. Song , J. Wang , K. Zhang ,Y. Chen , J. Shi and H. Chen. Small, 2015, DOI: 10.1002/smll.201403249. L. Guo, I. Panderi, D. D. Yan, K. Szulak, Y. Li, Y. T. Chen, H. Ma, D. B. Niesen, N. Seeram, A. Ahmed, B. Yan, D. Pantazatos and W. Lu, Acs Nano, 2013, 7, 8780-8793. L. Cheng, C. Wang, L. Feng, K. Yang and Z. Liu, Chem. Rev., 2014, 114, 10869-10939. K. Dong, Z. Liu, Z. Li, J. Ren and X. Qu, Adv. Mater., 2013, 25, 4452-4458. V. Biju, Chem. Soc. Rev., 2014, 43, 744-764. P. Yang, S. Gai and J. Lin, Chem. Soc. Rev., 2012, 41, 3679-3698. G. Song, Q. Wang, Y. Wang, G. Lv, C. Li, R. Zou, Z. Chen, Z. Qin, K. Huo, R. Hu and J. Hu, Adv. Funct. Mater., 2013, 23, 4281-4292. X. Liu, Q. Wang, C. Li, R. Zou, B. Li, G. Song, K. Xu, Y. Zheng and J. Hu, Nanoscale, 2014, 6, 4361-4370. Z. Zhang, L. Wang, J. Wang, X. Jiang, X. Li, Z. Hu, Y. Ji, X. Wu and C. Chen, Adv. Mater., 2012, 24, 1418-1423. American National Standard Institute, ANSI Z136. 1-2007, American National Standard for Safe Use of Lasers, Orlando, Fl 2007. X. Liu, F. Fu, K. Xu, R. Zou, J. Yang, Q. Wang, Q. Liu, Z. Xiao and J. Hu, J. Mater. Chem. B, 2014, 2, 5358-5367. B. Chang, X. Zhang, J. Guo, Y. Sun, H. Tang, Q. Ren and W. Yang, J.Colloid Interface Sci., 2012, 377, 64-75. Y. Wan and D. Zhao, Chem. Rev., 2007, 107, 2821-2860. S. Shen, H. Tang, X. Zhang, J. Ren, Z. Pang, D. Wang, H. Gao, Y. Qian, X. Jiang and W. Yang, Biomaterials, 2013, 34, 3150-3158. G. Yang, S. Gai, F. Qu and P. Yang, Acs Appl. Mater. Interfaces, 2013, 5, 5788-5796. X. Kang, Z. Cheng, D. Yang, P. Ma, M. Shang, C. Peng, Y. Dai and J. Lin, Adv. Funct. Mater., 2012, 22, 1470-1481. V. Cauda, C. Argyo and T. Bein, J. Mater. Chem., 2010, 20, 86938699. A. L. de Lima, A. Mbengue, R. A. S. San Gil, C. M. Ronconi and C. J. A. Mota, Catal. Today, 2014, 226, 210-216. R. Dobrowolski, M. Oszust-Cieniuch, J. Dobrzynska and M. Barczak, Colloids Surf., A, 2013, 435, 63-70. M. Popova, A. Szegedi, K. Yoncheva, S. Konstantinov, G. P. Petrova, H. A. Aleksandrov, G. N. Vayssilov and P. Shestakova, Microporous Mesoporous Mater., 2014, 198, 247-255. H. Chen, L. Shao, T. Ming, Z. Sun, C. Zhao, B. Yang and J. Wang, Small, 2010, 6, 2272-2280. C. M. Hessel, V. P. Pattani, M. Rasch, M. G. Panthani, B. Koo, J. W. Tunnell and B. A. Korgel, Nano Lett., 2011, 11, 2560-2566. Q. Tian, F. Jiang, R. Zou, Q. Liu, Z. Chen, M. Zhu, S. Yang, J. Wang, J. Wang and J. Hu, Acs Nano, 2011, 5, 9761-9771. X. C. Qin, Z. Y. Guo, Z. M. Liu, W. Zhang, M. M. Wan and B. W. Yang, J. Photochem. Photobiol., B, 2013, 120, 156-162. C. Wang, H. Xu, C. Liang, Y. Liu, Z. Li, G. Yang, H. Cheng, Y. Li and Z. Liu, Acs Nano, 2013, 7, 6782-6795. W. Fang, S. Tang, P. Liu, X. Fang, J. Gong and N. Zheng, Small, 2012, 8, 3816-3822.
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A difunctional nanoplatform (CuS@mSiO2-PEG) acted as NIR light induced photothermal-triggered drug delivery system for efficient chemo-photothermal therapy.
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Xijian Liu,a,b Qilong Ren,b Fanfan Fu,c Rujia Zou,b,d Qian Wang,b,e Guobing Xin, Zhiyin Xiao,b Xiaojuan Huang,b Qian Liub and Junqing Hu*,b 5
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Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX DOI: 10.1039/b000000x We report a facile and low-cost approach to design a difunctional nanoplatform (CuS@mSiO2-PEG) as a near-infrared (NIR) light responsive drug delivery system for efficient chemo-photothermal therapy. The nanoplatform demonstrated good biocompatibility and colloidal stability, as well as high loading capacity for anticancer drug (26.5% wt for doxorubicin (DOX)). The CuS nanocrystals (core) within these CuS@mSiO2-PEG core-shell nanoparticles can effectively absorb and convert NIR light to fatal heat under NIR light irradiation for photothermal therapy, and the release of DOX from the mesoporous silica (shell) can be triggered by pH and the NIR light for chemotherapy. When the CuS@mSiO2-PEG/DOX nanocomposites were irradiated by 980 nm light, both chemotherapy and photothermal therapy were simultaneously driven, resulting in a synergistic effect for killing cancer cells. Importantly, compared with chemotherapy or photothermal treatment alone, the combined therapy significantly improved the therapeutic efficacy.
