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Gadolinium-functionalized nanographene oxide for combined drug and microRNA delivery and magnetic resonance imaging
Biomaterials 35 (2014) 6534e6542
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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Gadolinium-functionalized nanographene oxide for combined drug and microRNA delivery and magnetic resonance imaging Hung-Wei Yang a,1, Chiung-Yin Huang b,1, Chih-Wen Lin a,1, Hao-Li Liu c, Chia-Wen Huang b, Shih-Sheng Liao a, Pin-Yuan Chen b, d, Yu-Jen Lu b, Kuo-Chen Wei b, d, **, Chen-Chi M. Ma a, * a
Department of Chemical Engineering, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsin-chu 30013, Taiwan, ROC Department of Neurosurgery, Chang Gung Memorial Hospital, Linkou, 5 Fu-Shing Road, Kuei-Shan, Tao-Yuan 33305, Taiwan, ROC Department of Electrical Engineering, Chang Gung University, 259 Wen-Hwa 1st Road, Kuei-Shan, Tao-Yuan 33302, Taiwan, ROC d School of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kuei-Shan, Tao-Yuan 33302, Taiwan, ROC b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 March 2014 Accepted 12 April 2014 Available online 6 May 2014
The delivery of anti-cancer therapeutics to tumors at clinically effective concentrations, while avoiding nonspecific toxicity, remains a major challenge for cancer treatment. Here we present nanoparticles of poly(amidoamine) dendrimer-grafted gadolinium-functionalized nanographene oxide (Gd-NGO) as effective carriers to deliver both chemotherapeutic drugs and highly specific gene-targeting agents such as microRNAs (miRNAs) to cancer cells. The positively charged surface of Gd-NGO was capable of simultaneous adsorption of the anti-cancer drug epirubicin (EPI) and interaction with negatively charged Let-7g miRNA. Using human glioblastoma (U87) cells as a model, we found that this conjugate of Let-7g and EPI (Gd-NGO/Let-7g/EPI) not only exhibited considerably higher transfection efficiency, but also induced better inhibition of cancer cell growth than Gd-NGO/Let-7g or Gd-NGO/EPI. The concentration of Gd-NGO/Let-7g/EPI required for 50% inhibition of cellular growth (IC50) was significantly reduced (to the equivalent of 1.3 mg/mL EPI) compared to Gd-NGO/EPI (3.4 mg/mL EPI). In addition, Gd-NGO/Let-7g/EPI could be used as a contrast agent for magnetic resonance imaging to identify the location and extent of bloodebrain barrier opening and quantitate drug delivery to tumor tissues. These results suggest that Gd-NGO/Let-7g/EPI may be a promising non-viral vector for chemogene therapy and molecular imaging diagnosis in future clinical applications. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Nanographene oxide Dendrimer Drug delivery miRNA delivery MR imaging
1. Introduction Gene therapy could potentially be used for cancer treatment by introduction of a tumor-suppressor gene into the target area to inhibit tumor growth. However, the safe and efficient delivery of genes into cells has remained a major challenge to clinical applications of gene therapy [1]. Many powerful gene delivery tools have been developed, including viruses, liposomes, polymer nanoparticles, carbon nanomaterials, and external physical forces [2e4].
* Corresponding author. Department of Chemical Engineering, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsin-chu 30013, Taiwan, ROC. Tel.: þ886 3 5713058; fax: þ886 3 5715408. ** Corresponding author. Department of Neurosurgery, Chang Gung Memorial Hospital, Linkou, 5 Fu-Shing Road, Kuei-Shan, Tao-Yuan 33305, Taiwan, ROC. Tel.: þ886 3 3281200x2412; fax: þ886 3 3285818. E-mail addresses: [email protected] (K.-C. Wei), [email protected] (C.-C.M. Ma). 1 Contributed equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2014.04.057 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.
One of the most promising candidates for targeting specific genes is microRNA (miRNA). miRNAs are a class of endogenous, noncoding small RNA molecules that participate in gene regulation and various physiological processes. Altered miRNA expression has been detected in human cancers: tumor suppressive miRNAs are downregulated while oncogenic miRNAs are upregulated. Nine members of the Let-7 miRNA family have been detected in human tissue. Let-7 miRNAs function as tumor suppressors by decreasing expression of the Ras oncogene family [5,6]. Downregulation of Let-7 has been observed in lung cancer, colon cancer, and melanoma and is correlated with poor survival [7e9]. Conversely, Ras proteins (K-Ras, N-Ras and H-Ras) are known to be upregulated in these cancers. Overexpression of Let-7 has also been shown to inhibit cancer cell growth [11]. In human brain tumor cells, overexpressed Let-7 miRNA leads to a decrease in Ras proteins, inhibition of cell proliferation and reduced tumor growth, without harming normal astrocytes [10]. This selective growth inhibition of brain tumor cells suggests Let-7 miRNA as an attractive candidate for gene-targeting therapy to treat malignant brain
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tumors. The development of a safe and effective method for delivery of these miRNAs to cancer cells has therefore emerged as a critical goal of current cancer therapeutic research. Nanomaterials have been attracting increasing interest for their potential use as tumor-selective drug delivery vehicles. Tumor vessels are usually leaky, with large endothelial interstitial gap junctions of about 100e600 nm [12]. Nanomaterials can reach tumors by the enhanced permeability and retention (EPR) effect [13]. Such systems are controlled by a number of parameters including pH, redox environment, light, heat, ultrasound, and magnetic and electrical fields [2,14e16]. Functionalized nanoscale graphene oxide (NGO) is an example of a nanomaterial that is capable of delivering oligonucleotides into cells and protecting them from enzymatic cleavage [17]. In addition, pep stacking between