Biomaterials 35 (2014) 5847e5861
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
A tumor-targeting near-infrared laser-triggered drug delivery system based on GO@Ag nanoparticles for chemo-photothermal therapy and X-ray imaging Jinjin Shi, Lei Wang, Jing Zhang, Rou Ma, Jun Gao, Yan Liu, Chaofeng Zhang, Zhenzhong Zhang* School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou 450001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 February 2014 Accepted 10 March 2014 Available online 17 April 2014
In this study, a GO@Ag nanocomposite was synthesized by chemical deposition of Ag nanoparticles onto graphene oxide (GO) through a hydro thermal reaction, and doxorubicin (DOX), one of the most effective drugs against a wide range of cancers, was employed as the model drug and linked to GO@Ag via ester bonds with a very high drug loading efficiency (w82.0%, weight ratio of DOX/GO@Ag), then GO@Ag-DOX was functionalized by DSPE-PEG2000-NGR, giving GO@Ag-DOX with active tumor-targeting capacity and excellent stability in physiological solutions. The release profiles of DOX from GO@Ag-DOX-NGR showed strong dependences on near-infrared (NIR) laser and the SPR effect of Ag nanoparticles. Compared with free DOX in an in vivo murine tumor model, GO@Ag-DOX-NGR afforded much higher antitumor efficacy without obvious toxic effects to normal organs owing to 8.4-fold higher DOX uptake of tumor and 1.7fold higher DOX released in tumor with NIR laser than the other tissues. Besides, in this work, GO@Ag-DOX-NGR not only served as a powerful tumor diagnostic X-ray contrast agent, but also as a strong agent for photothermal ablation of tumor, the ability of GO@Ag-DOX-NGR nanoparticles to combine the local specific chemotherapy with external photothermal therapy (PTT) significantly improved the therapeutic efficacy. GO@Ag-DOX-NGR showed excellent chem-photothermal therapeutic efficacy, tumor-targeting property, NIR laser-controlled drug releasing function and X-ray imaging ability, demonstrating that there is a great potential of GO@Ag-DOX-NGR for cancer diagnosis and therapy. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Controlled drug release Tumor-targeting GO@Ag X-ray imaging
1. Introduction A holy grail in cancer therapy is to deliver high dose of drug to tumor sites for maximum treatment efficacy while minimizing side effects to normal organs [1,2]. Nanostructured materials on systemic injection can accumulate in tumor tissues by escaping through abnormally leaky tumor blood vessels [2e6], making them useful for drug delivery applications. Nanostructured materials sensitive to certain physiological variables or external physicochemical stimuli, often referred as “intelligent” or “smart” materials, can be used for building controlled drug release systems [7,8], and the abilities to control drug dosing in terms of quantity, location, and time are the key goals for drug delivery science. The controlled drug systems responsive to a stimulus such as temperature, pH, applied magnetic or electrical field, ultrasound, light, or
* Corresponding author. Tel.: þ86 371 67781910; fax: þ86 371 67781908. E-mail address: [email protected] (Z. Zhang). http://dx.doi.org/10.1016/j.biomaterials.2014.03.042 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.
enzymatic action have been proposed as triggered delivery systems [9]. Light-responsiveness is receiving in creasing attention owing to the possibility of developing materials sensitive to electromagnetic radiation or light (mainly in the UV and visible range), which can be applied on demand at well delimited sites of the body [10e12]. The photo-regulated release of drug has been reported widely, however complicated preparation for drug carriers, the traditional utilization of ultraviolet radiation and the poor tissue penetration capability of visible light limit its applications [13,14]. Most intrinsic tissue chromophores, including oxyhemoglobin, deoxyhemoglobin and melanin, have relatively weak absorbance in the near infrared (NIR) spectral range (700e900 nm) [15] and this weak absorption makes the NIR light have the capability of deep tissue penetration depth, making it optimal for biomedical applications that utilize light [16]. The interaction of NIR light with noble metal nanoparticles results in collective oscillations of the free electrons in the metal known as localized surface plasmons resonances (SPR) [17]. Recently, NIR light-response drug delivery system based on SPR of the noble metal nanoparticles (AuNPs and
