Acta Biomaterialia 16 (2015) 196–205

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Specific aptamer-conjugated mesoporous silica–carbon nanoparticles for HER2-targeted chemo-photothermal combined therapy Kaiyuan Wang b, Hui Yao a, Ying Meng b, Yi Wang c,⇑, Xueying Yan b,⇑, Rongqin Huang a,⇑ a

Department of Pharmaceutics, School of Pharmacy, Key Laboratory of Smart Drug Delivery, Ministry of Education, Fudan University, Shanghai 201203, China School of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, China c Center of Analysis and Measurement, Fudan University, Shanghai 200433, China b

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Article history: Received 28 August 2014 Received in revised form 2 December 2014 Accepted 1 January 2015 Available online 14 January 2015 Keywords: HB5 aptamer Mesoporous silica–carbon nanoparticles Chemo-photothermal therapy HER2-positive Breast cancer

a b s t r a c t Tumor-specific therapeutic platforms designed for combined tumor therapy has recently received wide attention. In this work, a new HB5 aptamer-functionalized mesoporous silica–carbon based doxorubicin (DOX)-loaded system (MSCN-PEG-HB5/DOX) was successfully constructed and characterized for chemophotothermal combined therapy of human epithelial growth factor receptor 2 (HER2)-positive breast cancer cells. The in vitro release result showed that MSCN-PEG-HB5/DOX exhibited pH-sensitive and NIR-triggered release manner. HB5-modified nanoparticles showed significant higher cellular uptake in HER2-positive breast cancer cells (SK-BR-3) but not in normal breast epithelial cells (MCF-10A), compared to unmodified counterparts. The intracellular uptake of functional nanoparticles was mainly based on the receptor-mediated mechanism which was energy-dependent. Cytotoxicity experiments demonstrated that combined therapy induced highest cell killing effect compared to chemotherapy and photothermal therapy alone. The combination index (CI) was 0.253 indicating the synergistic effect of chemotherapy and photothermal therapy. These findings suggested that MSCN-PEG-HB5/DOX was a potential chemo-photothermal therapeutic platform targeting to HER2-positive breast cancers. Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction The delivery of cytotoxic agents to cancer cells via passive or active targeting has proved to be an effective method for minimal influence to normal cells and improving the therapy. Recently, various ligands that can specifically bind to receptors overexpressed on cancer cells, such as peptide [1,2], antibodies [3,4] and aptamers [5,6], have been incorporated with nano-materials for the design and construction of novel targeting drug delivery systems. Among these ligands, nucleic acid aptamers, as single-stranded DNA or RNA oligonucleotides that can fold into a three-dimensional structure, are now of great interest owing to their stability, non-immunogenicity and ease of manufacture [7]. It can be recognized by their targeted molecules with high affinity. For example, AS1411 is an intensively studied DNA aptamer ligand which shows high binding affinity to nucleolin, a kind of protein highly expressed in the plasma membrane of tumor cells [8,9]. Furthermore, AS1411 has been applied in phase II clinical trials for relapsed or ⇑ Corresponding authors. Tel./fax: +86 21 65643010 (Y. Wang). Tel./fax: +86 451 87266907 (X. Yan). Tel./fax: +86 21 51980078 (R. Huang). E-mail addresses: [email protected] (Y. Wang), [email protected] (X. Yan), [email protected] (R. Huang). http://dx.doi.org/10.1016/j.actbio.2015.01.002 1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

refractory acute myeloid leukemia and for renal cell carcinoma [10], suggesting the great potential of aptamer ligands for clinical applications. Therefore, further developing novel aptamer targeted delivery system are still of high demand for cancer therapy, especially for some specific proteins overexpressed cancer. Human epithelial growth factor receptor 2 (HER2)-positive breast cancer is a most commonly diagnosed one that brings significant health problem among women worldwide. The overexpression of HER2 protein can be found in approximately 20–30% of breast cancers and causes poor survival due to high proliferation, invasiveness and metastasis rates [11]. In most cases, HER2-positive breast cancer can rarely present significant therapeutic efficiency only through conventional methods, such as surgery and radiotherapy, which could generally lead to resistance and give rise to relapse. Although chemotherapy has been reported to play an important role in disease treatment, chemotherapeutic drugs themselves are non-targeted, which significantly limits the drug accumulation within cancer cells and induces undesired toxic side effects toward normal tissues [12,13]. Chemo-photothermal therapy could be more effective to destroy cancer cells than either therapy alone [14]. It has been reported that chemotherapy often causes systemic side effects due to unspecific drug delivery and produce drug resistance which