anti-cancer drugs and NGO allows high-efficiency loading and controlled release of anti-cancer drugs [18]. Polyethylene glycol (PEG)-functionalized NGO was initially developed as a nanocarrier and evaluated for its in vitro cellular uptake in the Dai laboratory [19]. Anticancer drugs are loaded into these PEG-NGOs via noncovalent physisorption. Yang et al. investigated the loading and release behaviors of doxorubicin hydrochloride (DOX) on NGO [18]. They found that the weight ratio of loaded drug to NGO carrier could reach 200%. Besides, the NGO also can be used for gene delivery; some cationic polymers such as polyethylenimine (PEI), chitosan, and Polyamidoamine (PAMAM), high molecular weight branched PEI (HMW BPEI) or low molecular weight branched PEI (LMW BPEI) were conjugated to the inorganic materials (i.e. silica, iron oxide, and carbon materials) to enhance cellular uptake and transfection efficiency [20,21]. However, the polymers with high molecular weight would induce cytotoxicity toward the cells [22]. Therefore, we modified the NGO with low molecular weight PAMAM and GdDTPA as drug/gene carriers which can be monitored by MRI to enhance the transfection efficiency with ultralow cytotoxicity. In
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addition to its use for drug and gene delivery, graphene oxide (GO) has also been extensively explored for bioimaging, biosensing, and many other biomedical applications. This versatility results from the excellent physiochemical, optical, and electrical properties of GO [23e26]. In this study, we report the design, synthesis, and evaluation of a new delivery system consisting of PAMAM-dendrimer and gadolinium (Gd)-functionalized NGO (Gd-NGO) loaded with miRNA (Let7g) and Epirubicin (EPI) (Gd-NGO/Let-7g/EPI) and designed for chemo/gene therapy and magnetic resonance imaging (MRI) (Fig. 1). This system could potentially allow safe delivery of other potent and toxic drugs into tumor cells and provide high transfection efficiency for gene delivery (miRNA, siRNA, pDNA) to overcome the therapeutic challenges of some tumors which are currently considered difficult to treat. We suggest that, with these new functionalities, Gd-NGO can be considered important therapeutic and diagnostic targets for the treatment and detection of cancers. 2. Materials and methods 2.1. Materials Graphite platelets (model xGnP; width 100 mm, thickness 5e15 nm) were obtained from XG Sciences Inc. (East Lansing, MI). Acrylic acid, potassium persulfate, sulfuric acid (H2SO4, 98%), sodium sulfate (Na2SO4), potassium permanganate (KMnO4), hydrogen peroxide solution (H2O2), and ammonia were purchased from Showa Chemical Co. (Tokyo, Japan). U87 human glioblastoma cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Hank’s balanced salt solution and RPMI-1640 medium were purchased from Gibco (Grand Island, NY). Fetal bovine serum was purchased from Biological Industries (Beit-Haemek, Israel). Gentamicin, penicillin, and streptomycin were obtained from MDBio. Minimum essential medium was purchased from Invitrogen (Carlsbad, CA). Epirubicin (EPI), glutaraldehyde, 1-ethyl-3-(3dimethylaminepropyl) carbodiimide hydrochloride (EDC), 2-(N-morpholino)ethanesulfonic acid hydrate (MES), sulfanilamide, N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), diethylenetriaminepentaacetic acid gadolinium (III) dihydrogen
Fig. 1. Schematic of the procedure for preparation of Gd-NGO/Let-7g/EPI.
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salt hydrate (Gd-DTPA), ethylenediamine, transfection reagent (Lipofectamine 2000), and poly(amidoamine) (PAMAM) dendrimer (generation 4.0 solution; Mw: 516.68) were purchased from Sigma (St Louis, MO). Synthetic Let-7g (50 -uga ggu agu agu uug uac agu u-30 ), fluorescein amidite (FAM)-labeled Let-7g, and a scrambled control sequence were purchased from MDBio, Inc (Taiwan). All reagents were of analytical grade and were used without any further purification. Deionized (DI) water was used in all experiments.
2.7. EPI and Let-7g release study Gd-NGO/Let-7g/EPI samples were placed in dialysis bags containing 10 mL of phosphate buffered saline (PBS) at pH 7.4 and 6.0, respectively, after adjusting with HCl. Drug release was assumed to start as soon as the dialysis bags were placed into the reservoir. The release reservoir was constantly stirred, and at various time points, the dialysis bags were taken out for characterization. The concentrations of Let-7g and EPI released from Gd-NGO/Let-7g/EPI into the distilled water of the reservoir were quantified using ELISA and HPLC, respectively.
2.2. Preparation of carboxylic nanographene oxide (NGO-COOH) NGO-COOH was prepared by a modified Hummers’ method. Graphite platelets (250 mg), NaNO3 (0.125 g), and H2SO4 (12 mL 98%) were magnetically mixed in a 100 mL flask in an ice bath, followed by slow addition of 0.75 g KMnO4, while keeping the temperature below 5 C. The flask was then heated to 100 C, followed by slow addition of 12 mL DI. The temperature of the solution was increased to 98 C for a 30 min incubation period. A 50 mL volume of 10% H2O2 was added to the solution until the cessation of gas evolution. The solution was centrifuged at 11,000 rpm and washed several times with DI to remove impurities and obtain large-scale GO (LGO). The precipitate was subjected to ultrasonication for 8 h at 800 W using an Ultrasonic Liquid Processor 2020 from Misonix (Farmingdale, NY) and centrifuged at 14,000 rpm for 0.5 h. The supernatant was filtered three times with Acrodisc 25 mm syringe filters (0.2 mm Supor membrane), and the NGO collected in the filtrate was subjected to further modification. NaOH (10 mL, 12 mg/ mL) was added to an aqueous suspension of the NGO (10 mL, 2 mg/mL) followed by sonication at 800 W for 2 h to convert OH groups to COOH, resulting in NGO-COOH. X-ray photoelectron spectroscopy (XPS) was used to investigate the structure, COOH conversion, and functional groups of NGO.