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AgNPs) have been received in creasing attentions [11,18,19] for its novel capacity of controlling drug release. In this study, AgNPs were used for controlling drug release in deep tissues with NIR light. Some problems of AuNPs or AgNPs based drug delivery systems still unresolved. Such as the drug loading content is still low because of the small specific surface area and weak adsorption capacity to small molecular drugs of AuNPs [14,20]. Graphene oxide (GO), a one-atom-thick two-dimensional (2D) layer of sp2-bonded carbon with a large amount of carboxyl and hydroxyl functionalities, extraordinary electrical, thermal, mechanical, structural properties [21e24], and low systemic toxicity [25], having shown tremendous promise in target-specific delivery of drugs in the body. Recently, great efforts have also been devoted to explore potential applications of GO in biomedicine [26e28]. A large number of groups have utilized functionalized nanoscale graphene oxide as nanocarriers for drug and gene delivery [29e31]. In order to achieved a high drug loading content in this study, GO was used as a antitumor drug platform. Angiogenic tumor vessels are important elements for tumor growth and metastasis, and extensive laboratory data suggest that angiogenesis plays an essential role in breast cancer development, invasion and metastasis [32,33]. Aminopeptidase N (APN)/CD13 isoform, the tumor vascular antigen [34,35], is selectively overexpressed in tumor vasculature and certain tumor cells, it plays multiple functions as a regulator of hormones and cytokines, protein degradation, antigen presentation, cell proliferation, cell migration and angiogenesis [36]. According to the report, the AsnGly-Arg (NGR) peptide motif can selectively recognize CD13 isoform and has been used as a potent tumor-targeting ligand for target drugs to improve biodistribution [37e40]. NGR has been certified useful for delivering chemotherapeutic drugs, apoptotic peptides and liposomes to tumor vessels [39e41]. Thence NGR was conjugated to GO forming a novel tumor targeting drug carrier. Photothermal therapy (PTT) uses light absorbing agents to convert optical energy into heat, leading to the thermal ablation of cancer cells. In recent years, PTT as a minimally invasive, controllable, and highly efficient treatment method has drawn widespread
attention [42,43]. AgNPs and Ag-based nanomaterials exhibit strong visible to NIR absorbance owing to the surface plasmon resonance (SPR) effect, making them powerful agents in PTT cancer treatment [44,45]. Carbon nanomaterials, such as carbon nanotubes (CNTs), carbon nanohorns, and graphene, are another type of extensively studied PTT agents [46e48]. In this study, AgNPs and GO were simultaneously used as PTT agents for in vitro and in vivo PTT. X-ray computed tomography are widely used for screening and diagnosis of various pathologies including cancer. The ability to visualize deep structures in the body is the main advantage of X-ray imaging [49]. Just like gold, silver also has a higher atomic number and a higher absorption coefficient than standard iodized contrast agents and hence provides greater contrast per unit weight [50]. Therefore a lower concentration of the contrast agent can be used leading to higher sensitivity of the imaging technique. In this study, X-ray imaging was used to observe the accumulation of targeted drug delivery system in the tumor. Despite the exciting recent progresses in the development of controlled release drug delivery system (DDS), decorating GO with noble metal with PTT and X-ray imaging properties for a NIRtriggered DDS has not yet been explored to our best knowledge. Therefore, in this work, a GO@Ag nanocomposite was synthesized by chemical deposition of Ag nanoparticles onto GO through a hydro thermal reaction, and doxorubicin (DOX), one of the most effective drugs against a wide range of cancers, was employed as the model drug and linked to GO@Ag via ester bonds, then GO@AgDOX was functionalized by DSPE-PEG2000-NGR, giving GO@AgDOX with active tumor targeting capacity and excellent stability in physiological solutions. Taking advantages of the NIR-triggered SPR effect and potential in PTT of GO@Ag, a tumor targeted drug delivery system (GO@Ag-DOX-NGR) (Fig. 1) was developed and characterized by transmission electron microscopy (TEM), dynamic laser scattering (DLS), Fourier transmission infrared spectroscopy (FTIR), elemental analyzer (HAADF-STEM-EDS) and X-ray diffractometer (XRD). NIR-triggered DOX release, PTT and tumor targeting efficacy of GO@Ag-DOX-NGR was evaluated using MCF-7 cells and
Fig. 1. Scheme of GO@Ag-DOX-NGR and its biofunctions.
J. Shi et al. / Biomaterials 35 (2014) 5847e5861
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tumor-bearing mice models, furthermore, in vivo X-ray imaging of tumor-bearing mice using GO@Ag-DOX-NGR is also realized. 2. Materials and methods 2.1. Materials Graphene oxide (GO, purity>95％) were purchased from Chengdu Xianfeng Chemicals Co. Ltd. Doxorubicin (DOX, purity>98％) was gotten from Beijing Yi-He Biotech Co. Ltd. NGR peptides (CNGRCK2HK3HK11) were synthesized by Shanghai Botai Biotechnology Co. Ltd. Silver nitrate(AgNO3), p-nitrophenylchloroformate, anhydrous pyridine, catalytic N,N-dimethylaminopyridine and DPM were obtained from SigmaeAldrich Co. LLC. Sulforhodamine B (SRB), DMEM cell culture medium, penicillin, streptomycin, fetal bovine serum (FBS), and heparin sodium were bought from Gibco Invitrogen. Other reagents were acquired from China National Medicine Corporation Ltd. The dialysis bags (MWCO ¼ 10, 00) were from Spectrum Laboratories Inc.