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leads to treatment failure [15]. In contrast, photothermal therapy can promote the release of chemotherapeutic drugs and increase their accumulation inside tumor cells, enhancing chemotherapeutic efficacy [16]. The combination of chemotherapy and photothermal therapy could realize optimized cancer treatment efficacy in controllable manner. Moreover, the most important issue for effective combined therapy is to design suitable drug carriers, which could be easily synthesized and functionalized. The mesoporous silica–carbon nanoparticles (MSCN) with semi-graphitized carbon (sGC) on the inner wall which can effectively convert NIR light into heat has been proved to be an effective chemo-photothermal therapeutic platforms due to many favorable features including large surface area and tunable pore size for drug loading, high dispersed sGC hotspots for photothermal heating and pH responsive release [17]. However, the application of pristine MSCN in cancer therapy is far enough due to the non-specificity and the inorganic surfaces. Therefore, in this work, a novel DNA aptamer (HB5) which is based on 86 nucleotides [18] and capable of binding to HER2 receptor was successfully conjugated on MSCN surfaces for chemo-photothermal combined tumor-targeted therapy of HER2positive breast cancer cells. DOX was selected as a model antitumor drug. Series studies including targeting capability, mechanism of cellular uptake and combined therapeutic ability were performed to evaluate the new therapeutic system. 2. Materials and methods The MSCN vector was synthesized in our lab as reported previously [17]. Doxorubicin (DOX) was obtained from Huafeng United Technology Co., Ltd. (Beijing, China). The thiol group modified DNA aptamer (HB5) (Ap, sequence: 50 -AACCGCCCAAATCCCTAAGAGTCTGCACTTGTCATTTTGTATATGTATTTGGTTTTTGGCTCTCACAGACACACTACACACGCACA-30 , 86 bp) was synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). a-Maleimidyl-x-N-hydroxysuccinimidyl polyethyleneglycol (NHS-PEG-MAL, MW 3500) which acted as a linker was purchased from JenKem Technology Co., Ltd. (Beijing, China). (3-aminopropyl)trimethoxysilane (APTMS) and isothiocyanate (FITC) were obtained from Sigma–Aldrich (Shanghai, China). Matrigel was bought from Becton Dickinson Medical Devices Co., Ltd. (Shanghai, China). Live-Dead Cell Staining Kit was purchased from Molecular Probes (Eugene, OR, U.S.A). Cell Counting Kit-8 (CCK-8) was bought from Dojindo Laboratories (Japan). Triethylamine (TEA) was obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDC) and Nhydroxysuccinimide (NHS) were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China). SK-BR-3 cells (HER2-positive human breast cancer cell line) and MCF-10A cells (non-cancerous breast epithelial cell line) were obtained from the America Type Culture Collection (ATCC), and incubated in RPMI-1640 and DMEM medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin, 1% streptomycin and 1% L-glutamine, respectively. All the reagents used in cell culture were purchased from Gibco (Tulsa, OK, U.S.A.). 2.1. Synthesis of different nanoparticles MSCN was synthesized according to template semi-graphitized methods in our previous study [17]. Briefly, surfactant cetyltrimethylammonium bromide (CTAB) as template co-condensed with tetraethyl orthosilicate (TEOS) under the condition of alkali catalysis, then was in situ graphitized under nitrogen temperature programming. The amine modification of the prepared MSCN by 3aminopropyl groups was conducted according to the method described [19]. MSCN needs to be dehydrated at 65 °C overnight

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to remove moisture in advance, then it was dispersed in dried toluene, and APTMS was used as the amine source. The mixed solution was stirred and refluxed in an oil bath at 100 °C. After reacting for 12 h, the NH2-functionalized MSCN (MSCN-NH2) was obtained after the toluene evaporated. The precipitate was washed several times with isopropanol and water, and then re-dispersed in water and freeze-dried overnight. The preparation of HER2 targeted aptamer (HB5)-functionalized MSCN (MSCN-PEG-HB5) was carried out by bringing in NHSPEG3500-MAL. PEG served as a linker between amine-functionalized MSCN and thiol group-modified HB5, to enhance the biocompatibility. PEG coated MSCN (MSCN-PEG) was prepared by mixing MSCN-NH2 with bifunctional PEG chain in phosphate buffered solution (PBS, pH 8.0) at the molar ratio of 1:1, and reacting for 2 h. The obtained MSCN-PEG was collected by centrifuging at 15,000 rpm for 10 min. Then the precipitate was dispersed in PBS (pH 7.0) and stirred with HB5-SH for 24 h. Thus, the resulting MSCN-PEG-HB5 was obtained after centrifuging at 15,000 rpm for 10 min. The synthesis process above was performed at room temperature. All the nanoparticles were freshly prepared for the following experiments. Quantitative detection of amino groups on MSCN was conducted using fluorescamine. MSCN-NH2 nanoparticles dispersed in sodium hydroxide (NaOH) solution (0.02 M) stirring for 60 h. After centrifugation, the supernatant including dissociated APTMS was collected for amine detection. PBS buffer (0.2 M, pH 8.0) and analyte solution were mixed. Then 140 ll fluorescamine solution (0.3 g/L in acetone) was added to the mixture stirring for 20 min for detection. The standard curve was obtained by reacting fluorescamine with different concentrations of APTMS solution (dissolved in NaOH solution) with the excitation at 394 nm and emission at 481 nm. For the synthesis of FITC-labeled nanoparticles, carboxyl unit on FITC was activated with NHS and EDC in PBS in the dark for 0.5 h. Then, MSCN-NH2 dispersed in PBS was added to the mixture and reacted for another 1.5 h. The amount of FITC that added in the reaction system was only 1% (mol/mol) of amines on MSCN. After centrifugation and washing with water, FITC-labeled MSCNNH2 was obtained for further conjugation with PEG and HB5 as mentioned above. 2.2. Characterization of synthesized nanoparticles The synthesized MSCN-based nanoparticles were characterized by Fourier transform infrared spectroscopy (FT-IR) with the type of Nicolet-670 FT-IR spectrometer. Transmission Electron Microscope (TEM) was used to investigate the morphology and size of MSCN-PEG-HB5, which was performed on JEOL a JEM-2010 instrument with an acceleration voltage of 200 kV. Raman spectra (LR) of MSCN-PEG-HB5 nanoparticles were taken on a Labram-1B (Dilor, France) Raman spectrometer with a 632.8-nm wavelength incident laser light. 2.3. Photothermal heating effect of nanoparticles To compare the photothermal heating effect of different nanoparticles, 200 ll of these nanoparticles with a certain MSCN concentration placed in a 96-well plate were irradiated by an 808 nm near infrared (NIR) laser of 6 W/cm2 for 5 min. To determine impacts of irradiation intensity, 200 ll MSCN-PEG-HB5 were irradiated by an 808 nm NIR laser with various intensity at 3, 4.5, 6 and 7.5 W/cm2 for 5 min. The solution temperature was monitored by an accurate digital thermometer. Initial temperature was recorded in order to calculate temperature increment of these nanoparticles.