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Chemotherapy is one of the most commonly used cancer treatment strategies, but it shows many limitations, such as systemic side effects, low efficacy, and drug resistance1. In order to improve anticancer efficacy and optimize therapy, nanocarriers were used for drug delivery2, 3. Particularly, stimuli-responsive drug delivery system using “smart” nanocarriers has become a promising modality for enhancing effects of the chemotherapy due to the ability of drug release with control of the area, time, and dosage4-6. To date, various strategies, such as hydrophobicity, pH, temperature, biomolecular reactions, and light have been developed to control the drug delivery for carriers3, 7. Among them, near-infrared (NIR) light responsive release is a very important method due to the non-invasive nature, high spatial resolution, and minimal damage to normal tissues of NIR light4, 6. Importantly, the NIR light responsive system for drug delivery combines the chemotherapy and photothermal therapy, exhibiting synergistic effect for cancer therapy due to enhanced cytotoxicity of anticancer drug doxorubicin hydrochloride (DOX) at elevated temperatures and higher heat sensitivity for the cells exposed to the DOX8, 9. Copper chalcogenides are a new class of photothermal agent which has been considered as a prospective candidate for photothermal therapy10-15. Moreover, CuS nanocrystals can be biodegradable and have lower long-time toxicity than most used photothermal agent-gold nanorod16. Zhou and coworkers have prepared CuS nanocrystals and used them for photothermal therapy, which exhibited good cancer treatment efficacy10, This journal is © The Royal Society of Chemistry [year]
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however, the exorbitant power density of the laser (12 W/cm2) were used in photothermal therapy that probably harmed skins and normal tissues. To solve this problem, combination of photothermal therapy of CuS nanocrystals with chemotherapy can be adopted due to the fact that the combined therapy can effectively enhance cancer treatment efficacy and reduce the power density of laser in photothermal therapy. And numerous good results showed that the combination of photothermal therapy and chemotherapy had better effects on destroying cancer cells than each separate treatment17.For example, hollow CuS nanoparticles combined photothermal therapy and chemotherapy that achieved enhanced tumoricidal efficacy with a lower drug dose and mild irradiation conditions due to the synergistic effects18. However, the biocompatibility of CuS nanoparticles at high concentration is alarming. Mesoporous silica nanoparticles are widely used for drug controllable release in chemotherapy due to excellent stability, good biocompatibility, large surface and cavity volumes, tunable porosity and facile modification2, 19, 20 . So the combination of chemotherapy and photothermal therapy in a platform can be achieved by mesoporous silica coating CuS nanocrystals for enhancing therapy effect. In our previous works, core (photothermal agents)-shell (mesoporous silica) nanocomposites based on hydrophobic inorganic nanoparticles, such as Cu9S5@mSiO2-PEG21, Cu2-xSe@mSiO2PEG22 that combined photothermal therapy and chemotherapy in a platform, exhibited remarkable cancer-cell killing efficiency due to the synergistic effect. Unfortunately, the synthesis process of Cu9S5 and Cu2-xSe nanocrystal is not convenient due to synthesis in high temperature, anhydrous and anaerobic condition, and the synthesis cost is not cheap due to use of [journal], [year], [vol], 00–00 | 1
Dalton Transactions Accepted Manuscript
CuS@mSiO2-PEG core-shell nanoparticles as a NIR light responsive drug delivery nanoplatform for efficient chemo-photothermal therapy
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oleylamine as solvent. In addition, it takes long time and complicated procedures to change Cu9S5 and Cu2-xSe nanocrystals from hydrophobicity to hydrophilcity before silica coating. Thus, it is a great need to develop a facile, low-cost and green synthetic approach to fabricate core (photothermal agents)shell (mesoporous silica) nanocomposites for chemophotothermal therapy. In this work, CuS@mSiO2-PEG core-shell nanoparticles were prepared by a facile, cheap and green way, and used as a NIR light responsive system for drug delivery. Compared with previous core-shell nanoparticles which combined photothermal therapy and chemotherapy, such as Au@SiO223, Cu9S5@mSiO2PEG21, Cu2-xSe@mSiO2-PEG22, the CuS@mSiO2-PEG coreshell nanoparticles had remarkable advantages. Not only the material cost for synthesis of the CuS nanocrystal is much cheaper than that of the Au nanorod, Cu9S5 and Cu2-xSe nanocrystal, but also the preparation of the CuS nanocrystal is more convenient than that of the Au nanorod, Cu9S5 and Cu2-xSe nanocrystal. The CuS nanocrystal can be synthesized in aqueous solution at low temperature only using cheap material (copper chloride, sodium sulphide, sodium citrate and water). Without any fussy pretreatment, mesoporous silica coating can be directly carried out after adding CTAB to CuS solution. Mesoporous silica shell as carrier for loading anticancer drug (DOX) and CuS nanocrystals core as photothermal agents, CuS@mSiO2-PEG core-shell nanoparticles can effectively convert the NIR light into fatal heat to kill cancer cells (photothermal therapy) under 980nm laser irradiation at a safe power density (0.72W/cm2)24, meanwhile the heat generated by the photothermal effect can lead to rapid release of the drug loaded inside the mesoporous SiO2 shells to kill cancer cells (chemotherapy). Importantly, the combination of photothermal therapy and chemotherapy significantly improved the therapeutic efficacy, and displayed better therapeutic effects for cancer treatment than individual therapies.