Synthesis of GO@Ag. First, AgNO3 (20 mg) and GO (30 mg) was added to 120 ml of water and sonicated for 1 h to form a homogeneous suspension, then 10 ml of NaOH (4 mol/l) was added dropwise into the above mixture at 70 C for 10 min, the result product (GO@Ag) was washed by centrifugation for several times with deionized (D.I.) water, and dried in vacuum for 12 h. Activation of GO@Ag. GO@Ag (50 mg) was suspended in anhydrous dimethylformamide (DMF) and sonicated for 1 h to form a homogeneous suspension. To the suspension, p-nitrophenylchloroformate (400 mg), anhydrous pyridine (2 ml), and catalytic N,N-dimethylaminopyridine were added, maintaining the temperature at 0 C. The solution was allowed to stir for 48 h, under nitrogen, along with 1 h sonication once every 8 h approximately. Product formation was indicated by increased solubility in DMF. The brown solid was precipitated out by the addition of diethyl ether and washed repeatedly with ether, dichloromethane, and isopropyl alcohol, respectively, and then dried in vacuum for 12 h. Attachment of DOX to GO@Ag. Activated GO@Ag (20 mg) was dissolved in anhydrous DMF and the solution was sonicated under N2 for 30 min. DOX (40 mg) was added at room temperature along with N,N-diisopropylethylamine and stirred for 24 h. After 24 h reaction, the resulting product (GO@Ag-DOX) was purified by washing 3 times with methanol and 3 times with D.I. water through a membrane filter to remove unreacted DOX and other reagents, and dried in vacuum for 12 h. Conjugation of NGR to GO@Ag-DOX. GO@Ag-DOX (50 mg) and DPM (10 mg) were added to ultra-pure water (20 ml), after sonicating for 2 h, a 4000 r/min centrifugation was done to remove big particles, then NGR (CNGRCK2HK3HK11) water solution was added to the GO@Ag-DOX suspension, and DPM with NGR and maleimide in a molar ratio of 1:20 [51]. After being left to stand overnight at room temperature, GO@Ag-DOX-NGR was obtained. The solid was washed with H2O through a membrane filter to remove excess NGR. After freeze-drying, the solid products were dried in vacuum at 30 C for 24 h and stored at 4 C until use. Thin layer silica gel chromatography using n-butanol-water-acetic acid (4:2:1) as the developing agent and ninhydrin solution 0.5% as the coloring agent were used to test for conjugation of NGR to GO@Ag-DOX [51]. 2.3. Characterization DLS (Zetasizer Nano ZS-90, Malvern, UK) and TEM (Tecnai G2 20, FEI) were used for characterizing particle size, zeta potential and morphological of GO@Ag-DOXNGR, respectively. The optical properties of GO@Ag-DOX-NGR were characterized using an ultra-violet-visible (UVeVIS) spectrometer (Lambda 35, PerkineElmer, USA). FT-IR spectra were recorded on a Nicolet iS10 spectrometer (Thermo). The phase and crystallographic structure of the samples were characterized by a X-ray diffractometer (Model: XD-3X, Beijing, China) with Cu Ka X-ray source (l ¼ 0.15406 nm) at a generator voltage 36 kV, a generator current 20 mA with the scanning rate 4 /min1. The in vivo X-ray images were conducted on a clinical X-ray scanner (SIEMENS). 2.4. Evaluation of NIR light sensitivity GO@Ag-DOX-NGR samples were suspended in 1 ml of water and sealed in dialysis membranes (MW cutoff 1000, Spectrapor). The dialysis bags were incubated in 10 ml PBS buffer at room temperature with gentle shaking. After NIR laser irradiating (808 nm laser, 1 W/cm2 or 2 W/cm2), A 200 ml portion of the aliquot was collected from the incubation medium at predetermined time intervals, and the released DOX was quantified by absorption spectroscopy recorded on UVevis spectrophotometer at 490 nm. The DOX release studies were performed in triplicate for each of the samples. 2.5. Photothermal effect of NIR on GO@Ag For photothermal therapy, a NIR laser (808 nm, 2 W/cm2) was used. Different samples of GO@Ag suspensions in water (0.2 ml) at 10 mg/ml were illuminated with a 808 nm, continuous-wave NIR with the power density of 2 W/cm2. The
Fig. 2. A schematic illustration of GO@Ag-DOX-NGR nanocomposite preparation.
2.2. Synthesis of GO@Ag-DOX-NGR
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Fig. 3. Characterization of GO@Ag A) UVeVIS spectrum of GO and GO@Ag; B) X-ray diffraction pattern of GO and GO@Ag; C) TEM images of a) GO, b) GO@Ag, c) HRTEM image of a nanoparticle on GO@Ag nanocomposite and d) Ag nanoparticles.
temperature was measured by a thermometer (HT-8878, ZhengZhou JinYangGuang Instrument Co. Ltd.).