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2.4. Drug loading content

2.9. Targeting capability and competitive inhibition

The preparation of DOX-loaded MSCN-PEG (MSCN-PEG/DOX) and DOX-loaded MSCN-PEG-HB5 (MSCN-PEG-HB5/DOX) was carried out in a mixed solution (DOXTEA solution: 1 mg/ml doxorubicin hydrochloride aqueous solution mixed with TEA at a molar ratio of 1:1, where TEA was added to neutralize HCl to expose hydrophobic DOX molecule partly). MSCN-based nanoparticles containing 300 lg MSCN were dispersed in 400 ll DOXTEA solution (400 lg) and stirred at room temperature for 24 h. The product was collected by centrifuging at 15,000 rpm for 10 min and was washed twice with water to remove the unreacted DOX. The supernatant DOX solution was collected to determine drug loading content by a UV–vis spectrophotometer at 500 nm. The DOX loading content was calculated based on the equation: Loading content = (weight of drug in vectors)/(weight of vectors).

The targeting capability and competitive inhibition studies were conducted with FITC-labeled MSCN-PEG and MSCN-PEGHB5. To evaluate cell selectivity of nanoparticles, HER2-positive breast cancer cells SK-BR-3 and non-cancerous breast epithelial cells MCF-10A were employed, which were seeded in 96-well plates at a density of 1.5  104/well and 1.3  104/well, respectively, for 48 h. After being washed, cells were treated with 100 ll FITC-MSCN-PEG (100 lg/ml) and FITC-MSCN-PEG-HB5 (100 lg/ml) in the incubator under dark condition for 1 h. The cells were rinsed with PBS, fixed with 4% formaldehyde for 15 min, and then analyzed using a DMI4000B inverted fluorescent microscope (Leica, Germany). For competition inhibitive experiment, SP13 peptide which has been demonstrated as a type of HER2 targeting ligand [20], was exploited as the inhibitive agent. Similar procedures were conducted except that cells were pretreated with excessive SP13 peptide with a concentration of 1 mg/ml (50 ll) for 20 min. Then 50 ll FITC-labeled nanoparticles (200 lg/ml) were added to gain the final volume of 100 ll and the final concentration of 100 lg/ ml, which was equal with that in the targeting capability experiment, for further incubation of 1 h. Confocal microscopy was performed to verify the intracellular localization of nanoparticles. SK-BR-3 cells were seeded in glass bottom cell culture dishes at the density of 1.8  104/well for 48 h. The cells were rinsed with PBS before treatments. For the energy-dependent cellular uptake, the cells were treated with FITC-MSCN-PEG-HB5 nanoparticles at 4 °C and 37 °C for 20 min, respectively. The competition inhibitive study was conducted as mentioned above. X/y sections were obtained along the z plane from the bottom of the cell culture dishes.