2.8. In vitro gene transfection studies For Western blot analysis, U87 cells were added to 24-well plates at a density of about 1 105 cells per well, 24 h before transfection. Transfection was carried out by adding 150 mL of Gd-NGO, Gd-NGO/Let-7g, or Gd-NGO/Let-7g/EPI into each well. After incubation for 12 h at 37 C, the media containing different transfection complexes were removed. Cells were washed three times with serum-free DMEM medium before addition of 750 mL formal DMEM growth medium containing 10% FBS. An additional 48 h incubation period was needed for efficient reaction of Let-7g inside the cells. Pure Let-7g without any transfection agent was used as a control. Expression of Pan-ras, K-ras, and N-ras proteins were determined by Western blot. For fluorescence microscopy, U87 cells were added to 8-well chamber slides at a density of about 5 103 cells per well, 24 h before transfection. Transfection was carried out by adding 50 mL of Gd-NGO/Let-7g or Gd-NGO/GFP-pDNA to each well. After incubation for 12 h at 37 C, the media containing different transfection complexes were removed. After another 36 h of incubation, cells were washed three times with serum-free DMEM medium. Fluorescence images of cells were captured using a laser scanning confocal fluorescence microscope (Leica TCS SP2, Germany). All images were collected using the same parameters.
2.3. Dendrimer-functionalization and Gd-DTPA conjugation to NGO-COOH Twenty-four milligrams of EDC and 27 mg of sulfo-NHS were dissolved in 2 mL of 0.5 M MES buffer (pH ¼ 6.3) in the dark. A 0.2 mL aliquot of this solution was mixed with 0.2 mL of Gd-DTPA (5 mg/mL) at 25 C and reacted for 60 min in the dark to allow the formation of amide bonds between activated carboxyl groups. The activated Gd-DTPA was reacted with ethylenediamine at 25 C for 3 h in the dark to form amine-functionalized Gd-DTPA. A 0.2 mL aliquot of the EDC/sulfo-NHS solution was mixed with 0.2 mL of NGOCOOH (3 mg/mL) at 25 C and reacted for 30 min in the dark to activate the carboxylic groups of NGO-COOH. Activated NGO-COOH was washed with 0.8 mL 0.1 M MES buffer, resuspended in 0.2 mL of MES buffer, and then mixed with 0.05 mL of amine-functionalized Gd-DTPA (2 mg/mL) and 0.15 mL of dendrimer (2 mg/mL) at 25 C by vortexing for 12 h. The product was then separated from the solution, washed with DI water and centrifuged at 14,000 rpm for 0.5 h to remove both MES buffer and unbound amine-functionalize Gd-DTPA and dendrimer, and finally dispersed in DI water to obtain Gd-conjugated NGO (Gd-NGO). 2.4. MRI phantom studies with Gd-DTPA and Gd-NGO For in vitro measurements, Gd-DTPA and Gd-NGO were diluted with physiological saline to 0.000625, 0.00125, 0.0025, 0.005, 0.01, and 0.02 mM. Circular wells (inner diameter ¼ 5 mm) were filled with 200 mL of contrast agent sample or physiological saline as control and were placed in the MR scanner (Clinscan, Bruker, Germany; 7 T). Spin-lattice relaxivity maps were calculated from two T1-weighted images with different flip angles (gradient recalled echo sequence, TR/ TE ¼ 2.3 ms/0.76 ms, slide thickness ¼ 0.8 mm, matrix ¼ 132 192, and flip angle ¼ 5 /20 ). The correlation between R1 (¼1/T1) mapping and Gd-DTPA concentration was determined [27].
2.9. Western blots Treated U87 glioma cells were washed twice in ice-cold PBS and lysed on ice in ice-cold T-PER tissue protein extraction reagent (Pierce, Rockford, IL, USA) containing protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Lysates were cleared by centrifugation, and total protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA, USA). Protein samples (20 mg/lane) were separated on 12% polyacrylamide gels by SDSePAGE and transferred to polyvinylidene difluoride membranes (Millipore). Blots were blocked overnight in TBS (20 mM TriseHCl, 150 mM NaCl, 0.1% Tween-20, 0.5 mM EDTA, pH 7.4) containing 5% nonfat dry milk. Blots were then incubated for 2 h with primary antibodies (K-Ras 1:1000, Pan-Ras 1:1000, K-Ras 1:500; Santa Cruz), followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG (1:20,000; PerkinElmer) for 1 h. Ras proteins were detected using the Western Lightning kit (PerkinElmer) according to the manufacturer’s instructions. Beta-actin antibody purchased from Cell Signal was used as an internal control. 2.10. In vitro cytotoxicity assay U87 cells were cultured in DMEM supplemented with 2.2 mg/mL sodium carbonate, 10% fetal bovine serum, 50 mg/mL gentamicin, 50 mg/mL penicillin, and 50 mg/mL streptomycin at 37 C and 5% CO2. Approximately 10,000 cells (i.e., 150 mL of a suspension of 6.67 104 cells/mL) were placed in each well of a 96-well culture plate and incubated in a humidified chamber at 37 C and 5% CO2 for 24 h. Different concentrations of samples (50 mL each) were added to the medium, and incubation was continued for 48 h. Before counting, the culture medium was removed, and the cells were incubated in 120 mL of XTT solution for 3 h. Subsequently, 100 mL of the XTT solution was removed from each well and transferred to a 96-well counting dish. The cytotoxicity toward U87 cells in vitro was evaluated by measuring the OD at 490 nm using an ELISA reader.
