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Reactive Oxygen Species-Manipulated Drug Release from a Smart Envelope-Type Mesoporous Titanium Nanovehicle for Tumor Sonodynamic-Chemotherapy Jinjin Shi,†,‡ Zhaoyang Chen,† Binghua Wang,† Lei Wang,† Tingting Lu,† and Zhenzhong Zhang*,†,‡ †
School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, P.R. China Collaborative Innovation Center of New Drug Research, Safety Evaluation, Henan Province, Zhengzhou 450001, P.R. China
‡
S Supporting Information *
ABSTRACT: Despite advances in drug delivery systems (DDSs), the stimuli-responsive controlled release DDSs with high spatial/temporal resolution are still the best choice. Herein, a novel type of envelope-type mesoporous titanium dioxide nanoparticle (MTN) was developed for one-demand drug delivery platform. Docetaxel (DTX) was loaded in the pores of MTN with a high drug loading efficiency (∼26%). Then β-cyclodextrin (β-CD, a bulky gatekeeper) was attached to the outer surface of MTN via a reactive oxygen species (ROS) sensitive linker to block the pores (MTN@DTX-CD). MTN@DTX-CD could entrap the DTX in the pores and allow the rapid release until a focused ultrasound (US) emerged. A large number of ROS were generated by MTN under US radiation, leading to the cleavage of the ROS-sensitive linker; thus, DTX could be released rapidly since the gatekeepers (β-CD) were detached. Besides, the generation of ROS could also be used for tumor-specific sonodynamic therapy (SDT). Studies have shown the feasibility of MTN@DTX-CD for US-triggered DTX release and sonodynamicchemotherapy. In the in vitro and in vivo studies, by integrating SDT and chemotherapy into one system, MTN@DTX-CD showed excellent antitumor efficacy. More importantly, this novel DDS significantly decreased the side effects of DTX by avoiding the spleen and hematologic toxicity to tumor-bearing mice. KEYWORDS: mesoporous titanium dioxide nanoparticle, ROS-manipulated drug release, sonodynamic therapy, combination therapy, nanomedicine
1. INTRODUCTION Until now, chemotherapy is still an important component of cancer therapies used to most cancers. Most of the cytotoxic chemotherapeutic agents are limited in their clinical applications due to the serious toxicities.1−5 In the past decade, with © 2015 American Chemical Society
Received: October 18, 2015 Accepted: November 20, 2015 Published: November 20, 2015 28554
DOI: 10.1021/acsami.5b09937 ACS Appl. Mater. Interfaces 2015, 7, 28554−28565
Research Article
ACS Applied Materials & Interfaces
Figure 1. Scheme of MTN@DTX-CD and its biofunctions.
dextrin (CD) is a common gatekeeper, for example, CD-gated MSNs are able to entrap different cargos efficiently.29−31 Therefore, in this study, after drug/dye uptake into the mesopores of MTN, β-CD was used and acted as a bulky gatekeeper to block the mesopores. To allow the burst release of drugs, β-CD was attached to the outer surface of MTN via a ROS-sensitive linker. The ROS-sensitive linker is selectively recognized and broken by ROS for oxidation (MTN-S-CH2−SCD).32 However, much attention has been drawn toward the photodynamic therapy (PDT) and sonodynamic therapy (SDT) applications of TiO2 nanoparticles; the employment of TiO2 nanoparticles for tumor-specific controlled drug delivery is still at an early stage of development. This year, Wang et al. attached polyethylenimine (PEI) to MTN and achieved controlled release by UV light.23 However, the above DDS still needs to improve. First, UV light could not penetrate deeply and could cause the damage to skin in vivo. Second, PEI modification could lead to an additional toxicity and influence the biodistribution of DDS. Third, drug release controlled by the photocatalytic of PEI was not sensitive enough. In this work, we developed a novel type of CD-capped MTN to develop the US-triggered drug delivery. As shown in Figure 1, docetaxel (DTX) was loaded in the pores of MTN. Then βCD was attached to the outer surface of MTN via a ROSsensitive linker (MTN@DTX-CD). MTN@DTX-CD was able to entrap the DTX in the pores; in contrast, when the system was irradiated by a focused US, DTX could be released rapidly. As illustrated in Figure 1, upon US excitation, a large number of ROS were created by MTN, leading to the breaking of the ROS-sensitive linker, and drugs could be released quickly from the DDS since the CD (gatekeeper) was detached. On the other hand, the ROS generated by MTN under US radiation could also lead to apoptosis of tumor cells, achieved a dual effect of combining SDT and chemotherapy. These dual therapeutic effects of the combine therapy were remotely controlled by the focused US.