2.6. Cellular experiments 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 DMEM 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. Cellular uptake. Intracellular uptake of GO@Ag-DOX and GO@Ag-DOX-NGR were performed with MCF-7 cells. MCF-7 cells were seeded at 5 104 cells per well on glass cover slips in 6-well plates. When cells reached 70% confluence, they were treated with GO@Ag-DOX (DOX concentration: 10 mg/ml and GO@Ag concentration: 12.2 mg/ml) and GO@Ag-DOX-NGR (DOX concentration: 10 mg/ml and GO@Ag-NGR concentration: 12.2 mg/ml) for 1 and 3 h, respectively. Cell nuclei were stained with DAPI for 30 min. After washing three times with PBS, the cells were imaged by a laser confocal microscope (Olympus FV1100, Japan). Cytotoxicity and phototoxicity assay. The cytotoxicity of GO@Ag-DOX-NGR against MCF-7 cells was assessed by using the standard SRB assay. The MCF-7 cells were cultured and lifted as described above before seeding (1 104) into 96well plates and incubating for 24 h. The medium then was replaced with fresh medium containing various concentrations of free GO, GO@Ag, GO@Ag-NGR, DOX, GO@Ag-DOX and GO@Ag-DOX-NGR for 24 h, the cells were or were not irradiated with 808 nm NIR laser (Changchun laser research center) with the power density of 2 W/cm2 for 3 min. NIR laser controlled DOX release in vitro. For the NIR sensitivity release in vitro, GO@Ag-DOX-NGR (DOX: 10 mg/ml, GO@Ag-NGR: 12.2 mg/ml) was incubated with MCF-7 cells for 2 h, after incubation, cells were washed twice with PBS, and then a NIR laser (808 nm, 2 W/cm2, 3 min) was delivered to the center of the culture dish. After irradiation, the cellular uptake and apoptosis of the MCF-7 cells were acquired using a Fluorescence Microscope (Zeiss LSM 510).
2.7. In vivo experiments 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 saline into the right shoulder of female BALB/c mice (18e20 g, Henan laboratory animal center). The mice were used when the tumor volume reached 60e100 mm3 (w6 days after tumor inoculation).
Biodistribution studies. 0.2 ml of DOX (5 mg/kg), GO@Ag-DOX (DOX: 5 mg/kg, GO@Ag: 6.1 mg/kg) and GO@Ag-DOX-NGR (DOX: 5 mg/kg, GO@Ag-NGR: 6.1 mg/kg) were intravenous injected into tumor-bearing mice (3 mice per group). After injection for different time points, the mice were killed to collect heart, liver, spleen, lung, kidney and tumor, then the collected tissues were made into frozen sections, and imaged by a Fluorescence Microscope (Zeiss LSM 510). In vivo antitumor effect. For the in vivo antitumor experiments, the tumorbearing mice were divided into eight groups (six mice per group), minimizing the differences of weights and tumor sizes in each group. The mice were administered with (1) saline (0.1 ml), (2) control/NIR (808 nm laser, 2 W/cm2, 3 min), (3) GO@AgNGR (6.1 mg/kg), (4) GO@Ag-NGR/NIR (6.1 mg/kg, 808 nm laser, 2 W/cm2, 3 min), (5) DOX (5 mg/kg), (6) GO@Ag-DOX (DOX: 5 mg/kg, GO@Ag: 6.1 mg/kg), (7) GO@AgDOX-NGR (DOX: 5 mg/kg, GO@Ag-NGR: 6.1 mg/kg) and (8) GO@Ag-DOX-NGR/NIR (DOX: 5 mg/kg, GO@Ag-NGR: 6.1 mg/kg, 808 nm laser, 2 W/cm2, 3 min) were intravenous injected into mice via the tail vein every 2 days, respectively, the NIR groups were exposed to 808 nm laser at 3 h post-injection. 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 ¼ (tumor length) (tumor width)2/2. After treatment for 14 days, the mice were killed to collect tumor tissue for H&E staining. Morphological changes were observed and under microscope (Zeiss LSM 510). NIR laser controlled DOX release in vivo. For the NIR sensitivity release in vivo, 0.2 ml of GO@Ag-DOX-NGR (DOX: 5 mg/kg, GO@Ag-NGR: 6.1 mg/kg) were intravenous injected into tumor-bearing mice (6 mice per group) and tumors were exposed to 808 nm NIR laser for 1, 2 and 3 min at 3 h post-injection. After NIR irradiating, the mice were killed and tumors were collected, weighed, and homogenized in buffer (methanol to saline ratio, 1:1). DOX in tumors were determined by high performance liquid chromatography (HPLC, 1100 Agilent, USA) under the following chromatographic conditions: an Eclipse XDB-C18 column (150 mm 4.6 mm, 5.0 mm); mobile phase sodium acetate solution (0.02 mol/L)/ acetonitrile 80:20; column temperature 40 C; fluorescence detector with the excitation and emission wavelengths set at 475 nm and 525 nm, respectively; flowrate 1.0 mL/min; and injection volume 20 mL. In vivo X-ray imaging. For in vivo X-ray imaging, the tumor-bearing mice were intravenously injected with GO@Ag-DOX-NGR (DOX: 20 mg/kg, GO@Ag-NGR: 24.4 mg/kg), after injection for 3 h, X-ray imaging was conducted on a clinical Xray scanner. Statistical analysis Quantitative data are expressed as mean SD and analyzed by use of Student’s t test. P values
Contents lists available at ScienceDirect
Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