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2.1 Chemicals and reagents All reagents were used without further purification. Copper (II) chloride (CuCl2·2H2O), sodium citrate (C6H5Na3O7·2H2O), cetyltrimethylammonium bromide (CTAB), sodium hydroxide (NaOH) and anhydrous ethanol are analytically pure, which were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China), Tetraethylorthosilicate (TEOS, GR) and sodium sulfide (Na2S·9H2O) were obtained from Aladdin, and 2[Methoxy(polyethyleneoxy)propyl]-trimethoxysilane (PEG-silane, MW = 596-725 g/mol, 9-12 EO) was obtained from Gelest (Morrisville, PA) and doxorubicin hydrochloride (DOX) was got from Huafeng United Technology CO., Ltd. (Beijing, China) 2.2 Characterization Sizes, morphologies, and microstructures of the nanoparticles were measured by a transmission electron microscope (TEM; JEM-2100F). The surface area, pore size, and pore-size distribution of the products were determined by BrunauerEmmett-Teller (BET) nitrogen adsorption-desorption and BarettJoyner-Halenda (BJH) methods (Micromeritics, ASAP2020). Powder X-ray diffraction (XRD) was determined by a D/max2550 PCX-ray diffractometer (Rigaku, Japan). UV-visible absorption spectra were determined on an UV-Vis 1901
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Spectrophotometer (Phoenix) using quartz cuvettes with an optical path of 1 cm. Fourier transform infrared (FTIR) spectra were measured using attenuated total reflectance (ATR) methods by an IRPRESTIGE-21 spectrometer (Shimadzu). 29Si CP (crosspolarization)/MAS(magic-angle spinning) solid state NMR measurement was carried out on a Bruker Avance 400 spectrometer (9.4T), operating at Larmor frequencies 79.51 MHz (spinning at 5 kHz, 4 ms contact time and 2 s repetition time). 2.3 Synthesis of CuS nanocrystals The CuS nanocrystals was synthesized according to previous methods10. 0.2 mmol Copper (II) chloride and 0.136 mmol sodium citrate were dissolved in 180 mL deionized water under stirring, then 20 mL sodium sulfide solution (10 mmol/L) was dropwise added. The reaction mixture turned dark brown after stirring for 5 min at room temperature, then was heated to 90 oC and maintained for 15 min. Finally, a dark-green solution of citrate-coated CuS nanocrystals was obtained and cooled with ice-cold water, then stored at 4 oC for later use. 2.4 Synthesis of CuS@mSiO2-PEG core-shell nanoparticles 0.7 g of CTAB and 65 mL of CuS nanocrystals’ solution were added to a round flask, then heated to 40 oC and maintained 2 h under stirring. 3 mL of ethanol and 100 µL of NaOH solution (30 mg/mL) were added, subsequently 100 µL of TEOS was dropped into this mixture. The mixture maintained 40 oC under continuously stirring for 1.5 h, then 100 µL of PEG-silane was added and the mixture maintained at 40 oC for another 6.5 h upon stirring. Finally, CuS@mSiO2-PEG core-shell nanoparticles were collected by centrifugation (12000 rpm, 10 min), and washed three times with ethanol. Then the products were dispersed in the ethanol solution of NH4NO3 (50 mL, 10 mg/mL), and the mixture stirred at 50 °C for 2 h to remove the template of CTAB by ion exchange. CuS@mSiO2-PEG core-shell nanoparticles were further washed three times with ethanol, then dispersed in deionized water and stored at 4 °C for further use. 2.5 Measurement of photothermal performance Photothermal performance was measured according to our previous method25. Aqueous dispersions of the CuS@mSiO2PEG core-shell nanoparticles with different concentrations (10, 25, 50, 100, 200 mg/mL) were illuminated by 980 nm laser (power density ~ 0.72 W/cm2). The temperature of the dispersions was measured by a digital thermometer (with an accuracy of 0.1 oC) using a thermocouple probe every 5 s. 2.6 DOX loading and in vitro release 3 mg of CuS@mSiO2-PEG nanoparticles was dispersed in 5 mL of PBS solution (pH = 7.4) containing DOX (0.24 mg/mL), and stirred for 48 h in darkness at room temperature to reach the equilibrium state. The DOX-loaded CuS@mSiO2-PEG nanocomposites (CuS@mSiO2-PEG/DOX) were collected by centrifugation and were washed three times with PBS solution to remove the unbound DOX. All the supernatant solution was collected together and measured by a UV-Vis spectrophotometer at 482 nm to calculate the amount of DOX payload in the CuS@mSiO2-PEG nanoparticles. The encapsulation efficiency of the CuS@mSiO2-PEG nanoparticles = (weight of DOX loaded into the CuS@mSiO2-PEG)/(initial weight of DOX). The loading content of the CuS@mSiO2-PEG nanoparticles = (weight of DOX loaded into the CuS@mSiO2-PEG)/(weight of the CuS@mSiO2-PEG + DOX loaded into the CuS@mSiO2-PEG). This journal is © The Royal Society of Chemistry [year]