2.5. Adsorption of Let-7g or GFP-pDNA on Gd-NGO Gd-NGO/Let-7g and Gd-NGO/GFP-pDNA complexes were freshly prepared prior to use. Briefly, 0.1 mL of Gd-NGO aqueous solution (1 mg/mL) was mixed with 0.1 mL of Let-7g (2 nmol/mL) or 0.1 mL of green fluorescent protein-expressing plasmid DNA (GFP-pDNA, 1 mg/mL) by gentle vortexing, and incubated at room temperature for 30 min. Complexes were separated from the solution, washed with DI water, centrifuged at 14,000 rpm for 0.5 h to remove free Let-7g or GFP-pDNA, and finally dispersed in DI water to obtain Gd-NGO/Let-7g or Gd-NGO/GFP-pDNA. 2.6. Drug loading onto Gd-NGO/Let-7g Epirubicin (EPI) loading onto Gd-NGO/Let-7g was accomplished by simply mixing EPI (0.5 mL in 0.5% dendrimer solution, 1 mg/mL) with a solution of Gd-NGO/ Let-7g (0.5 mL, 1 mg/mL) at pH 8 and 4 C overnight. Unbound EPI was removed by repeated washing with DI water. The resulting Gd-NGO/Let-7g/EPI complexes were resuspended and stored at 4 C. The concentration of EPI loaded onto Gd-NGO/Let7g was measured by high-performance liquid chromatography (HPLC) on a SUPELCOSILTM LC-18 column (4.6 250 mm) using an L-2130 pump and an L-2400 UV-detector (Hitachi). The mobile phase of the HPLC was a 50/15/35 (v/v/v) mixture of DI water, acetonitrile and methanol with a flow rate of 1.5 mL/min and data were collected at 256 nm.
2.11. In vivo MRI of Gd-DTPA and Gd-NGO In the animal experimental group, focused ultrasound (FUS)-induced BBB opening was monitored by MRI with a 7-T magnetic resonance scanner and a 4channel surface coil. The mouse was placed in an acrylic holder, positioned in the center of the magnet, and anesthetized with isoflurane gas (1e2%) at 50e70 breaths/ min during the entire MRI procedure. The distribution of Gd-DTPA (n ¼ 3) or GdNGO (n ¼ 3) leakage was investigated immediately after conducting FUS-BBB opening. After BBB opening, the Gd-DTPA and Gd-NGO were injected into animals then the animals were relocated into the MRI scanning room for imaging after 10 min of injection. Contrast-enhanced T1-weighted images with different flip angles were acquired to calculate spin-lattice relaxivity maps by transferring two images with different flip angles (gradient recalled echo sequence, TR/TE ¼ 2.3 ms/ 0.76 ms, slice thickness ¼ 0.8 mm, slice number ¼ 14, matrix ¼ 132 192, and flip angle ¼ 5 /20 ). 2.12. Histology and microscopy Animals were sacrificed 12 h after post-injection of Gd-NGO/FAM-labeled Let7g. Slides were stained with DAPI (Sigma) to detect nuclei. Briefly, brain sections mounted on slides were stained in a 1:1 mixture of 2% potassium ferrocyanide and
H.-W. Yang et al. / Biomaterials 35 (2014) 6534e6542 2% hydrochloric acid for 30 min at room temperature. The slides were rinsed with distilled water, dehydrated, and photographed. Nuclei were stained with the fluorescent dye DAPI. Fluorescent microscopic observations were performed using TCS SP2 confocal spectral microscopy using Leica QWin software (Leica).
3. Results and discussion 3.1. Synthesis and characterization of Gd-NGO To avoid serious obstructions in major organs and blood vessels, the original GO-COOH was shattered by sonication to produce nanometer-scale GO-COOH (NGO-COOH), following a previously reported protocol [15,16]. Positively charged Gd-functionalized NGO was synthesized by carbodiimide-catalyzed amide formation, by sonicating an aqueous solution of NGO-COOH in the presence of amine-functionalized Gd-DTPA and dendrimer solution. The concentration of Gd3þ ions conjugated on the surface of NGOCOOH was 168 29 mg/mg NGO-COOH. Dendrimer drugs are emerging as ideal candidates for gene delivery with reasonable transfection efficiency and minimal cytotoxicity. Gd-NGO was then used for association with Let-7g and EPI (Fig. 1). The sheet sizes of NGO-COOH and Gd-NGO were determined to be between 140 and 150 nm by atomic force microscopy (AFM) imaging (Fig. 2A, B). AFM imaging also showed that the thicknesses (T) of NGO-COOH and Gd-NGO were 1.77 0.21 nm and 2.95 0.39 nm, respectively. The
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average roughness (Ra) of Gd-NGO was 0.36 0.06 nm, which was higher than that of NGO-COOH (0.18 0.05 nm), likely owing to covalent conjugation of the Gd-DTPA and dendrimer on the rougher surface of NGO-COOH sheets (Fig. 2C). Synthesized compounds were characterized by X-ray photoelectron spectroscopy (XPS) (Fig. 3A). The NGO-COOH sample showed C1s and O1s peaks at 285 eV and 532 eV, respectively. After conjugation of Gd-DTPA and dendrimer, N1s and Gd peaks appeared at 399 eV and 147 eV, respectively [28], confirming covalent binding of Gd-DTPA and dendrimer to NGO-COOH. The surface composition, structure and functional groups present on NGO-COOH and Gd-NGO were also investigated. The NGO-COOH C1s XPS spectrum revealed a significant degree of oxidation indicated by numerous oxygen-containing groups; peaks at 284.2, 284.8, 285.6, 286.5, and 288.5 eV were assigned to carbon atoms in C]C, CeC, CeOH, CeOeC, and OeC]O, respectively (Fig. 3B). After modification, the C1s XPS spectrum of the resulting Gd-NGO showed significantly decreased carboxyl acid groups (288.5 eV). Additional peaks at 287.4 eV for Gd-NGO originated from CeN bonds [29]. We concluded that Gd-DTPA and dendrimer had been successfully grafted onto NGO carboxyls and the conjugation ratio was about 22.8% (Fig. 3C). Thermogravimetric analysis (TGA) was used to investigate the thermal stability of the graphene based-materials and determine the quantity of grafted organic substances, based on thermal
Fig. 2. (A) AFM image of NGO-COOH deposited on mica substrate. (B) AFM image of Gd-NGO deposited on mica substrate. (C) Thickness distribution of NGO-COOH and Gd-NGO. T ¼ thickness, Ra ¼ average roughness.