A tumor-targeting near-infrared laser-triggered drug delivery system based on GO@Ag nanoparticles for chemo-photothermal therapy and X-ray imaging Jinjin Shi, Lei Wang, Jing Zhang, Rou Ma, Jun Gao, Yan Liu, Chaofeng Zhang, Zhenzhong Zhang* School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou 450001, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 February 2014 Accepted 10 March 2014 Available online 17 April 2014
In this study, a GO@Ag nanocomposite was synthesized by chemical deposition of Ag nanoparticles onto graphene oxide (GO) through a hydro thermal reaction, and doxorubicin (DOX), one of the most effective drugs against a wide range of cancers, was employed as the model drug and linked to GO@Ag via ester bonds with a very high drug loading efficiency (w82.0%, weight ratio of DOX/GO@Ag), then GO@Ag-DOX was functionalized by DSPE-PEG2000-NGR, giving GO@Ag-DOX with active tumor-targeting capacity and excellent stability in physiological solutions. The release profiles of DOX from GO@Ag-DOX-NGR showed strong dependences on near-infrared (NIR) laser and the SPR effect of Ag nanoparticles. Compared with free DOX in an in vivo murine tumor model, GO@Ag-DOX-NGR afforded much higher antitumor efficacy without obvious toxic effects to normal organs owing to 8.4-fold higher DOX uptake of tumor and 1.7fold higher DOX released in tumor with NIR laser than the other tissues. Besides, in this work, GO@Ag-DOX-NGR not only served as a powerful tumor diagnostic X-ray contrast agent, but also as a strong agent for photothermal ablation of tumor, the ability of GO@Ag-DOX-NGR nanoparticles to combine the local specific chemotherapy with external photothermal therapy (PTT) significantly improved the therapeutic efficacy. GO@Ag-DOX-NGR showed excellent chem-photothermal therapeutic efficacy, tumor-targeting property, NIR laser-controlled drug releasing function and X-ray imaging ability, demonstrating that there is a great potential of GO@Ag-DOX-NGR for cancer diagnosis and therapy. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Controlled drug release Tumor-targeting GO@Ag X-ray imaging
1. Introduction A holy grail in cancer therapy is to deliver high dose of drug to tumor sites for maximum treatment efficacy while minimizing side effects to normal organs [1,2]. Nanostructured materials on systemic injection can accumulate in tumor tissues by escaping through abnormally leaky tumor blood vessels [2e6], making them useful for drug delivery applications. Nanostructured materials sensitive to certain physiological variables or external physicochemical stimuli, often referred as “intelligent” or “smart” materials, can be used for building controlled drug release systems [7,8], and the abilities to control drug dosing in terms of quantity, location, and time are the key goals for drug delivery science. The controlled drug systems responsive to a stimulus such as temperature, pH, applied magnetic or electrical field, ultrasound, light, or
* Corresponding author. Tel.: þ86 371 67781910; fax: þ86 371 67781908. E-mail address: [email protected] (Z. Zhang). http://dx.doi.org/10.1016/j.biomaterials.2014.03.042 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.
enzymatic action have been proposed as triggered delivery systems [9]. Light-responsiveness is receiving in creasing attention owing to the possibility of developing materials sensitive to electromagnetic radiation or light (mainly in the UV and visible range), which can be applied on demand at well delimited sites of the body [10e12]. The photo-regulated release of drug has been reported widely, however complicated preparation for drug carriers, the traditional utilization of ultraviolet radiation and the poor tissue penetration capability of visible light limit its applications [13,14]. Most intrinsic tissue chromophores, including oxyhemoglobin, deoxyhemoglobin and melanin, have relatively weak absorbance in the near infrared (NIR) spectral range (700e900 nm) [15] and this weak absorption makes the NIR light have the capability of deep tissue penetration depth, making it optimal for biomedical applications that utilize light [16]. The interaction of NIR light with noble metal nanoparticles results in collective oscillations of the free electrons in the metal known as localized surface plasmons resonances (SPR) [17]. Recently, NIR light-response drug delivery system based on SPR of the noble metal nanoparticles (AuNPs and
5848