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The release of DOX from the CuS@mSiO2-PEG/DOX nanocomposites with or without the 980 nm NIR laser irradiation was studied. The CuS@mSiO2-PEG/DOX nanocomposites were dispersing in PBS solution at pH 7.4 and pH 5.0 with ultrasonic, respectively. The laser-triggered drug release experiments were performed by irradiating (980 nm, 0.72W/cm2) the dispersion liquid for 5 min under stirring. Then the dispersion liquid was centrifuged and supernatant was collected, and bottom CuS@mSiO2-PEG/DOX nanocomposites were dispersed in fresh PBS solution again and the dispersion liquid was placed in room temperature with stirring. As the controls, dispersion liquid of the CuS@mSiO2-PEG/DOX under stirring without laser irradiation was placed in room temperature with stirring and was centrifuged every hour or two hour intervals to collect supernatant. The amount of released DOX in the supernatant was determined by UV-Vis-NIR spectrophotometer. At a certain time intervals, above operation was repeated. 2.7 CLSM Imaging. The intracellular DOX uptake was qualitatively confirmed by CLSM(Confocal Laser Scanning Microscopy). HeLa cells were seeded in a 12-well plate and cultured for 24 h to allow the HeLa cells to attach onto the coverslips. Before CLSM imaging, the HeLa cells attached onto the coverslips were incubated with 1 mL of fresh cell medium containing the CuS@mSiO2-PEG/DOX nanocomposites (DOX concentration 5 µg/mL) for 4 h and 8 h, respectively. Then the cells were washed with PBS several times. Then the cells were fixed with glutaraldehyde for 15 min at 4 °C and counterstained with Hoechst 33342 for 15 min at 37 °C using a standard
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procedure. Finally, samples were imaged by CLSM (Leica TCS SP5, Germany). 2.8 Cytotoxicity of the CuS@mSiO2-PEG nanoparticles The in vitro cytotoxicity was determined by the MTT assay in human cervical carcinoma cell line (HeLa)13. HeLa cells were plated into a 96-well plate in a complete medium at 37 oC and 5% CO2 for 24 h before the experiments. The culture medium was replaced and cells were incubated with complete medium containing the CuS@mSiO2-PEG nanoparticles at a series of concentrations at 37 oC with 5% CO2 for further 24 h. Relative cell viabilities were determined by the standard MTT assay. 2.9 Chemo-photothermal therapy Chemo-photothermal therapy experiments were referenced by our previous method22. HeLa cells were plated into a 96-well plate in a complete medium for 24 h before the experiments. The culture medium was replaced with fresh medium or complete medium containing the DOX, CuS@mSiO2-PEG and CuS@mSiO2PEG/DOX nanocomposites at a series of concentrations. Especially, at each equivalent DOX point, CuS@mSiO2-PEG has the same Cu concentration as the CuS@mSiO2-PEG/DOX. After 4 h of incubation, the cells were washed with PBS and replaced with fresh medium. The cells were treated with or without irradiation of 980 nm light (0.72 W/cm2) for 5 min, respectively, and then incubated for further 24 h. The standard MTT assay was carried out to quantify the cell viabilities.
3. Results and Discussions
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Fig. 1 Schematic illustration of the synthesis of CuS@mSiO2-PEG core-shell nanoparticles The typical procedure of fabricating CuS@mSiO2-PEG core-shell potential of CuS nanocrystals is -42.8 mV due to the citric acid nanoparticles was illustrated in Fig. 1. As-prepared CuS 45 ligands. After adding CTAB, the Zeta potential of the CuS/CTAB nanocrystals were capped by hydrophilic citric acid ligands in nanoparticles increases to 31.2 mV, indicating a change from aqueous solution, thus the nanoparticles were electronegative due negatively charge to positively charge. After removal of CTAB template, the Zeta potential decreases to -20.0 mV, attributing to to citric acid ligands. Mesoporous silica shell can’t coat directly on such electronegative nanoparticles because of the hydrolysate the OH groups on the surface. of TEOS with negative charge. So the surfactant-CTAB was used to change the chemistry of the surfactant head groups on the surface of the CuS nanocrystals. The bilayer of cationic CTAB coats around the CuS nanocrystals by electrostatic interaction and hydrophobic interaction, and changes the nanocrystals from negatively charge to positively charge26. The micelles formed by excess CTAB and bilayer of CTAB around the nanocrystals were used as a soft template, and the TEOS hydrolyzed and condensed under basic conditions and SiO2 grew around the template due to electrostatic interaction (between electropositive CTAB and anionic silicate species)27, forming CuS@SiO2 core-shell nanoparticles. In order to further improve the biocompatibility of the nanoparticles, the PEG was grafted onto the surface of SiO2 shell. The template CTAB was removed by treatment with NH4NO3 ethanol solution, and CuS@mSiO2-PEG core-shell nanoparticles with mesoporous structure were obtained. CuS nanocrystals capped with citric acid were first prepared according to the literature10. The transmission electron microscope (TEM) images of the obtained CuS nanocrystals showed an average diameter of ~12 nm and high crystallinity (Fig S1). The interplanar spacing is ~0.305 nm, corresponding to d spacing for the (102) crystal planes of standard hexagonal structured CuS crystal.
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Fig. 2 Low magnified TEM image (a), medium magnified TEM image (b), HRTEM image (c) and magnified HRTEM image (d) of CuS@mSiO2-PEG core-shell nanoparticles. In the silica-coating process, CTAB acted as an organic template for preparing CuS@SiO2 core-shell nanoparticles. The CuS@SiO2-PEG core-shell nanoparticles were obtained by grafting with PEG onto the SiO2 shell. After removal of CTAB template by ion exchange of NH4NO3, the CuS@mSiO2-PEG core-shell nanoparticles were obtained. The Zeta potentials of CuS nanocrystals, CuS/CTAB and CuS@mSiO2-PEG nanoparticles displayed the surface charge change of nanoparticles before and after silica coating (Fig S3). The Zeta 4 | Journal Name, [year], [vol], 00–00