the rapid development of nanotechnology, nanoparticles have been used as the carriers for efficient drug delivery.6−8 Such drug delivery systems (DDSs) have shown advantageous features in improving accumulation of drugs at tumor sites and reducing systemic toxicity.9−11 However, the release profiles in many of the reported DDSs rely on spontaneous degradation of the carriers in vivo and do not allow for controlled drug release.12,13 Moreover, traditional DDSs always suffer from the unexpected drug release during circulation. To maximize the therapeutic effects and minimize the side effects, a highly toxic antitumor drug should be zero release in the noncancerous sites, and then once arriving at tumor cells or tissues, the drugs should be burst release. Therefore, stimuliresponsive controlled-release DDSs are the best choice for onedemand drug delivery platform. Mesoporous nanoparticles with stable mesostructure, large surface areas, and tailorable pore size are suitable for stimuliresponsive controlled drug release system.14−17 The mesoporous silica (MSN) is one of the leaders. Although, MSN had been successfully explored, the applications in drug delivery, MSN, itself could not be activated by the external stimulus and could not play a therapeutic role in cancer treatment. Besides, the success of MSN encouraged the exploration of the other mesoporous nanoparticles for biomedical applications. Recently, TiO2, a typical semiconductor material, has been widely used in many fields including biomedicine.18−20 Just as MSN, the mesoporous TiO 2 nanoparticle (MTN) with low cytotoxicity and suitable pore size for drug loading is another ideal carrier for stimuli-responsive DDS.21−23 In addition, TiO2 nanoparticles could strongly absorb ultraviolet (UV) light or untrasound (US) and generate a lot of reactive oxygen species (ROS, such as hydrogen peroxide, hydroxyl radicals, and super oxides). Therefore, TiO2 nanoparticles can be used for photodynamic therapy (PDT)20,24,25 or sonodynamic therapy (SDT).24−27 The above reports also provide a theoretical foundation for UV light or US-triggered drug release. Furthermore, an ideal DDS should be able to encapsulate drugs efficiently before reaching the tumor.28 Therefore, MTN should be coated with a monolayer of gatekeepers. Cyclo28555
DOI: 10.1021/acsami.5b09937 ACS Appl. Mater. Interfaces 2015, 7, 28554−28565
Research Article
ACS Applied Materials & Interfaces
nm; flow rate 1.0 mL/min; and injection volume 20 μL.33 mloaded DTX = 20 mg − mfree DTX. 2.2.7. β-CD Capping. MTN@DTX-AD (20 mg) and β-CD (8 mg) were added to 20 mL of water, stirred at room temperature in the dark for 2 h, and the as-prepared MTN@DTX-CD were collected by centrifugation. The amount of β-CD attached onto MTN@DTX-AD was measured by HPLC with the following conditions: an Eclipse XDB-C18 column (150 mm × 4.6 mm, 5.0 mm); mobile phase, methanol/water 15:85; column temperature 30 °C; a refractive index detector; flow rate 1.0 mL/min; and injection volume 20 μL (the conditions were referred to Chinese Pharmacopoeia). 2.3. Characterization. DLS (Zetasizer Nano ZS-90, Malvern, UK) and TEM (Tecnai G2 20, FEI) were used for characterizing zeta potential, particle size, and morphology of MTN@DTX-CD, respectively. A surface area and pore size analyzer (V-sorb 2800P, Gold App Instruments, CN) was used to measure the pore characteristics of the samples. The optical properties of MTN@ DTX-CD were characterized using an ultraviolet−visible (UV−vis) spectrometer (Lambda 35, PerkinElmer, USA). Fourier transform infrared (FT-IR) spectra were recorded on a Nicolet iS10 spectrometer (Thermo). The relative amount of organics linked to MTNs was tested using a thermal gravimetric analysis (TGA, PerkinElmer) with the following conditions of scanning from 25− 800 °C under nitrogen at a heating rate of 20 °C/min. 2.4. Evaluation of US Sensitivity. 