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AgNPs) have been received in creasing attentions [11,18,19] for its novel capacity of controlling drug release. In this study, AgNPs were used for controlling drug release in deep tissues with NIR light. Some problems of AuNPs or AgNPs based drug delivery systems still unresolved. Such as the drug loading content is still low because of the small specific surface area and weak adsorption capacity to small molecular drugs of AuNPs [14,20]. Graphene oxide (GO), a one-atom-thick two-dimensional (2D) layer of sp2-bonded carbon with a large amount of carboxyl and hydroxyl functionalities, extraordinary electrical, thermal, mechanical, structural properties [21e24], and low systemic toxicity [25], having shown tremendous promise in target-specific delivery of drugs in the body. Recently, great efforts have also been devoted to explore potential applications of GO in biomedicine [26e28]. A large number of groups have utilized functionalized nanoscale graphene oxide as nanocarriers for drug and gene delivery [29e31]. In order to achieved a high drug loading content in this study, GO was used as a antitumor drug platform. Angiogenic tumor vessels are important elements for tumor growth and metastasis, and extensive laboratory data suggest that angiogenesis plays an essential role in breast cancer development, invasion and metastasis [32,33]. Aminopeptidase N (APN)/CD13 isoform, the tumor vascular antigen [34,35], is selectively overexpressed in tumor vasculature and certain tumor cells, it plays multiple functions as a regulator of hormones and cytokines, protein degradation, antigen presentation, cell proliferation, cell migration and angiogenesis [36]. According to the report, the AsnGly-Arg (NGR) peptide motif can selectively recognize CD13 isoform and has been used as a potent tumor-targeting ligand for target drugs to improve biodistribution [37e40]. NGR has been certified useful for delivering chemotherapeutic drugs, apoptotic peptides and liposomes to tumor vessels [39e41]. Thence NGR was conjugated to GO forming a novel tumor targeting drug carrier. Photothermal therapy (PTT) uses light absorbing agents to convert optical energy into heat, leading to the thermal ablation of cancer cells. In recent years, PTT as a minimally invasive, controllable, and highly efficient treatment method has drawn widespread
attention [42,43]. AgNPs and Ag-based nanomaterials exhibit strong visible to NIR absorbance owing to the surface plasmon resonance (SPR) effect, making them powerful agents in PTT cancer treatment [44,45]. Carbon nanomaterials, such as carbon nanotubes (CNTs), carbon nanohorns, and graphene, are another type of extensively studied PTT agents [46e48]. In this study, AgNPs and GO were simultaneously used as PTT agents for in vitro and in vivo PTT. X-ray computed tomography are widely used for screening and diagnosis of various pathologies including cancer. The ability to visualize deep structures in the body is the main advantage of X-ray imaging [49]. Just like gold, silver also has a higher atomic number and a higher absorption coefficient than standard iodized contrast agents and hence provides greater contrast per unit weight [50]. Therefore a lower concentration of the contrast agent can be used leading to higher sensitivity of the imaging technique. In this study, X-ray imaging was used to observe the accumulation of targeted drug delivery system in the tumor. Despite the exciting recent progresses in the development of controlled release drug delivery system (DDS), decorating GO with noble metal with PTT and X-ray imaging properties for a NIRtriggered DDS has not yet been explored to our best knowledge. Therefore, in this work, a GO@Ag nanocomposite was synthesized by chemical deposition of Ag nanoparticles onto GO through a hydro thermal reaction, and doxorubicin (DOX), one of the most effective drugs against a wide range of cancers, was employed as the model drug and linked to GO@Ag via ester bonds, then GO@AgDOX was functionalized by DSPE-PEG2000-NGR, giving GO@AgDOX with active tumor targeting capacity and excellent stability in physiological solutions. Taking advantages of the NIR-triggered SPR effect and potential in PTT of GO@Ag, a tumor targeted drug delivery system (GO@Ag-DOX-NGR) (Fig. 1) was developed and characterized by transmission electron microscopy (TEM), dynamic laser scattering (DLS), Fourier transmission infrared spectroscopy (FTIR), elemental analyzer (HAADF-STEM-EDS) and X-ray diffractometer (XRD). NIR-triggered DOX release, PTT and tumor targeting efficacy of GO@Ag-DOX-NGR was evaluated using MCF-7 cells and
Fig. 1. Scheme of GO@Ag-DOX-NGR and its biofunctions.
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tumor-bearing mice models, furthermore, in vivo X-ray imaging of tumor-bearing mice using GO@Ag-DOX-NGR is also realized. 2. Materials and methods 2.1. Materials Graphene oxide (GO, purity>95％) were purchased from Chengdu Xianfeng Chemicals Co. Ltd. Doxorubicin (DOX, purity>98％) was gotten from Beijing Yi-He Biotech Co. Ltd. NGR peptides (CNGRCK2HK3HK11) were synthesized by Shanghai Botai Biotechnology Co. Ltd. Silver nitrate(AgNO3), p-nitrophenylchloroformate, anhydrous pyridine, catalytic N,N-dimethylaminopyridine and DPM were obtained from SigmaeAldrich Co. LLC. Sulforhodamine B (SRB), DMEM cell culture medium, penicillin, streptomycin, fetal bovine serum (FBS), and heparin sodium were bought from Gibco Invitrogen. Other reagents were acquired from China National Medicine Corporation Ltd. The dialysis bags (MWCO ¼ 10, 00) were from Spectrum Laboratories Inc.