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Fig. 3 (a) XRD patterns of the standard CuS (lower), as-prepared CuS (middle) and CuS@mSiO2-PEG nanoparticles (upper). (b) The FTIR spectra of as-prepared CuS@mSiO2 and CuS@mSiO2PEG nanoparticles (Inset: photo of as as-prepared dispersion of CuS@mSiO2-PEG nanoparticles). As shown in Fig. 2a-b, the CuS nanocrystals are completely encapsulated into mesoporous SiO2 shell with an estimated thickness of ~13 nm, and CuS@mSiO2-PEG nanoparticles have a diameter of ~37 nm. The diameter of the CuS@mSiO2-PEG coreshell nanoparticles in the water was also measured by dynamic light scattering (Zetasizer Nano Z). As shown in Fig. S4, there is a peak around 90 nm, and the average diameter is ~ 179.7 nm, suggesting that the CuS@mSiO2-PEG core-shell nanoparticles have slight aggregation in the water. As shown in the HRTEM images of CuS@mSiO2-PEG core-shell nanoparticles, the interplanar spacing of the core is ~0.189 nm, corresponding to the d spacing for the (110) crystal planes of standard hexagonal structured CuS crystal, which suggests that the phase structure of CuS nanocrystals well retained after silica coating. The phase structures of as-obtained CuS nanocrystals and CuS@mSiO2-PEG core-shell nanoparticles were examined by This journal is © The Royal Society of Chemistry [year]
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Fig. 4 (a) UV-Vis-NIR absorbance spectra of as-prepared CuS@mSiO2-PEG nanoparticles. (b) Plots of linear fitting absorbance at 980 nm versus the concentration of CuS@mSiO2PEG nanoparticles. PEG modification of the silica shells can improve colloidal stability, decrease immunogenicity and enhance biocompatibility12, 28, thus the CuS@mSiO2 nanoparticles were further grafted with PEG on the outside surface of silica shell. The Fourier transform infrared (FTIR) spectra of the CuS@mSiO2 and CuS@mSiO2-PEG core-shell nanoparticles are shown in Fig 3b. The CuS@mSiO2 and CuS@mSiO2-PEG coreshell nanoparticles both show typical stretching vibration peaks of Si-O-Si at 1074 cm-1 and 962 cm-1, and a vibration peak of H2O molecule adsorbed by mesoporous silica at 1634 cm-1 29, 30. Compared with the CuS@mSiO2 nanoparticles, the additional adsorption peak of the CuS@mSiO2-PEG at 2928 cm-1 is assigned to stretching vibration of methylene (CH2) in the long alkyl chain of the backbone of PEG, and at 1350 cm-1 is corresponded to deformation vibration of backbone of PEG31. These results indicate that the PEG was successfully grafted on the mesoporous silica shell. The 29Si NMR spectrum of CuS@mSiO2-PEG nanoparticles is shown in Fig. S5. The signal This journal is © The Royal Society of Chemistry [year]
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at -64.2 ppm can be assigned to Si of C-Si(OSi)3 (T3 sites)32-34, which further proves the PEG was successfully grafted on the mesoporous silica shell. The CuS@mSiO2-PEG core-shell nanoparticles show excellent colloidal stability due to the PEG modification. After aqueous dispersion of the CuS@mSiO2-PEG core-shell nanoparticles standing for a week, no aggregation was discovered (Fig 3b, inset).
Fig.5 (a) Temperature change of the aqueous solution containing the CuS@mSiO2-PEG nanoparticles with different concentrations under irradiation of the 980 nm laser (0.72 W/ cm2). (b) Plot of temperature elevation over a period of 360 s versus the concentration of the CuS@mSiO2-PEG nanoparticles. As shown in Fig. S2a, as-synthesized CuS@mSiO2-PEG coreshell nanoparticles after removal of CTAB have a BrunauerEmmett-Teller (BET) surface area as high as 802 m2/g and exhibit the type IV isotherm. The pore size distribution exhibits a sharp peak centered at a mean value of 3.6 nm (Fig. S2b), and the Barrett-Joyner-Halenda (BJH) pore volume of the CuS@mSiO2PEG core-shell nanoparticles is calculated to be 0.829 m3/g. Thus, a large surface area, great pore volume and appropriate pore size insure high drug loading capacity of the CuS@mSiO2-PEG coreshell nanoparticles. Ideal photothermal agents should have strong adsorption in NIR region, so the optical property of the aqueous dispersion of the CuS@mSiO2-PEG nanoparticles was examined by UV-VisNIR spectroscopy. As shown in Fig. 4a, the CuS@mSiO2-PEG aqueous dispersion exhibits a strong absorption in the NIR region due to the localized surface plasma resonances (SPR) of valenceband free carriers (positive holes) of CuS core10. Moreover, the Journal Name, [year], [vol], 00–00 | 5
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XRD pattern (Fig. 3a). The XRD patterns of both samples match well with the standard hexagonal phase of CuS (JCPDS card no: 79-2321). In addition, CuS@mSiO2-PEG nanoparticles have a wide peak at ~ 23°, which is clearly due to the amorphous silica coating. These results further prove that the phase structures of CuS nanocrystals were well retained after silica coating.
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absorptions at 980 nm were lineally enhanced with the increase of the concentration (Fig. 4b). Particularly, the absorbance peak locates at 975 nm, and is very close to the wavelength (980 nm) of excitation laser, which ensures that the CuS@mSiO2-PEG aqueous dispersion can convert the absorbed light into heat more effectively and nearly gain the highest temperature under irradiation of 980 nm laser35.