2.4.1. ROS Generation Assay. Cell-free experiments were performed in 96-well plates. MTN-CD solutions were diluted to a final concentration of 10 μg/mL per well in water, and singlet oxygen sensor green (SOSG) (Molecular Probes, Invitrogen, Bedford, MA) was added to each well at a final concentration of 5.0 μM. Each experimental group contained three wells. After US irradiation (Ultrasonic physiotherapy instrument: Chattanooga group 4714 Adams Road Hixson, TN 37343, power density: 0−10W, probes area: 2 or 5 cm2), a microplate reader (Spectra Max M5) was used for acquisition of fluorescence signal; fluorescence emission at 525 nm was measured upon excitation at 505 nm using a 2.0 nm monochromator band-pass for both excitation and emission. 2.4.2. US-Response DTX Release. MTN@DTX-CD samples were suspended in water (1 mL) and then sealed in dialysis membranes. The dialysis bags were incubated in 50 mL of sodium dodecyl sulfate solution (SDS, 0.5%) at 37 °C with gentle shaking, respectively. After US irradiating (1 W/cm2, 40 s), a 200 μL portion of the aliquot was collected from the incubation medium at predetermined time intervals, and the released DTX was quantified by HPLC under the chromatographic conditions described in section 2.2. 2.5. Cellular Experiments. 2.5.1. Cell Culture. MCF-7 human breast cancer cell line was obtained from Chinese Academy of Sciences Cell Bank (Catalog No. HYC3204). Cells were cultured in normal RPMI 1640 culture medium with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in 5% CO2 and 95% air at 37 °C in a humidified incubator. 2.5.2. US-Response Release in Vitro. FITC (fluorescent label, green) was loaded into MTN-AD (MTN@FITC-AD); after that, βCD was also attached to the outer surface as a bulky gatekeeper (MTN@FITC-CD). MCF-7 cells were seeded at 5 × 104 cells per well on glass coverslips in six-well plates. When cells reached 70% confluence, they were treated with MTN@FITC-AD or MTN@FITCCD for 1, 2, 3, 4, 5, 6, and 8 h, respectively. The MTN@FITC-CD group was irradiated by a focused US (1 W/cm2, 40 s) at 4 h incubation. After being washed three times with PBS, the cells were imaged by a fluorescence microscope (Zeiss LSM 510). 2.5.3. Intracellular ROS Detection. Intracellular ROS production following US was detected using DCFH-DA Reactive Oxygen Species Assay Kit. MCF-7 cells were seeded at 5 × 104 cells per well in six-well plates. Following incubation with MTN-CD (10 μg/mL) for 24 h, DCFH-DA was loaded into the cells. After incubation for 30 min, cells were washed twice with PBS and then exposed to US (1 W/cm2, 40 s) irradiation; after incubation for 1 h, fluorescence images of treated cells were acquired using a fluorescence microscope (Zeiss LSM 510).
2. MATERIALS AND METHODS 2.1. Materials. Titanium(IV) isopropoxide (TIP) (95%) was obtained from Alfa Aesar (Lancs, UK). Docetaxel (DTX, purity >98%) was obtained from Beijing Yi-He Biotech Co. Ltd. Pluronic F68, (3aminopropyl)triethoxysilane, 1-amino, amantadine (AD), β-CD, IR 783, HS-IR783 dye, and dimethyl sulfoxide (DMSO) were obtained from Sigma-Aldrich Co. LLC. ROS-sensitive linker (HOOC-S-CH2− S-COOH) was homemade. Sulforhodamine B (SRB), RPMI 1640 cell culture medium, penicillin, streptomycin, fetal bovine serum (FBS), and heparin sodium were bought from Gibco Invitrogen. Fluoresceine isothiocyanate (FITC), DCFH-DA, JC-1, and ethidium bromide (EB) were supplied by Beyotime Biotechnology Co. Ltd. Other reagents were acquired from China National Medicine Corporation Ltd. The dialysis bags (MWCO = 1000) were from Spectrum Laboratories Inc. 2.2. Synthesis of US-Responsive MTNs (MTN@DTX-CD). 2.2.1. Fabrication of MTN. The MTN nanoparticles were synthesized via a sol−gel route using F68 as a structure-directing agent and TIP as a TiO2 source. In detail, 4 g of F68 was completely dissolved in 200 mL of ethanol, and then 0.6 mL of water was added to this solution with stirring. Then 3.4 mL of TIP was added to the solution under vigorous stirring. When the clear solution turned into a milky white suspension, the solution was stirred continuously for 1 h, and then the solution was kept at room temperature without stirring to stand for 12 h. After centrifugation, the precipitate was dispersed in 200 mL of ethanol and then stirred for 2 h at 70 °C. After stirring, as-prepared MTNs were collected by centrifugation. To completely remove F68, the MTN was washed three times in the same way and finally dried at 100 °C for 48 h to remove ethanol. 