2.5. Fluorescence quenching experiment Fluorescence spectrum was used to investigate the location of DOX in MSCN-PEG-HB5. Briefly, 200 lg MSCN-PEG-HB5 nanoparticles were dispersed in DOX aqueous solution (1 ml, 200 lg/ml) and stirred for 12 h at room temperature. The mixture and the pure DOX solution were measured by fluorescent spectrophotometer with equal dilution. Then, the mixture was centrifuged at 15,000 rpm for 10 min, and was washed twice with water. Both the collected supernatant and re-dispersed precipitate (MSCNPEG-HB5/DOX) were measured by fluorescent spectrophotometer at the equal concentration with the two solutions mentioned above. 2.6. In vitro release To determine pH- and heat-sensitive release of MSCN-PEGHB5/DOX, drug release experiments were conducted in NIR-irradiated group and normal group, in PBS with pH 7.4, pH 6.0 and pH 5.0 in sequence. For the NIR-irradiated group, 100 lg of MSCNPEG-HB5/DOX nanoparticles were dispersed in 500 ll PBS 7.4 stirred at 300 rpm. At one hour interval, the solution was irradiated with an 808 nm NIR laser (6 W/cm2) for 5 min, and then was centrifuged at 15,000 rpm for 10 min to obtain the supernatant. At the first three hours, 200 ll supernatant was taken out for measurement and an equal volume of PBS 7.4 was added back. At the fourth hour, 450 ll the supernatant was removed and the solution was replaced with 450 ll PBS 6.0. As described above, the PBS was replaced every four hours. For the normal group, similar procedures were carried out but without NIR laser irradiation. The amounts of the released DOX were measured by a fluorescence spectrophotometer (excitation at 488 nm, emission at 593 nm). 2.7. Zeta potential measurements Zeta potentials of different MSCN-based nanoparticles were measured by dynamic light scattering (DLS) using a Malvern NanoZS Zeta Potential/Particle Sizer.

2.10. In vivo imaging analysis Female Balb/c nude mice with the age of 4–5 weeks and weight of 20–22 g were maintained under specific pathogen free conditions at the Department of Experimental Animals, Fudan University (Shanghai, China). To obtain SK-BR-3 tumor models, about 4  106 cells in 200 ll mixed solution (serum-free RMPI-1640 medium and matrigel at the volume ratio of 1:1) were injected under the right shoulder. The SK-BR-3 tumor-bearing mice were treated when the tumor volume reached 100 mm3. After anesthetized, the mice were intravenously injected with 200 ll of 5 mg/ml MSCN-PEG/DOX and MSCN-PEG-HB5/DOX solution, respectively. The fluorescence of DOX released from nanoparticles was measured. Fluorescent image of each mouse was obtained using an IVIS Spectrum in Vivo imaging system at selected time points. After 6 h, mice were sacrificed and major organs and tumors were carefully excised for further fluorescent imaging analysis. The tumor volume was calculated as following formula: width2  length/2. All animal experiments were carried out in accordance with guidelines evaluated and approved by the ethics committee of Fudan University. 2.11. Cell death imaging

2.8. Concentration-dependent cellular uptake SK-BR-3 cells were seeded in 96-well plates for 1.2  104/well and further cultured for 48 h. The medium was removed and the cells were washed once with PBS. After that, MSCN-PEG-HB5/ DOX at different concentrations (20, 50, 100 and 150 lg/ml) were added and incubated for 1 h. Then cells were rinsed with PBS, and fixed with 4% formaldehyde for 15 min. Fluorescent images were obtained by a DMI4000B inverted fluorescent microscope (Leica, Germany).

To discuss the chemo-photothermal therapeutic effect, LiveDead Kits were applied to distinguish cell death conditions. SKBR-3 cells were seeded in a 96-well plate at the density of 1  104 cells/well for 48 h. After washing with PBS, MSCN-PEG-HB5 and MSCN-PEG-HB5/DOX with the concentration of 50 and 100 lg/ml were added and incubated for 1 h at 37 °C. For photothermal therapy, corresponding wells were irradiated by an 808 nm NIR laser (6 W/cm2) for 5 min. After washed, the cells were incubated with fresh media for another 12 h. Finally, cells were stained using

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Live-Dead Kits according to the manufacturer’s instructions and then observed by a laser scanning confocal microscopy. 2.12. Cytotoxicity study and combination index calculation In vitro viability against breast cancer cells of different treatments were determined by CCK assay. SK-BR-3 cells at the density of 1.5  104 cells/well were seeded in 96-well plates and incubated for 48 h. The medium was removed, and cells were treated by MSCN-PEG-HB5 and MSCN-PEG-HB5/DOX with concentration range from 1 to 150 lg/ml for 4 h in the dark. For thermal therapy, corresponding wells were irradiated by an 808 nm NIR laser with the density of 6 W/cm2 for 5 min. Then fresh media were added to the cell wells and continued to incubate for another 12 h. After that, 10 ll CCK-8 solution was added to each well and cultivated for 3 h at 37 °C. The absorbance of each well was measured by a microplate reader at 450 nm. The therapeutic effect of combining the chemotherapy and photothermal therapy can be evaluated by the combination index (CI) according to previous study [21]. The value of CI 1 means antagonism, and =1 shows additive effects. 2.13. Statistical analysis Data were presented as mean ± S.E.M. The statistical analysis was performed via the one-way ANOVA using GraphPad Prism 6 software. 3. Results and discussion 3.1. Synthesis and characterization of nanoparticles MSCN was obtained by the calcination under N2 atmosphere at the high temperature. In order to make it easily conjugate with functional molecules for desired biological applications, a posttreatment was first used to create amino-groups on MSCN (see Section 2), and the content of ANH2 groups grafted on MSCN was 1.64 lmol/mg. Then, bifunctional NHS-PEG-MAL was conjugated to the nanoparticles via the specific reaction of ANHS and ANH2 in aminated MSCN, resulting MSCN-PEG. Eventually, MSCN-PEG-