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Fig. 6 (a) Absorption spectra of the DOX and the CuS@mSiO2PEG/DOX dispersion (Inset: photo of as as-prepared CuS@mSiO2-PEG/DOX nanocomposites’ dispersion). (b) The cumulative release kinetics of the DOX from the CuS@mSiO2PEG/DOX nanocomposites in PBS (pH 7.4) and acetate buffer (pH 5.0) at room temperature with or without the NIR laser irradiation at a safe power density (0.72 W/cm2). To investigate the photothermal effect generated by the NIR laser irradiation, the temperatures of the CuS@mSiO2-PEG aqueous dispersion were measured under irradiation of 980 nm light with a safe power density (0.72 W/cm2). As shown in Fig. 5b, only a negligible temperature increase is observed from the control experiment of pure water (0 µg/mL) under the NIR irradiation. In contrast, the temperatures of the CuS@mSiO2-PEG aqueous dispersion were raised rapidly during the irradiation process under the same condition. For example, the temperature of the aqueous dispersion at the concentration of 200 µg/mL raised from 20.0 ℃ to 40.4 ℃ after 360 s of NIR irradiation. If the normal body temperature is 37 ℃, so the final temperature can reach 57.4 ℃ that is high enough to kill the cancer cells. Moreover, the temperature elevation increased as the concentration of the CuS@mSiO2-PEG nanoparticles increased 6 | Journal Name, [year], [vol], 00–00
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(Fig.5b). The photothermal conversion efficiency of CuS@mSiO2-PEG core-shell nanoparticles was also measured according to the references (see ESI for details) 13, 36, 37. As shown in Fig.S6, the photothermal conversion efficiency of CuS@mSiO2-PEG core-shell nanoparticles under irradiation of 980 laser (0.72W/cm2) can be calculated to be 29.5%, which is higher than those of previously reported Au rods (~ 21.0%, 800 nm laser), Cu2-xSe nanocrystals (~ 22%, 800 nm laser) and Cu9S5 nanocrystals (~ 25.7%,980 nm laser)37. These data illustrate that the CuS@mSiO2-PEG nanoparticles can effectively absorb and convert the NIR light into fatal heat due to their strong SPR absorption peak approximately at the wavelength (980 nm) of illumination laser, so the CuS@mSiO2-PEG core-shell nanoparticle is an excellent photothermal agent. These CuS@mSiO2-PEG core-shell nanoparticles are suitable for drug delivery due to the mesoporous silica shell. The anticancer drug DOX could be loaded into the CuS@mSiO2-PEG core-shell nanoparticles by simply mixing phosphate-buffer saline (PBS) solutions containing DOX with the nanoparticles. As shown in Fig. 6a, the CuS@mSiO2-PEG/DOX nanocomposites not only exhibit strong NIR absorbance from the CuS nanocrystals, but also show typical absorption peak near 480 nm region due to DOX, which indicates that the DOX has been successfully incorporated into the CuS@mSiO2-PEG nanopaticles. The color the CuS@mSiO2-PEG/DOX dispersion turned red brown (from dark yellow-green) due to loading DOX (Fig. 6a, inset). Because of large surface area and pore volume, appropriate pore size, the encapsulation efficiency and loading content of the DOX into the CuS@mSiO2-PEG core-shell nanoparticles are determined to be as high as 90.1％ and 26.5％ (by weight), respectively. Release of the DOX from the CuS@mSiO2-PEG core-shell nanoparticles against buffer solution at pH 7.4 and 5.0 was studied to simulate normal physiological environment and cellular lysosome environment, respectively (Fig. 6b). The cumulative release amount of the DOX at pH 7.4 was quite small within 9 h due to strong electrostatic interactions between the DOX and dissociated silanols23, indicating that the CuS@mSiO2PEG/DOX nanocomposites released very little DOX molecules in normal physiological environment. However, the drug release rate became much faster and the cumulative release of drug reached up to 37.9 % at pH 5.0 due to the enhancement of the solubility of the DOX for protonated daunosamine group under acidic conditions38. This pH-dependent release behavior with more the DOX release at lower pH value for the CuS@mSiO2PEG/DOX nanocomposites is beneficial for the cancer therapy since the microenvironments of extracellular tissues of tumors and intracellular lysosomes and endosomes are acidic28, 39. Whether the photothermal effect originated from CuS core under the irradiation (0.72 W/cm2, 980 nm) for 5 min can trigger the DOX release from the CuS@mSiO2-PEG/DOX nanocomposites at pH 5.0 and 7.4 was studied. The DOX released rapid upon the NIR irradiation, while it released slowly when the laser was shut off. At pH 5.0, the cumulative release of the DOX under irradiation increased from 37.9% to 67.2% within 9 h. At pH 7.4, the cumulative release of the DOX was also enhanced by laser irradiation. These results demonstrate that the release of the DOX from the CuS@mSiO2-PEG/DOX This journal is © The Royal Society of Chemistry [year]
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nanocomposites could be triggered by the NIR irradiation, which is due to the heat generated by the photothermal effect of the CuS@mSiO2-PEG dissociated the strong interactions between DOX and SiO228, and thus more DOX molecules released. Since the light can be manipulated precisely, the release of the DOX from the CuS@mSiO2-PEG nanocomposites can be conveniently and accurately controlled by the NIR irradiation. Thus, CuS@mSiO2-PEG core-shell nanoparticles have successfully achieved the drug release with control of the area, time, and dosage by pH and NIR light.
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Fig. 7 (a) Viabilities of HeLa cells incubated for 24 h with different concentrations of the CuS@mSiO2-PEG nanoparticles. (b) Viabilities of HeLa cells after incubation with different concentrations of free DOX, CuS@mSiO2-PEG and CuS@mSiO2-PEG/DOX with or without 5 min of NIR irradiation (0.72 W/cm2, 980 nm), and HeLa cells (incubated with only medium) with 5 min of NIR irradiation. At the each equivalent DOX point, CuS@mSiO2-PEG has a same Cu concentration as the CuS@mSiO2-PEG/DOX. To further investigate the intracellular drug delivery by CuS@mSiO2-PEG/DOX nanoparticles, DOX fluorescence in HeLa cell was examined using CLSM. As shown in Fig.S7, red fluorescence (DOX) of the cells was relatively weak after 4 h of incubation, but the intensity of red fluorescence was significantly enhanced after 8 h of incubation. These results indicated that increasing amounts of DOX delivered by the CuS@mSiO2PEG/DOX gradually passed through the cytomembrane, entered