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Fig. 3. (A) The XPS survey spectra of NGO-COOH and Gd-NGO. The C1s peaks in the XPS spectra of (B) NGO-COOH and (C) Gd-NGO.
stripping of the covalent bonds that link the graphene sheet and its substituents in the 200e500 C temperature range [30]. Typical TGA thermograms indicated weight loss, demonstrating thermal stability of surface-functionalized NGO-COOH materials (Fig. 4A). Unlike NGO-COOH, the thermal degradation of Gd-NGO occurred in a single stage between 300 and 500 C, which we attributed to the thermal degradation of Gd-DTPA and dendrimer at the NGO-COOH surface. Gd-DTPA and dendrimer altered the surface properties of NGO-COOH, preventing weight loss at low temperatures (below 200 C). The results showed that amine-functionalized Gd-DTPA
and dendrimer were covalently conjugated on the surface of NGOCOOH. R1 relaxivities of Gd-DTPA and Gd-NGO were calibrated in vitro. The R1-signal increased in a highly linear manner with increased concentrations of Gd-DTPA or Gd-NGO (input concentrations of 0.000625, 0.00125, 0.0025, 0.005, 0.01, and 0.02 mM) as shown by the calibration curve (r2 ¼ 0.977 for Gd-DTPA, and r2 ¼ 0.998 for Gd-NGO). Both Gd-DTPA and Gd-NGO thus acted as dosedependent positive contrast agents. The relaxivity of Gd-NGO (7.59 mM1 s1) was higher than that of Gd-DTPA (2.65 mM1 s1)
Fig. 4. (A) TGA curves of NGO-COOH and Gd-NGO with a heating rate of 10 C/min in N2. (B) Dependence of R1 on Gd-DTPA and Gd-NGO concentrations; relaxivities were estimated as about 2.65 mM1 s1and 7.59 mM1 s1, respectively.
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at 7 T (Fig. 4B). This difference is most likely due to the strong interaction of a significant portion of Gd(III) ions with the entrapped NGO, which limits the water accessibility of the Gd(III) ions [31]. This calibration curve could be used for precise quantitation of Gd-NGO deposition in the brain. 3.2. Let-7g and EPI loading To detect loading of Let-7 and EPI onto Gd-NGO, we determined the zeta potential as a measure of the change in surface charge. GdNGO particles had a positive charge of 48.4 mV 5.8 mV. The charge changed to 18.7 mV 6.1 mV by adsorption of negatively charged Let-7g onto the surface of Gd-NGO. After coating with EPI and dendrimer, the charge returned to 33.4 mV 8.4 mV (Fig. 5A). These findings were consistent with successful loading of Let-7g and EPI onto the Gd-NGO, since Let-7g and EPI are negatively and positively charged, respectively. Drug delivery formulations with relatively small particles and positively charged surfaces have been shown to exhibit high cell penetration and accumulation rates [32]. We used FAM-labeled Let-7g to quantify the loading efficiency of Let-7g onto the Gd-NGO surface by fluorescence intensity. The excitation and emission wavelengths were 485 nm and 528 nm, respectively. The amount of loaded Let-7g reached saturation (0.15 nmole Let-7g/mg Gd-NGO) at 0.2 nmole of added Let-7g (up to 75% adsorption) (Fig. 5B). The loading efficiency was as high as 75% due to the high positive surface charge (48.4 mV) of Gd-NGO which favored adsorption of high quantities of negatively charged Let-7g. Loading of EPI onto Gd-NGO/Let-7g was investigated next, based on the characteristic UVevis absorbance peak of EPI at
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495 nm superimposed on the Gd-NGO/Let-7g/EPI absorption spectrum (Fig. 5C). The readout at 480 nm was compared with a standard curve generated from free EPI at different concentrations to calculate the unbound EPI concentration and thus the loading efficiency. The addition of 800 mg of EPI resulted in a saturating concentration of 606 mg EPI/mg Gd-NGO/Let-7g (up to 76%) (Fig. 5D). Efficient EPI loading onto NGO-COOH could be attributed to simple pep stacking under controlled pH conditions [18]. 3.3. Let-7g and EPI release study Another advantage of using Gd-NGO is pH-dependent drug release, based on weakening of hydrogen bonds at low pH. EPI was located on the outer layer, and was therefore expected to be released earlier than Let-7g. We found that about 15% of EPI was rapidly released from Gd-NGO/Let-7g during the first 18 h of incubation at pH 7.4, with essentially no further release over time (Fig. 6A). However, at pH 6.0 the amine groups of EPI were protonated, resulting in increased solubility and accelerated release from Gd-NGO/Let-7g [33]. The amount of EPI released reached nearly 41% within 24 h, and increased to 57% after another 60 h of incubation. Let-7g release from Gd-NGO at pH 7.4 was delayed until 10 h of incubation and only about 13% was released after 84 h. However, 39% was released at pH 6.0. Presumably Let-7g had to traverse the additional outer shell of EPI and dendrimer, which may have prevented contact with the surrounding acidic medium [34]. These data suggested that Let-7g would not be released from GdNGO/Let-7g/EPI too early, before reaching the cell. pH-dependent drug release by Gd-NGO/Let-7g/EPI could be exploited for
Fig. 5. (A) Zeta potential measurements. (B) Amount of Let-7g bound per 1 mg of Gd-NGO vs. amount of Let-7g added. Values are means SD (n ¼ 8). Inset, standard curve of fluorescence intensity of FAM-labeled Let-7g. (C) UVevis absorption spectra of NGO-COOH, Gd-NGO, Gd-NGO/Let-7g, and Gd-NGO/Let-7g/EPI. (D) Amount of EPI bound per 1 mg of Gd-NGO/Let-7g vs. amount of EPI added. Values are means SD (n ¼ 8).