2.2.2. Synthesis of MTN-NH2. MTN (50 mg) was suspended in toluene (20 mL) and sonicated for 1 h to form a homogeneous suspension. To the suspension, (3-aminopropyl) triethoxysilane (APTES, 4 mL) was added, and then the solution was allowed to stir at 80 °C for 24 h under N2. After 24 h, the resulting product (MTN-NH2) was purified by washing three times with ethanol and then dried in vacuum at 60 °C for 12 h. 2.2.3. Synthesis of the ROS-Sensitive Thioketal Linker. Three milliliters of acetone was added to 7 mL of thioglycolic acid solution (30% in water); after stirring at 50 °C for 48 h, the result product thioketal linker was obtained by repeating washes with ice water. Characterization of ROS-sensitive thioketal linker was shown in Supporting Information 1. 2.2.4. Conjugation of ROS-Sensitive Linker. MTN-NH2 (45 mg) was suspended in water (10 mL) and sonicated for 1 h to form a homogeneous suspension. EDC·HCl (45 mg) and NHS (30 mg) were then added to the suspension. The ROS-sensitive linker (HOOC-SCH2−S-COOH, 45 mg) dissolved in acetone (1 mL) was added dropwise to the above mixture, and then the mixture was allowed to stir at 50 °C for 48 h under N2. After 48 h, the resulting product (MTN-S-CH2−COOH) was purified by washing three times with acetone and three times with water to remove unreacted HOOC-SCH2−S-COOH and other reagents, and then it was dried in vacuum at 50 °C for 24 h. 2.2.5. Synthesis of MTN-AD. MTN-S-CH2−COOH (45 mg) was suspended in water (10 mL), EDC·HCl (90 mg), NHS (60 mg), and 1-amino; amantadine (AD, 90 mg) was then added. The mixture was allowed to stir at 50 °C for 48 h under N2. After 48 h, the resulting product (MTN-AD) was purified by washing ten times with water to remove unreacted AD and other reagents, and then it was dried in vacuum at 50 °C for 24 h. 2.2.6. DTX Loading. MTN-AD (50 mg) was added to DTX ethanol solution (10 mL) containing DTX (20 mg) and sonicated at room temperature for 2 h and then sonicated using an ultrasonic cell disruption system (400W, 10 times); finally the nanosuspension was centrifuged to remove free DTX. The amount of free DTX was determined by high-performance liquid chromatography (HPLC, 1100 Agilent, USA) with the following conditions: an Eclipse XDB-C18 column (150 mm × 4.6 mm, 5.0 mm); mobile phase acetonitrile/ water 50:50; column temperature 30 °C; detection wavelength: 231 28556
DOI: 10.1021/acsami.5b09937 ACS Appl. Mater. Interfaces 2015, 7, 28554−28565
Research Article
ACS Applied Materials & Interfaces
Figure 2. A schematic illustration of MTN@DTX-CD nanocomposite preparation. 2.5.4. Mitochondrial Membrane Potential Detection. Mitochondrial membrane potential was detected using JC-1 staining. MCF-7 cells were seeded at 5 × 104 cells per well in six-well plates. Following incubation with MTN-CD (10 μg/mL) for 24 h, cells were washed twice with PBS and exposed to US (1 W/cm2, 40 s) irradiation. After incubation for 1 h, JC-1(5 μg/mL) was loaded into the cells, and they were incubated for 30 min; after being washed three times with PBS, the cells were imaged by a fluorescence microscope (Zeiss LSM 510). 2.5.5. Comet Assay. MCF-7 cells were incubated with MTN-CD for 24 h; cells were washed twice with PBS and exposed to US (1 W/ cm2, 40 s) irradiation. After that, the cells were collected and resuspended in prewarmed low melting point agarose, and then 100 μL of cell LMA suspension was tiled on the slide, which was precoated with normal melting point agarose (NMA), and lysed in cold lysis solution (Beijing CoWin Bioscience Co., Ltd.) for 2 h; after lysis, the samples were then electrolyted for 25 min. Next, the samples were filled with neutralization buffer at room temperature in the dark for 45 min, then dried at room temperature and stained in the dark with 2% ethidium bromide; finally, the samples were observed by a fluorescence microscope (Zeiss LSM 510). 