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into cytoplasm, passed through the nucleus membrane and eventually assembled in nucleus with incubation time increasing. The undamaged nucleuses shown in Fig.S7d-f are much fewer than that of Fig.7Sa-c, indicating as the incubation time was prolonged the nucleus gradually collapsed and cells died due to DOX in nucleuses. Thus, the effective uptake of DOX indicates the effective delivery of DOX by the CuS@mSiO2- PEG/DOX nanoparticles to kill cancer cells. Although mesoporous silica has been widely reported to be suitable for drug delivery, we still investigated the cytotoxicity of CuS@mSiO2-PEG core-shell nanoparticles before biological applications to confirm such an argument. Thus, the standard MTT assay was carried out to determine the relative viabilities of HeLa cells after they were incubated with the CuS@mSiO2-PEG nanocomposites at various concentrations for 24 h. The results showed that the CuS@mSiO2-PEG core-shell nanoparticles exhibited no appreciable negative effect on the viability of cells, even at a high concentration up to 500 µg/mL (Fig. 7a), indicating the good biocompatibility of the CuS@mSiO2-PEG core-shell nanoparticles. The therapy effects of the photothermal therapy, chemotherapy, and their combination were studied in vitro. Free DOX and CuS@mSiO2-PEG/DOX nanocomposites have obvious cytotoxicity to Hela cells, which show an increasing inhibition ratio against HeLa cells with the increasing concentration (Fig. 7b). Moreover, the CuS@mSiO2-PEG/DOX nanoparticles demonstrated slightly higher cytotoxicity than free DOX at the all tested concentrations since DOX-loaded nanoparticles can enter cancer cells more easily than free DOX6, 40. Whether the NIR irradiation can improve cancer cell-killing efficacy was studied. There was hardly any negative effect to the cell viability when the cells were irradiated with NIR laser alone. However, NIR irradiation showed significant toxicity in the presence of the CuS@mSiO2-PEG nanoparticles due to their photothermal effect. The CuS@mSiO2-PEG/DOX nanocomposites under the NIR irradiation demonstrated a higher cell toxicity at all tested concentrations than the chemotherapy (without NIR irradiation) or the photothermal therapy (without DOX) alone (Fig.7b). For example, when the DOX concentration is 15 µg/mL, 63.0 % of the cells were inhibited by the CuS@mSiO2-PEG/DOX nanocomposites under the NIR irradiation, while 51.3% of the cells were killed by the CuS@mSiO2-PEG/DOX nanocomposites at the same concentration without the NIR illumination and only 30.9% of the cells were inhibited by the CuS@mSiO2-PEG nanocomposites (Cu concentration is identical as the CuS@mSiO2-PEG/DOX nanocomposites) in absence of DOX. These results demonstrated that the combination of chemotherapy and photothermal therapy by the CuS@mSiO2-PEG/DOX nanocomposites showed better therapy effect for cancer cells than individual therapy due to enhanced toxicity of DOX and hyperthermia8, 9. Therefore, as-prepared CuS@mSiO2-PEG nanoparticles can act as a difunctional drug delivery nanoplatform for combined chemo-photothermal therapy when driven by NIR irradiation.
In conclusion, we have developed a NIR light responsive drug delivery nanoplatform (CuS@mSiO2-PEG) for efficient chemoJournal Name, [year], [vol], 00–00 | 7
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photothermal therapy, which was fabricated by a facile, cheap and green way. CuS@mSiO2-PEG core-shell nanoparticles have good biocompatibility and colloidal stability, as well as a very high DOX loading content (26.5% wt). The CuS nanocrystals (core) can effectively absorb and convert NIR light to fatal heat under NIR light irradiation, and the release of DOX from the mesoporous silica (shell) can be triggered by pH and the NIR light. When the CuS@mSiO2-PEG/DOX nanocomposites were illuminated by NIR light, both chemotherapy and photothermal therapy were simultaneously driven, resulting in a synergistic effect for killing cancer cells. More importantly, the combination of therapies demonstrated significant enhancement of therapy effects for cancer cells than chemotherapy or photothermal therapy alone. Therefore, the NIR light responsive drug delivery nanoplatform has great potential in cancer chemo-photothermal therapy.
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Notes and references a College of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, Shanghai, 201620, China b State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University, Shanghai 201620, China; E-mail: [email protected] c College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China d Center of Super-Diamond and Advanced Films (COSDAF), Department of Physics and Materials Science, City University of Hong Kong, Hong Kong e Department of Orthopaedics, Shanghai First People’s Hospital, Shanghai Jiaotong University, 100 Haining Road, Hongkou District, Shanghai 200080, China
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Acknowledgements This work was financially supported by the National Natural Science Foundation of China (Grant no. 21171035, 51472049 and 51302035), the Key Grant Project of Chinese Ministry of Education (Grant no. 313015), the PhD Programs Foundation of the Ministry of Education of China (Grant no. 20110075110008 and 20130075120001), the National 863 Program of China (Grant no. 2013AA031903), the Science and Technology Commission of Shanghai Municipality (Grant no. 13ZR1451200), the Fundamental Research Funds for the Central Universities, the Program Innovative Research Team in University (IRT1221), the Shanghai Leading Academic Discipline Project (Grant no. B603) and the Program of Introducing Talents of Discipline to Universities (no. 111-2-04).
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C. Xu, D. R. Yang, L. Mei, Q. H. Li, H. Z. Zhu and T. H. Wang, Acs Appl. Mater. Interfaces, 2013, 5, 12911-12920. F. Tang, L. Li and D. Chen, Adv. Mater., 2012, 24, 1504-1534. T. L. Doane and C. Burda, Chem. Soc. Rev., 2012, 41, 2885-2911. W. Yin, L. Yan, J. Yu, G. Tian, L. Zhou, X. Zheng, X. Zhang, Y. Yong, J. Li, Z. Gu and Y. Zhao, Acs Nano, 2014, 8, 6922-6933. J. H. Park, G. von Maltzahn, L. L. Ong, A. Centrone, T. A. Hatton, E. Ruoslahti, S. N. Bhatia and M. J. Sailor, Adv. Mater., 2010, 22, 880-885. W. Fang, J. Yang, J. Gong and N. Zheng, Adv. Funct. Mater., 2012, 22, 842-848. W.D. Jang, D. Yim and I.H. Hwang, J. Mater. Chem. B, 2014, 2, 2202-2211.
8 | Journal Name, [year], [vol], 00–00
37. 38. 125
39. 40.