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Fig. 6. (A) Cumulative Let-7g and EPI release (%) from Gd-NGO/Let-7g/EPI at 37 C in PBS (pH ¼ 6.0 or 7.4). Values are expressed as means SD (n ¼ 5). (B) Fluorescence microscopic images of U87 cells treated with GFP-pDNA (left) and Gd-NGO/GFP-pDNA (right) at 37 C for 12 h. (C) Fluorescence microscopic images of U87 cells treated with FAM-labeled Let-7g (left) and Gd-NGO/FAM-labeled Let-7g (right) 37 C for 48 h (Blue: nuclei; Green: GFP; Red: FAM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
controlled drug and gene delivery since microenvironments in both the extracellular tissues of tumors and intracellular lysosomes and endosomes are acidic [35]. 3.4. In vitro transfection study Next we examined the ability of Gd-NGO to deliver pDNA or Let7g into brain tumor cells in vitro. U87 cells were transfected with free GFP-pDNA, free FAM-labeled Let-7g, Gd-NGO/GFP-pDNA, or Gd-NGO/Let-7g complexes. After 48 h of incubation, the cells were observed by fluorescence microscopy. The Gd-NGO/GFP-pDNA complexes showed a significantly higher transfection efficacy (12.7-fold increase) than free GFP-pDNA in U87 cells (Fig. 6B). Thus GFP-pDNA could be successfully carried into the cytoplasm by GdNGO, and then released from Gd-NGO to express GFP. FAM-labeled Let-7g was also delivered into the cells by Gd-NGO, and the miRNA was clearly visible in the nuclei, indicating the miRNA was successfully released from the carrier (Fig. 6C). The efficient transfection results were most likely due to increased electrostatic interactions between the many amino groups of dendrimers and negatively charged pDNA or miRNA, resulting in high adsorption onto the surface of Gd-NGO. In addition, Gd-NGO appeared to be taken up by an endocytotic pathway so the pDNA or miRNA could be delivered into the cells by any transfection protocol. 3.5. In vitro cytotoxicity study We investigated the effect of Let-7g carried into U87 cells by GdNGO after incubation for 48 h. Since the highly adsorptive surface of Gd-NGOs severely restricted RNA extraction, we were unable to use expression profiling to analyze the effect of miRNA. We therefore analyzed downstream protein targets of Let-7g to investigate its bioavailability and function when delivered by Gd-NGO. Since Let7g miRNA is known to reduce cell proliferation and invasion via
inhibition of the oncogenic Ras signaling pathway [5], the expression levels of Ras protein family members were analyzed (Fig. 7A). The expression levels of Pan-Ras proteins were unaltered by GdNGO alone, but were significantly reduced by Gd-NGO/Let-7g. The expression level was the same when EPI was co-incorporated (Gd-NGO/Let-7g/EPI), indicating that EPI was not involved in Ras protein inhibition. Incubation of U87 cells with naked Let-7g miRNA in the presence of transfection reagent had no effect on expression compared to the dramatic decrease of target protein expression with Gd-NGO-carried Let-7g, most likely the concentration of transfection reagent (Lipofectamine 2000) we used was too low to sufficiently transfect the Let-7g into U87 cells to inhibit the expression of Ras family proteins due to the overdose of Lipofectamine 2000 would cause cytotoxicity toward to the cells. We also investigated the viability of U87 cells treated with different materials. Gd-NGO and pure Let-7g were not cytotoxic toward U87 cells (Fig. 7B). Thus pure Let-7g was unable to enter the cells by itself to induce apoptosis. The concentration required for 50% inhibition of cellular growth (IC50) was higher for free EPI (greater than 6.4 mg/mL) than for Gd-NGO/EPI (equivalent of 3.4 mg/ mL EPI). The IC50 of Gd-NGO/Let-7g/EPI was significantly lower (equivalent of 1.3 mg/mL EPI and 16.3 nM Let-7g) than Gd-NGO/EPI (equivalent of 3.4 mg/mL EPI) and Gd-NGO/Let-7g (equivalent of 23.8 nM Let-7g), presumably because of inhibition of the oncogenic Ras signaling pathway by Let-7g. The combination of chemotherapy and gene-targeting therapy thus resulted in better inhibition of cancer cell growth at a lower dose, indicating that this delivery system could reduce the dose of chemotherapeutics and the accompanying serious side-effects. 3.6. In vivo MRI The bloodebrain barrier (BBB) prevents larger molecules from entering the brain parenchyma, thus protecting the brain from toxic
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Fig. 7. (A) Analysis of protein expression downstream of Let-7g miRNA. U87 human glioma cells were co-cultured with control (no substance added), Lipofectamine 2000 þ Let-7g (9.375 nM), Lipofectamine 2000 þ scrambled sequence, Gd-NGO, Gd-NGO/Let-7g (equivalent to 9.375 nM Let-7g), Gd-NGO/scrambled sequence, Gd-NGO/Let-7g/EPI (equivalent to 9.375 nM Let-7g and 1.5625 mg/mL EPI). Gd-NGO/Let-7g and Gd-NGO/Let-7g/EPI significantly reduced the expression of oncogenic Pan-Ras protein. (B) Relative cell viability of U87 cells for 24 h after drug treatment. Values are expressed as means SD (n ¼ 8).