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HB5 was synthesized due to the interaction between -MAL in PEG and -SH in HB5. As shown in FT-IR spectra (Fig. 1a), the absorption peaks of MSCN were observed at 1079 cm1 (SiAO stretching vibration), 451 cm1 (SiAO bending vibration), 958 cm1 (SiAOH bending vibration) and 805 cm1 (SiAOASi bending vibration) [22]. The existence of primary amine on MSCN was proved by CAN stretching vibration at 1385 cm1, suggesting the successful synthesis of MSCN-NH2. After PEG conjugation, the typical absorption peaks of CAH stretching vibration in PEG emerged at 2954 cm1 and 2885 cm1. Moreover, signals of primary amino groups disappeared and peaks of tertiary amino groups at about 680 cm1 appeared, further confirming the successful functionalization of PEG [23]. Upon the conjugation of HB5, a distinct peak emerged at 1259 cm1 which was mainly assigned to the CAH bending vibration in deoxyribose of DNA oligonucleotides. Thus, the successful decoration of cell-targeting aptamer HB5 on the nanoparticles was verified. TEM was employed to observe the morphology and size of MSCN-PEG-HB5, and also, an auxiliary method to prove the conjugation of PEG and HB5 (Fig. 1b). After HB5 conjugation, the nanoparticle size was increased to about 140 nm, which is larger than that of native MSCN (115 nm). Moreover, HB5-decorated nanoparticles still retained spherical shape while the surface was covered by a layer of thin film, which might be due to the shielding effect of organic materials [24]. The changes above demonstrated the successful conjugation of PEG and HB5. MSCN-PEG-HB5 nanoparticles contained sGC were further verified by Raman spectra, which presented obvious signals of D- and G-band (at 1330 cm1 and 1580 cm1) (Fig. 1c). It has been reported that MSCN possessed the photothermal capability due to the decoration of sGC on the inner wall. In this work, the organic materials-modified MSCN nanoparticles well inherited the NIR absorption property of MSCN, and the temperature significantly increased in 5 min under the laser irradiation with a power density of 6 W/cm2 (Fig. 2a). The results of the photothermal heating effect showed that MSCN-PEG-HB5 exhibited the strongest photothermal conversion efficiency. The temperature of MSCN-PEG-HB5 was raised by 36.1 °C within 5 min to reach 60.2 °C, compared with that of MSCN-PEG which was increased by 29 °C with the final temperature of 53.1 °C. Thus, the improved NIR-induced heat generation of MSCN-PEG-HB5 might be attrib-

Fig. 1. (a) The FT-IR spectra of different nanoparticles; (b) TEM image of MSCN-PEG-HB5, and (c) Raman spectra of MSCN-PEG-HB5.

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Fig. 2. (a) The temperature increment of different nanoparticles (100 lg/ml) under the NIR laser irradiation of 6 W/cm2 for 5 min. (b) The temperature increment of MSCNPEG-HB5 (100 lg/ml) under various laser intensity for 5 min. (c) UV–vis spectra of MSCN-NH2, MSCN-PEG, MSCN-PEG-HB5 and MSCN-PEG-HB5/DOX at the concentration of 100 lg/ml. H2O was used as negative control. Inset: UV–vis absorption curves of corresponding nanoparticles around 808 nm. (d) Fluorescence spectra of DOX in different solutions.

uted to the improvement of dispersity when targeting ligand HB5 was coupled to MSCN. The enhanced photothermal conversion efficiency and dispersity after HB5 conjugation made MSCN-PEG-HB5 an ideal photothermal therapeutic vector. Furthermore, the temperature of MSCN-PEG-HB5 raised in a laser power intensitydependent manner (Fig. 2b). For materials with efficient photothermal conversion efficiency, the high temperature can be reached in mice [25]. And, the temperature can be controlled by the intensity of NIR, the irradiation time and also the amount of nanomaterials that accumulated in the tumor.