Synthesis of GO@Ag. First, AgNO3 (20 mg) and GO (30 mg) was added to 120 ml of water and sonicated for 1 h to form a homogeneous suspension, then 10 ml of NaOH (4 mol/l) was added dropwise into the above mixture at 70 C for 10 min, the result product (GO@Ag) was washed by centrifugation for several times with deionized (D.I.) water, and dried in vacuum for 12 h. Activation of GO@Ag. GO@Ag (50 mg) was suspended in anhydrous dimethylformamide (DMF) and sonicated for 1 h to form a homogeneous suspension. To the suspension, p-nitrophenylchloroformate (400 mg), anhydrous pyridine (2 ml), and catalytic N,N-dimethylaminopyridine were added, maintaining the temperature at 0 C. The solution was allowed to stir for 48 h, under nitrogen, along with 1 h sonication once every 8 h approximately. Product formation was indicated by increased solubility in DMF. The brown solid was precipitated out by the addition of diethyl ether and washed repeatedly with ether, dichloromethane, and isopropyl alcohol, respectively, and then dried in vacuum for 12 h. Attachment of DOX to GO@Ag. Activated GO@Ag (20 mg) was dissolved in anhydrous DMF and the solution was sonicated under N2 for 30 min. DOX (40 mg) was added at room temperature along with N,N-diisopropylethylamine and stirred for 24 h. After 24 h reaction, the resulting product (GO@Ag-DOX) was purified by washing 3 times with methanol and 3 times with D.I. water through a membrane filter to remove unreacted DOX and other reagents, and dried in vacuum for 12 h. Conjugation of NGR to GO@Ag-DOX. GO@Ag-DOX (50 mg) and DPM (10 mg) were added to ultra-pure water (20 ml), after sonicating for 2 h, a 4000 r/min centrifugation was done to remove big particles, then NGR (CNGRCK2HK3HK11) water solution was added to the GO@Ag-DOX suspension, and DPM with NGR and maleimide in a molar ratio of 1:20 [51]. After being left to stand overnight at room temperature, GO@Ag-DOX-NGR was obtained. The solid was washed with H2O through a membrane filter to remove excess NGR. After freeze-drying, the solid products were dried in vacuum at 30 C for 24 h and stored at 4 C until use. Thin layer silica gel chromatography using n-butanol-water-acetic acid (4:2:1) as the developing agent and ninhydrin solution 0.5% as the coloring agent were used to test for conjugation of NGR to GO@Ag-DOX [51]. 2.3. Characterization DLS (Zetasizer Nano ZS-90, Malvern, UK) and TEM (Tecnai G2 20, FEI) were used for characterizing particle size, zeta potential and morphological of GO@Ag-DOXNGR, respectively. The optical properties of GO@Ag-DOX-NGR were characterized using an ultra-violet-visible (UVeVIS) spectrometer (Lambda 35, PerkineElmer, USA). FT-IR spectra were recorded on a Nicolet iS10 spectrometer (Thermo). The phase and crystallographic structure of the samples were characterized by a X-ray diffractometer (Model: XD-3X, Beijing, China) with Cu Ka X-ray source (l ¼ 0.15406 nm) at a generator voltage 36 kV, a generator current 20 mA with the scanning rate 4 /min1. The in vivo X-ray images were conducted on a clinical X-ray scanner (SIEMENS). 2.4. Evaluation of NIR light sensitivity GO@Ag-DOX-NGR samples were suspended in 1 ml of water and sealed in dialysis membranes (MW cutoff 1000, Spectrapor). The dialysis bags were incubated in 10 ml PBS buffer at room temperature with gentle shaking. After NIR laser irradiating (808 nm laser, 1 W/cm2 or 2 W/cm2), A 200 ml portion of the aliquot was collected from the incubation medium at predetermined time intervals, and the released DOX was quantified by absorption spectroscopy recorded on UVevis spectrophotometer at 490 nm. The DOX release studies were performed in triplicate for each of the samples. 2.5. Photothermal effect of NIR on GO@Ag For photothermal therapy, a NIR laser (808 nm, 2 W/cm2) was used. Different samples of GO@Ag suspensions in water (0.2 ml) at 10 mg/ml were illuminated with a 808 nm, continuous-wave NIR with the power density of 2 W/cm2. The
Fig. 2. A schematic illustration of GO@Ag-DOX-NGR nanocomposite preparation.
2.2. Synthesis of GO@Ag-DOX-NGR
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Fig. 3. Characterization of GO@Ag A) UVeVIS spectrum of GO and GO@Ag; B) X-ray diffraction pattern of GO and GO@Ag; C) TEM images of a) GO, b) GO@Ag, c) HRTEM image of a nanoparticle on GO@Ag nanocomposite and d) Ag nanoparticles.
temperature was measured by a thermometer (HT-8878, ZhengZhou JinYangGuang Instrument Co. Ltd.).