foreign substances. However, it also prohibits delivery of many potentially effective diagnostic or therapeutic agents or nanoparticles. Focused ultrasound (FUS) in the presence of circulating microbubbles can temporarily open the BBB of capillaries in the central nervous system (CNS) parenchyma. This FUS-induced BBB opening can be verified by MRI. Gd-NGO can be used as an effective MRI contrast agent to monitor the location and extent of FUS-induced BBB opening and to
quantify the concentration of EPI/Let-7g delivered into the brain. The Gd-NGO still can be significantly accumulated in the brain, although the Gd-NGO did not be conjugated specific targeting ligands (i.e. EGFR), likely because we used the FUS to open the BBB and allow the Gd-NGO can flow into the brain. Therefore, the BBBopened area was clearly visible at increased contrast intensity in T1-weighted images and R1 maps (Fig. 8A). Gd-NGO could also be observed in the same way as Gd-DTPA in the FUS BBB-opened areas
Fig. 8. (A) Typical MRI contrast-enhanced T1 images of Gd-DTPA and Gd-NGO before and after FUS exposure and R1 maps (after FUS exposure) in normal animals. (Yellow arrow: BBB-opened area). (B) Fluorescence images of tumor tissue: control group (without any treatment; top) and Gd-NGO/FAM-labeled Let-7g treated group. (green: FAM-labeled Let-7g; blue: nuclei). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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in subtracted T1 images. In addition, R1 maps could be used to quantify the concentration and determine the distribution of either Gd-NGO or Gd-DTPA. The signal of Gd-NGO was in fact stronger than that of Gd-DTPA, presumably because of the 2.9-fold higher contrast ability of Gd-NGO compared to Gd-DTPA, and because of the Gd-NGO would be easily accumulated in the brain. We next investigated the transfection of Let-7g in tumor tissue; the GdNGO/FAM-labeled Let-7g was injected in mice via tail vein after FUS treatment. The mice were sacrificed after 12 h of post-injection, the fluorescence microscopy of tumor tissue slides also confirmed that the FAM-labeled Let-7g (green color) could not only be delivered into brain tumor tissues but also successfully transfected in the tumor cells (Fig. 8B). Gd-NGO thus not only provides an important means to simultaneously deliver chemotherapeutics (EPI) and miRNA (Let-7g) for cancer treatment, but can also be monitored by MRI, permitting quantification of drug/miRNA delivery in real time in vivo. 4. Conclusion An MRI-detectable, drug and miRNA carrying vector (Gd-NGO) was developed to effectively load EPI and Let-7g miRNA (Gd-NGO/ Let-7g/EPI) and transfer them into cancer cells. The transfer process was monitored by fluorescence confocal microscopic imaging in glioma U87 cells. The net positive charge density of Gd-NGO resulted in concentrated binding and efficient delivery of GFPpDNA into cells. Gd-NGO was also capable of efficient and simultaneous delivery of EPI and Let-7g miRNA into cells to destroy the DNA and knockdown expression of Ras family proteins, respectively. Moreover, Gd-NGO could be detected by MRI to identify the tumor area and quantify the concentration of therapeutics within the tumor. Taken together, these promising results suggest the potential use of Gd-NGO complexes as efficient non-viral drug and gene delivery vectors for future chemogene therapy applications. Acknowledgements We thank the National Science Council of the Republic of China, National Health Research Institutes of Taiwan, Chang Gung Memorial Hospital, Industrial Technology Research Institute (103A0045J2), and National Tsing Hua University (103N2753E1) for financial support (NSC 102-2221-E-007-003-(102), NSC 1022622-E-007-024-CC3, NSC 102-2314-B-182A-068-MY3, NSC 1022120-M-182A-002-CC1, NHRI-EX103-10004NI, CMRPG3D0571, CMRPG3B1012, CMRPG392103, CORPG3C0041, CMRPG390071, CMRPG380571, 103A0045J2, 103N2753E1). The authors thank the Boost Program from Low Carbon Energy Research Center of National Tsing Hua University; Chang Gung Memorial Hospital Animal Molecular Imaging Center for assistance with 7T-MRI and Microscopy Core Laboratory. References [1] Niidome T, Huang L. Gene therapy progress and prospects: nonviral vectors. Gene Ther 2002;9:1647e52. [2] Wells DJ. Gene therapy progress and prospects: electroporation and other physical methods. Gene Ther 2004;11:1363e9. [3] Dong H, Ding L, Yan F, Ji H, Ju H. The use of polyethylenimine-grafted graphene nanoribbon for cellular delivery of locked nucleic acid modified molecular beacon for recognition of microRNA. Biomaterials 2011;32:3875e82. [4] Karlsen TA, Brinchmann JE. Liposome delivery of microRNA-145 to mesenchymal stem cells leads to immunological off-target effects mediated by Rig-I. Mol Ther 2013;21:1169e81. [5] Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, et al. RAS is regulated by the let-7 microRNA family. Cell 2005;120:635e47. [6] Roush S, Slack FJ. The let-7 family of microRNAs. Trends Cell Biol 2008;18: 505e16.