3.2. In vitro drug loading and release To investigate the in vitro drug loading and release from MSCNPEG-HB5, the widely studied chemotherapeutic drug DOX was selected as the model drug. The optimized amount of DOX for MSCN-based nanoparticles was obtained by the drug loading curve (ESI Fig. S1). The loading efficiency of DOX in MSCN-PEG-HB5 was calculated to be about 0.605 (lg/lg, DOX/MSCN). The UV–vis absorption spectrum of MSCN-PEG-HB5 showed the absorption peak at 260 nm, which may be assigned to the single-chain DNA structure (Fig. 2c). In comparison, a new absorption peak emerged in the spectrum of DOX-loaded MSCN-PEG-HB5 (MSCN-PEG-HB5/ DOX) at about 500 nm, indicating the presence of DOX molecules in the nanoparticles. Meanwhile, the inserted UV–vis absorption curve showed higher NIR absorbance of MSCN-PEG-HB5 than MSCN-NH2 and MSCN-PEG at 808 nm (Fig. 2c), which was corresponding to the tendency of photothermal conversion efficiency. To investigate the location of DOX in MSCN-PEG-HB5, fluorescence spectra of DOX in different solutions diluted in the same way were shown in Fig. 2d. Due to the energy transfer from DOX to semi-graphitized carbon on the inner wall of MSCN, fluorescence quenching of DOX will happen when loaded within MSCN. As shown in Fig. 2d, the fluorescence of unloaded DOX in supernatant after centrifugation was decreased and no obvious fluorescence was observed in DOX-loaded MSCN-PEG-HB5 nanoparticles, indicating that DOX molecules were loaded into MSCN.

The in vitro drug release behavior of MSCN-PEG-HB5/DOX was conducted in PBS 7.4, 6.0 and 5.0 buffers in sequence for 4 h in the dark (Fig. 3). MSCN contains plentiful mesopores with inner walls decorated with semi-graphitized carbon. Therefore, aromatic and hydrophobic DOX molecules (after neutralized by TEA) can be loaded in MSCN-PEG-HB5 via both p–p interaction and hydrophobic–hydrophobic interaction. And also, there existed electrostatic interaction between the positively charged DOX molecule and negatively charged MSCN-PEG-HB5. It could be found that both the two curves showed stepwise growth once PBS with lower pH was replaced, indicating the pH-responsive drug release mechanism. The main reason is that the interaction between DOX and carriers varied with the decrease of pH, leading to the release of large amount of DOX [26]. In the presence of NIR laser irradiation,

Fig. 3. Release profile of DOX from MSCN-PEG-HB5 with and without NIR irradiation. The release experiment was conducted in PBS with pH 7.4, pH 6.0 and pH 5.0 in sequence.

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the drug release rate became much faster than that in the normal group at any pH condition, demonstrating the NIR-triggered drug release mechanism. This result was considered to be that the absorption of NIR light by MSCN-PEG-HB5 converted light energy into heat energy efficiently, inducing more DOX release. The pHresponsive release of DOX from MSCN-PEG-HB5 was important for cancer therapy due to the lower pH of tumor microenvironment, and NIR-triggered drug release manner can further enhance the chemotherapeutic efficacy. The changes in surface charge could verify the successful modification of MSCN at every step (Fig. 4). Amine-functionalized MSCN was successfully synthesized by comparing the zeta potential of MSCN (29.23 ± 2.83 mV) with MSCN-NH2 (23.1 ± 1.67 mV). Then zeta potential got back to approximately neutral after PEGylation, demonstrating that the ANH2 on the surface was covered by PEG molecules. When negatively charged aptamer HB5 assembled, MSCN-PEG-HB5 (34.17 ± 1.58 mV) exhibited excellent dispersity due to the intense repulsion force between nanoparticles. By the loading of positively charged DOX, the zeta potential nanoparticles turned into 32.97 ± 1.19 mV, indicating successful preparation of MSCN-PEG-HB5/DOX [26].

3.3. Mechanism studies of cellular uptake Cellular uptake experiment was carried out by incubating different concentrations of MSCN-PEG-HB5/DOX with SK-BR-3 cells for 1 h. As illustrated in ESI Fig. S2, fluorescence signals increased along with the increase of the concentration, indicating that cellular uptake exhibited a distinct concentration-dependent pattern. When treated with 100 lg/ml nanoparticles, the cells not only took up plenty of nanoparticles, but also presented satiate morphology. Moreover, nanoparticles at this concentration possessed great photothermal effect. Thus, 100 lg/ml of MSCN-PEG-HB5 was considered to be a desirable concentration in the following experiment. It is essential to deliver drugs to specific cells in cancer therapy. SK-BR-3 cell line was chosen as a tumor cell model. As a negative control, MCF-10A cell line was introduced. FITC-modified MSCNPEG (FITC-MSCN-PEG) and FITC-modified MSCN-PEG-HB5 (FITCMSCN-PEG-HB5) were used to evaluate the cell selectivity (Fig. 5). As shown in the fluorescent microscope images, SK-BR-3 cells showed significant green fluorescence signals after 1 h incubation with FITC-MSCN-PEG-HB5 (Fig. 5g and h), much stronger than that of FITC-MSCN-PEG (Fig. 5c and d). It should be noticed that a number of FITC-MSCN-PEG-HB5 was internalized by HER2-overexpressed SK-BR-3 cells, while it could hardly be taken up by normal MCF-10A cells (Fig. 5e and f), suggesting that HB5modified system had an excellent targeting ability to HER2-positive tumor cells. A competitive inhibition experiment was conducted to confirm the specific interaction between aptamer HB5 modified nanoparticles and HER2 receptors. Peptide SP13, another type of HER2-targeting ligand [20], was introduced as a competitive inhibitive agent. The presence of SP13 had no influence on the uptake of FITC-MSCN-PEG in SK-BR-3 cancer cells (Fig. 5k

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Fig. 4. Zeta potential of different MSCN-based nanoparticles. The data were expressed as mean ± S.E.M. (n = 4).