2.6. Cellular experiments 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 DMEM 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. Cellular uptake. Intracellular uptake of GO@Ag-DOX and GO@Ag-DOX-NGR were performed with MCF-7 cells. MCF-7 cells were seeded at 5 104 cells per well on glass cover slips in 6-well plates. When cells reached 70% confluence, they were treated with GO@Ag-DOX (DOX concentration: 10 mg/ml and GO@Ag concentration: 12.2 mg/ml) and GO@Ag-DOX-NGR (DOX concentration: 10 mg/ml and GO@Ag-NGR concentration: 12.2 mg/ml) for 1 and 3 h, respectively. Cell nuclei were stained with DAPI for 30 min. After washing three times with PBS, the cells were imaged by a laser confocal microscope (Olympus FV1100, Japan). Cytotoxicity and phototoxicity assay. The cytotoxicity of GO@Ag-DOX-NGR against MCF-7 cells was assessed by using the standard SRB assay. The MCF-7 cells were cultured and lifted as described above before seeding (1 104) into 96well plates and incubating for 24 h. The medium then was replaced with fresh medium containing various concentrations of free GO, GO@Ag, GO@Ag-NGR, DOX, GO@Ag-DOX and GO@Ag-DOX-NGR for 24 h, the cells were or were not irradiated with 808 nm NIR laser (Changchun laser research center) with the power density of 2 W/cm2 for 3 min. NIR laser controlled DOX release in vitro. For the NIR sensitivity release in vitro, GO@Ag-DOX-NGR (DOX: 10 mg/ml, GO@Ag-NGR: 12.2 mg/ml) was incubated with MCF-7 cells for 2 h, after incubation, cells were washed twice with PBS, and then a NIR laser (808 nm, 2 W/cm2, 3 min) was delivered to the center of the culture dish. After irradiation, the cellular uptake and apoptosis of the MCF-7 cells were acquired using a Fluorescence Microscope (Zeiss LSM 510).
2.7. In vivo experiments 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 saline into the right shoulder of female BALB/c mice (18e20 g, Henan laboratory animal center). The mice were used when the tumor volume reached 60e100 mm3 (w6 days after tumor inoculation).
Biodistribution studies. 0.2 ml of DOX (5 mg/kg), GO@Ag-DOX (DOX: 5 mg/kg, GO@Ag: 6.1 mg/kg) and GO@Ag-DOX-NGR (DOX: 5 mg/kg, GO@Ag-NGR: 6.1 mg/kg) were intravenous injected into tumor-bearing mice (3 mice per group). After injection for different time points, the mice were killed to collect heart, liver, spleen, lung, kidney and tumor, then the collected tissues were made into frozen sections, and imaged by a Fluorescence Microscope (Zeiss LSM 510). In vivo antitumor effect. For the in vivo antitumor experiments, the tumorbearing mice were divided into eight groups (six mice per group), minimizing the differences of weights and tumor sizes in each group. The mice were administered with (1) saline (0.1 ml), (2) control/NIR (808 nm laser, 2 W/cm2, 3 min), (3) GO@AgNGR (6.1 mg/kg), (4) GO@Ag-NGR/NIR (6.1 mg/kg, 808 nm laser, 2 W/cm2, 3 min), (5) DOX (5 mg/kg), (6) GO@Ag-DOX (DOX: 5 mg/kg, GO@Ag: 6.1 mg/kg), (7) GO@AgDOX-NGR (DOX: 5 mg/kg, GO@Ag-NGR: 6.1 mg/kg) and (8) GO@Ag-DOX-NGR/NIR (DOX: 5 mg/kg, GO@Ag-NGR: 6.1 mg/kg, 808 nm laser, 2 W/cm2, 3 min) were intravenous injected into mice via the tail vein every 2 days, respectively, the NIR groups were exposed to 808 nm laser at 3 h post-injection. 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 ¼ (tumor length) (tumor width)2/2. After treatment for 14 days, the mice were killed to collect tumor tissue for H&E staining. Morphological changes were observed and under microscope (Zeiss LSM 510). NIR laser controlled DOX release in vivo. For the NIR sensitivity release in vivo, 0.2 ml of GO@Ag-DOX-NGR (DOX: 5 mg/kg, GO@Ag-NGR: 6.1 mg/kg) were intravenous injected into tumor-bearing mice (6 mice per group) and tumors were exposed to 808 nm NIR laser for 1, 2 and 3 min at 3 h post-injection. After NIR irradiating, the mice were killed and tumors were collected, weighed, and homogenized in buffer (methanol to saline ratio, 1:1). DOX in tumors were determined by high performance liquid chromatography (HPLC, 1100 Agilent, USA) under the following chromatographic conditions: an Eclipse XDB-C18 column (150 mm 4.6 mm, 5.0 mm); mobile phase sodium acetate solution (0.02 mol/L)/ acetonitrile 80:20; column temperature 40 C; fluorescence detector with the excitation and emission wavelengths set at 475 nm and 525 nm, respectively; flowrate 1.0 mL/min; and injection volume 20 mL. In vivo X-ray imaging. For in vivo X-ray imaging, the tumor-bearing mice were intravenously injected with GO@Ag-DOX-NGR (DOX: 20 mg/kg, GO@Ag-NGR: 24.4 mg/kg), after injection for 3 h, X-ray imaging was conducted on a clinical Xray scanner. Statistical analysis Quantitative data are expressed as mean SD and analyzed by use of Student’s t test. P values