H. Park, J. Yang, J. Lee, S. Haam, I. H. Choi and K. H. Yoo, Acs Nano, 2009, 3, 2919-2926. S. M. Lee, H. Park and K. H. Yoo, Adv. Mater., 2010, 22, 40494053. M. Zhou, R. Zhang, M. Huang, W. Lu, S. Song, M. P. Melancon, M. Tian, D. Liang and C. Li, J. Am. Chem. Soc., 2010, 132, 1535115358. Q. Tian, M. Tang, Y. Sun, R. Zou, Z. Chen, M. Zhu, S. Yang, J. Wang, J. Wang and J. Hu, Adv. Mater., 2011, 23, 3542-3547. Q. Xiao, X. Zheng, W. Bu, W. Ge, S. Zhang, F. Chen, H. Xing, Q. Ren, W. Fan, K. Zhao, Y. Hua and J. Shi, J. Am. Chem. Soc., 2013, 135, 13041-13048. X. Liu, B. Li, F. Fu, K. Xu, R. Zou, Q. Wang, B. Zhang, Z. Chen and J. Hu, Dalton Trans., 2014, 43, 11709-11715. Y. Li, W. Lu, Q. Huang, M. Huang, C. Li and W. Chen, Nanomedicine, 2010, 5, 1161-1171. J. Mou , P. Li , C. Liu , H. Xu , L. Song , J. Wang , K. Zhang ,Y. Chen , J. Shi and H. Chen. Small, 2015, DOI: 10.1002/smll.201403249. L. Guo, I. Panderi, D. D. Yan, K. Szulak, Y. Li, Y. T. Chen, H. Ma, D. B. Niesen, N. Seeram, A. Ahmed, B. Yan, D. Pantazatos and W. Lu, Acs Nano, 2013, 7, 8780-8793. L. Cheng, C. Wang, L. Feng, K. Yang and Z. Liu, Chem. Rev., 2014, 114, 10869-10939. K. Dong, Z. Liu, Z. Li, J. Ren and X. Qu, Adv. Mater., 2013, 25, 4452-4458. V. Biju, Chem. Soc. Rev., 2014, 43, 744-764. P. Yang, S. Gai and J. Lin, Chem. Soc. Rev., 2012, 41, 3679-3698. G. Song, Q. Wang, Y. Wang, G. Lv, C. Li, R. Zou, Z. Chen, Z. Qin, K. Huo, R. Hu and J. Hu, Adv. Funct. Mater., 2013, 23, 4281-4292. X. Liu, Q. Wang, C. Li, R. Zou, B. Li, G. Song, K. Xu, Y. Zheng and J. Hu, Nanoscale, 2014, 6, 4361-4370. Z. Zhang, L. Wang, J. Wang, X. Jiang, X. Li, Z. Hu, Y. Ji, X. Wu and C. Chen, Adv. Mater., 2012, 24, 1418-1423. American National Standard Institute, ANSI Z136. 1-2007, American National Standard for Safe Use of Lasers, Orlando, Fl 2007. X. Liu, F. Fu, K. Xu, R. Zou, J. Yang, Q. Wang, Q. Liu, Z. Xiao and J. Hu, J. Mater. Chem. B, 2014, 2, 5358-5367. B. Chang, X. Zhang, J. Guo, Y. Sun, H. Tang, Q. Ren and W. Yang, J.Colloid Interface Sci., 2012, 377, 64-75. Y. Wan and D. Zhao, Chem. Rev., 2007, 107, 2821-2860. S. Shen, H. Tang, X. Zhang, J. Ren, Z. Pang, D. Wang, H. Gao, Y. Qian, X. Jiang and W. Yang, Biomaterials, 2013, 34, 3150-3158. G. Yang, S. Gai, F. Qu and P. Yang, Acs Appl. Mater. Interfaces, 2013, 5, 5788-5796. X. Kang, Z. Cheng, D. Yang, P. Ma, M. Shang, C. Peng, Y. Dai and J. Lin, Adv. Funct. Mater., 2012, 22, 1470-1481. V. Cauda, C. Argyo and T. Bein, J. Mater. Chem., 2010, 20, 86938699. A. L. de Lima, A. Mbengue, R. A. S. San Gil, C. M. Ronconi and C. J. A. Mota, Catal. Today, 2014, 226, 210-216. R. Dobrowolski, M. Oszust-Cieniuch, J. Dobrzynska and M. Barczak, Colloids Surf., A, 2013, 435, 63-70. M. Popova, A. Szegedi, K. Yoncheva, S. Konstantinov, G. P. Petrova, H. A. Aleksandrov, G. N. Vayssilov and P. Shestakova, Microporous Mesoporous Mater., 2014, 198, 247-255. H. Chen, L. Shao, T. Ming, Z. Sun, C. Zhao, B. Yang and J. Wang, Small, 2010, 6, 2272-2280. C. M. Hessel, V. P. Pattani, M. Rasch, M. G. Panthani, B. Koo, J. W. Tunnell and B. A. Korgel, Nano Lett., 2011, 11, 2560-2566. Q. Tian, F. Jiang, R. Zou, Q. Liu, Z. Chen, M. Zhu, S. Yang, J. Wang, J. Wang and J. Hu, Acs Nano, 2011, 5, 9761-9771. X. C. Qin, Z. Y. Guo, Z. M. Liu, W. Zhang, M. M. Wan and B. W. Yang, J. Photochem. Photobiol., B, 2013, 120, 156-162. C. Wang, H. Xu, C. Liang, Y. Liu, Z. Li, G. Yang, H. Cheng, Y. Li and Z. Liu, Acs Nano, 2013, 7, 6782-6795. W. Fang, S. Tang, P. Liu, X. Fang, J. Gong and N. Zheng, Small, 2012, 8, 3816-3822.
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A difunctional nanoplatform (CuS@mSiO2-PEG) acted as NIR light induced photothermal-triggered drug delivery system for efficient chemo-photothermal therapy.
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