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Fig. 5. The targeting capability and competitive inhibition were evaluated in SKBR-3 breast cancer cells (c, d, g, h, k, l, o, p) and normal MCF-10A cells (a, b, e, f, i, j, m, n). SP13 is a breast cancer ligand used to compete with aptamer HB5. Fluorescence microscope images showed FITC-MSCN-PEG (a–d), FITC-MSCN-PEGHB5 (e–h), FITC-MSCN-PEG with SP13 (i–l) and FITC-MSCN-PEG-HB5 with SP13 (m– p), respectively. Scale bars = 100 lm.

Fig. 6. Mechanism studies of FITC-labeled MSCN-PEG-HB5 nanoparticles. SK-BR-3 cells were treated with (a) FITC-MSCN-PEG-HB5 nanoparticles at 4 °C, (b) FITCMSCN-PEG-HB5 nanoparticles at 37 °C, (c) FITC-MSCN-PEG-HB5 nanoparticles pretreated with excessive SP13 peptide at 37 °C, respectively. 2D confocal microscopy images were presented as x/y sections. The 2.5D images were obtained from the middle of z planes. Scale bars = 20 lm.

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+ HB5

Fig. 7. (a) Fluorescent images of SK-BR-3 tumor-bearing mice after intravenous injection of MSCN-PEG/DOX (labeled as HB5) and MSCN-PEG-HB5/DOX (labeled as +HB5) were obtained at 1, 2, 4 and 6 h. (b) Fluorescent images of excised organs and tumors after 6 h. The fluorescence of DOX released from nanoparticles was measured.

and l). However, the fluorescence signals of FITC-MSCN-PEG-HB5 in SK-BR-3 cells turned negligible with the existence of competitive SP13 (Fig. 5o and p). These results confirmed that the uptake of FITC-MSCN-PEG-HB5 was a receptor-mediated process. Besides, energy-dependent experiment was conducted at 4 °C and 37 °C, respectively. Fluorescent microscopy images showed distinct fluorescence at 37 °C and ignorable fluorescence at 4 °C, indicating an energy consuming process of the cellular uptake (ESI Fig. S3). In order to observe intracellular localization of nanoparticles clearly, a through confocal microscopy images were performed. As shown in Fig. 6, fluorescence was mainly observed in the cell membranes at 4 °C, indicating the binding interaction of MSCNPEG-HB5 with cells at low temperature [27]. When the temperature was elevated to 37 °C, apparent fluorescence was observed within cells, demonstrating the endocytosis of nanoparticles, which process could be significantly inhibited by excessive SP13 peptide. This is considered as typical receptor-mediated inhibition. Furthermore, the intracellular localization of nanoparticles can be clearly observed, especially in the 2.5D images which were obtained from the middle of the z plane. A group of thorough x/y sections of SK-BR-3 cells internalized with FITC-MSCN-PEG-HB5 nanoparticles were presented in ESI Fig. S4, which also demonstrated the intracellular localization of HB5-conjugated nanoparti-

cles. These findings further confirmed that the uptake of MSCNPEG-HB5 was mainly based on the receptor-mediated mechanism.

3.4. Targeting ability in vivo To confirm targeting ability of HB5-modified drug delivery system in vivo, fluorescence images of SK-BR-3 tumor-bearing mice were captured after intravenous injection with MSCN-PEG/DOX and MSCN-PEG-HB5/DOX (Fig. 7a). Breast cancer is a kind of superficial tumor mainly locating at the same spatial position, the whole-body images could be used for observation of relative distribution of DOX within tumor sites. At each time point, MSCN-PEGHB5/DOX showed much higher DOX accumulation at tumor site than MSCN-PEG/DOX, which was further proved by fluorescent images of excised tumors (Fig. 7b). Time-dependent tumor accumulation of MSCN-PEG-HB5/DOX was observed, the strongest fluorescent signal of which was determined at 4 h. In addition, the modification of HB5 significantly decrease the drug accumulation within liver and kidney, which might reduce the toxicity in these organs. Notably, no fluorescence was observed in heart, showing that MSCN-based nanoparticles can avoid the cardiac toxicity of free DOX.