2.5.6. Cell Viability Measurements. MCF-7 cells were plated in 96well plates and then incubated for 24 h. After incubating, the medium was replaced with fresh medium containing various concentrations of free DTX, MTN, MTN-CD, and MTN@DTX-CD for 24 and 48 h, and the cells were or were not irradiated with US (1 W/cm2, 30 S). The cells were also treated with different concentrations of nonloaded MTNs for 24 h to investigate cytotoxicity of the blank delivery system; standard SRB assay was carried out to determine cell viabilities. 2.5.7. Cell Apoptosis Assay. Apoptosis was monitored by an Annexin-V-Fluos Staining kit (Sigma-Aldrich Co. LLC). MCF-7 cells were treated with free DTX (2 μg/mL), MTN-CD (10 μg/mL), and MTN@DTX-CD (DTX, 2 μg/mL; MTN-CD, 7.7 μg/mL) for 24 h; in US irradiation groups, cells were irradiated with a focused US (1 W/ cm2, 30 S) and then trypsinized, washed with PBS, and resuspended in binding buffer (10 μM HEPEs/NaOH, pH 7.4, 140 μM NaCl, and 2.5 μM CaCl2). After cell density was adjusted to 1 × 106 cell/mL, 1 μL of recombinant human anti-Annexin V-FITC and 2 μL of propidium iodide (PI) were added to 100 μL of cell suspension, mixed by a vortex, and incubated at room temperature in the dark for 15 min. Finally, 400 μL of binding buffer was added to the above cells and subsequently analyzed by flow cytometry (FCM, Epics XL.MCL). 2.6. In Vivo Experiments. 2.6.1. Xenograft Tumor Mouse Model. All animal experiments were performed under a protocol approved by Henan laboratory animal center. The S180 tumor models were generated by subcutaneous injection of 2 × 106 cells in 0.1 mL of saline into the right shoulder of female BALB/c mice (18−20 g, Henan laboratory animal center). The mice were used when the tumor volume reached 60−100 mm3 (∼6 days after tumor inoculation).
2.6.2. NIR Imaging. A near-infrared dye (HS-IR783) was used to mark MTN@DTX-CD via the high affinity between HS and Ti on the surface of MTN. In detail, HS-IR783 (20 μg) was added to MTN@ DTX-CD nanosuspension (1 mg/mL, 5 mL) and then stirred for 6 h to obtain MTN@DTX-CD-IR783. Excess HS-IR783 was removed by Sephadex G-25 column (Sigma-Aldrich Co. LLC). A sample of 0.2 mL of MTN@DTX-CD-IR783 was intravenously injected into tumorbearing mice, and the whole body fluorescence imaging was performed at 0.5, 1, 2, 4, 8, 12, 24, and 48 h after injection using a small animal imaging system (Xtreme, Bruke). 2.6.3. DTX Levels in Tumor and Spleen. A sample of 0.2 mL of DTX (5 mg/kg) or MTN@DTX-CD (DTX, 5 mg/kg; MTN-CD, 18.9 mg/kg) was intravenously injected into tumor-bearing mice, respectively. Tumors were exposed to a focused US (1 W/cm2, 40 s) at 12 h postinjection. At predetermined time intervals, the mice were sacrificed. The tumors and spleens were collected, weighed, and homogenized in buffer (acetonitrile-to-saline ratio, 1:1). DTX in tumor/spleen was determined by HPLC under the chromatographic conditions described in section 2.2. 2.6.4. In Vivo Antitumor Effect. For the in vivo antitumor experiments, the tumor-bearing mice were divided into seven groups (six mice per group), minimizing the differences of weights and tumor sizes in each group. The mice were administered with (1) PBS (0.1 mL), (2) PBS+US (0.1 mL, 1 W/cm2, 40 s), (3) MTN-CD (18.9 mg/ kg, 0.2 mL), (4) MTN-CD+US (18.9 mg/kg, 0.2 mL, 1 W/cm2, 40 s), (5) DTX (DTX: 5 mg/kg, 0.2 mL), (6) MTN@DTX-CD (DTX, 5 mg/kg; MTN-CD, 18.9 mg/kg; 0.2 mL), and (7) MTN@DTX-CD +US (DTX, 5 mg/kg; MTN-CD, 18.9 mg/kg; 0.2 mL, 1 W/cm2, 40 s) and were intravenously injected into mice via the tail vein every 2 days, respectively. The US irradiating groups were exposed to a focused US at 24 h postinjection. The mice were observed daily for clinical symptoms, and the tumor sizes were measured by a caliper every other day and calculated as the volume = (tumor length) × (tumor width)2/ 2. After treatment for 15 days, the mice were sacrificed to collect heart, liver, spleen, lung, kidney, brain, and tumor for hematoxylin and eosin (H&E) staining. Morphological changes were observed under microscope (Zeiss LSM 510). 2.7. Statistical Analysis. Quantitative data are expressed as mean ± SD and analyzed by use of Student’s t test. P-values