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BF

Live

Dead

Merge

(a)

(b)

(c)

(d)

(e)

Fig. 8. Live-Dead staining of SK-BR-3 cells under different treatments. (a) MSCN-PEG-HB5, (b) MSCN-PEG-HB5+NIR, (c) MSCN-PEG-HB5/DOX, (d) MSCN-PEG-HB5/DOX+NIR with the MSCN concentration at 100 lg/ml, and (e) MSCN-PEG-HB5/DOX+NIR with the MSCN concentration at 50 lg/ml. Scale bars = 100 lm.

Fig. 9. Cell viability of SK-BR-3 cells under different treatments. The data expressed as mean ± S.E.M. (n = 4). Statistical significance (p < 0.001) was obtained between all the groups within the same concentration.

3.5. Synergistic effect of chemo-photothermal therapy To prove synergistic effect of chemo-photothermal therapy, cell viability following different treatments was identified qualitatively

using the Live-Dead Kit. As shown in Fig. 8, MSCN-PEG-HB5/DOX exposed to the NIR laser (chemo-photothermal therapy) showed significantly decreased cell viability (Fig. 8d) compared to the photothermal therapy (Fig. 8b) and chemotherapy alone (Fig. 8c). At the same time, the material, MSCN-PEG-HB5, did not present detectable cell cytotoxicity, indicating its high biocompatibility (Fig. 8a). Moreover, the cancer cell killing efficiency was increased with the concentration of MSCN-PEG-HB5/DOX for combined therapy (Fig. 8d and e). To further investigate the combined therapeutic capacity, cell viability measured via Cell Counting Kit-8 (CCK-8) was carried out for quantitative evaluation (Fig. 9). MSCN-PEG-HB5/DOX with the NIR irradiation showed lower cell viability compared to chemotherapy and photothermal therapy alone in all tested concentrations. Similarly, no obvious cytotoxicity was observed in the presence of MSCN-PEG-HB5. Furthermore, the result of hemolytic test showed that the hemolytic percentage of MSCN-based nanoparticles was less than 5% even at the highest concentration (500 lg/ml), demonstrating excellent blood compatibility of the tested materials (ESI Fig. S5). IC50 of breast cancer cells with different treatments and calculated CI value were exhibited in Table 1. The data presented the IC50 of DOX (34.81 lg/ml) in chemotherapy and the IC50 of MSCN (217.82 lg/ml) in photothermal therapy. It should be noticed that a significant decrease of IC50 in combined

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Table 1 IC50 (lg/ml) under different treatments on SK-BR-3 cells and the calculated CI. MSCN-PEG-HB5

MSCN-PEG-HB5+NIR

MSCN-PEG-HB5/DOX

MSCN-PEG-HB5/DOX+NIR

MSCN

MSCN

DOX

MSCN

DOX

20043.85

217.82

34.81

11.08

6.71

therapy (with DOX 6.71 lg/ml and MSCN 11.08 lg/ml) was achieved. Furthermore, the calculated CI value was 0.253, indicating the synergistic effect of chemotherapy and photothermal therapy (Table 1). This synergistic effect was probably ascribed to the enhanced DOX release via pH/NIR dual-triggered manner, increased intracellular DOX delivery and cell deaths via NIR laser irradiation. On one hand, MSCN-PEG-HB5/DOX irradiated by NIR laser can absorb the NIR light and convert it into heat. Hyperthermia could not only promote molecular vibration, but also dissociate the strong interaction between DOX and nanoparticles, which resulted in significant DOX release to improve the chemotherapeutic efficacy, and at the same time induced cell deaths itself [28]. On the other hand, the cell membrane permeability increased with photothermal heating treatment may further enhance the intracellular DOX delivery [29,30]. Based on the synergistic effects of chemo-photothermal therapy in vitro, it could be expected that the combination therapy in vivo systems would also be more efficient than single chemotherapy or photothermal therapy. 4. Conclusions In summary, DOX-loaded HB5-modified mesoporous silica–carbon based drug delivery system for combined therapy has been successfully prepared. This novel system did not only exhibit synergistic effect of chemotherapy and photothermal therapy, which was mainly attributed to the increased DOX release triggered by both pH decrease and NIR laser irradiation, but also had significantly increased accumulation within HER2-overexpressed SKBR-3 cells due to specific aptamer HB5 modification. The targeting capability and synergistic therapeutic effect of MSCN-PEG-HB5/ DOX together led to significant cytotoxicity to HER2-positive breast cancer cells. Our findings demonstrated that specific aptamer conjugated drug delivery system MSCN-PEG-HB5/DOX is a great platform for combined chemo-photothermal therapy targeted to HER2-positive cancers. Acknowledgements This work was supported by the grants from National Key Basic Research Program (2013CB932502) of China (973 Program), National Natural Science Foundation of China (21303022), ‘‘Zhuo Xue’’ Talent Plan of Fudan University, Natural Science Foundation of Shanghai City of China (13ZR1451400) and Sino-German Research Project (GZ995). Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 1–9 are difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2015. 01.002. Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2015.01. 002.

CI

0.253

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