[7] Akao Y, Nakagawa Y, Naoe T. Let-7 microRNA functions as a potential growth suppressor in human colon cancer cells. Biol Pharm Bull 2006;29: 903e6. [8] Schultz J, Lorenz P, Gross G, Ibrahim S, Kunz M. MicroRNA let-7b targets important cell cycle molecules in malignant melanoma cells and interferes with anchorage-independent growth. Cell Res 2008;18:549e57. [9] Trang P, Medina PP, Wiggins JF, Ruffino L, Kelnar K, Omotola M, et al. Regression of murine lung tumors by the let-7 microRNA. Oncogene 2010;29: 1580e7. [10] Lee ST, Chu K, Oh HJ, Im WS, Lim JY, Kim SK, et al. Let-7 microRNA inhibits the proliferation of human glioblastoma cells. J Neurooncol 2011;102:19e24. [11] Yu F, Yao H, Zhu P, Zhang X, Pan Q, Gong C, et al. Let-7 regulates self renewal and tumorigenicity of breast cancer cells. Cell 2007;131:1109e23. [12] Yuan F, Dellian M, Fukumura D, Leunig M, Berk DA, Torchilin VP, et al. Vascular permeability in a human tumor xenograft: molecular size dependence and cutoff size. Cancer Res 1995;55:3752e6. [13] Allen TM, Cullis PR. Drug delivery systems: entering the mainstream. Science 2004;303:1818e22. [14] Yang XZ, Du JZ, Dou S, Mao CQ, Long HY, Wang J. Sheddable ternary nanoparticles for tumor acidity-targeted siRNA delivery. ACS Nano 2012;6: 771e81. [15] Yang HW, Lu YJ, Lin KJ, Hsu SC, Huang CY, She SH, et al. EGRF conjugated PEGylated nanographene oxide for targeted chemotherapy and photothermal therapy. Biomaterials 2013;34:7204e14. [16] Yang HW, Hua MY, Hwang TL, Lin KJ, Huang CY, Tsai RY, et al. Non-invasive synergistic treatment of brain tumors by targeted chemotherapeutic delivery and amplified focused ultrasound-hyperthermia using magnetic nanographene oxide. Adv Mater 2013;25:3605e11. [17] Lu CH, Zhu CL, Li J, Liu JJ, Chen X, Yang HH. Using graphene to protect DNA from cleavage during cellular delivery. Chem Commun 2010;46:3116e8. [18] Yang X, Zhang X, Liu Z, Ma Y, Huang Y, Chen Y. 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Biomaterials journal homepage: www.elsevier.com/locate/biomaterials
Gadolinium-functionalized nanographene oxide for combined drug and microRNA delivery and magnetic resonance imaging Hung-Wei Yang a,1, Chiung-Yin Huang b,1, Chih-Wen Lin a,1, Hao-Li Liu c, Chia-Wen Huang b, Shih-Sheng Liao a, Pin-Yuan Chen b, d, Yu-Jen Lu b, Kuo-Chen Wei b, d, **, Chen-Chi M. Ma a, * a
Department of Chemical Engineering, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsin-chu 30013, Taiwan, ROC Department of Neurosurgery, Chang Gung Memorial Hospital, Linkou, 5 Fu-Shing Road, Kuei-Shan, Tao-Yuan 33305, Taiwan, ROC Department of Electrical Engineering, Chang Gung University, 259 Wen-Hwa 1st Road, Kuei-Shan, Tao-Yuan 33302, Taiwan, ROC d School of Medicine, Chang Gung University, 259 Wen-Hwa 1st Road, Kuei-Shan, Tao-Yuan 33302, Taiwan, ROC b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 March 2014 Accepted 12 April 2014 Available online 6 May 2014
The delivery of anti-cancer therapeutics to tumors at clinically effective concentrations, while avoiding nonspecific toxicity, remains a major challenge for cancer treatment. Here we present nanoparticles of poly(amidoamine) dendrimer-grafted gadolinium-functionalized nanographene oxide (Gd-NGO) as effective carriers to deliver both chemotherapeutic drugs and highly specific gene-targeting agents such as microRNAs (miRNAs) to cancer cells. The positively charged surface of Gd-NGO was capable of simultaneous adsorption of the anti-cancer drug epirubicin (EPI) and interaction with negatively charged Let-7g miRNA. Using human glioblastoma (U87) cells as a model, we found that this conjugate of Let-7g and EPI (Gd-NGO/Let-7g/EPI) not only exhibited considerably higher transfection efficiency, but also induced better inhibition of cancer cell growth than Gd-NGO/Let-7g or Gd-NGO/EPI. The concentration of Gd-NGO/Let-7g/EPI required for 50% inhibition of cellular growth (IC50) was significantly reduced (to the equivalent of 1.3 mg/mL EPI) compared to Gd-NGO/EPI (3.4 mg/mL EPI). In addition, Gd-NGO/Let-7g/EPI could be used as a contrast agent for magnetic resonance imaging to identify the location and extent of bloodebrain barrier opening and quantitate drug delivery to tumor tissues. These results suggest that Gd-NGO/Let-7g/EPI may be a promising non-viral vector for chemogene therapy and molecular imaging diagnosis in future clinical applications. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Nanographene oxide Dendrimer Drug delivery miRNA delivery MR imaging
1. Introduction Gene therapy could potentially be used for cancer treatment by introduction of a tumor-suppressor gene into the target area to inhibit tumor growth. However, the safe and efficient delivery of genes into cells has remained a major challenge to clinical applications of gene therapy [1]. Many powerful gene delivery tools have been developed, including viruses, liposomes, polymer nanoparticles, carbon nanomaterials, and external physical forces [2e4].
* Corresponding author. Department of Chemical Engineering, National Tsing Hua University, 101, Section 2, Kuang-Fu Road, Hsin-chu 30013, Taiwan, ROC. Tel.: þ886 3 5713058; fax: þ886 3 5715408. ** Corresponding author. Department of Neurosurgery, Chang Gung Memorial Hospital, Linkou, 5 Fu-Shing Road, Kuei-Shan, Tao-Yuan 33305, Taiwan, ROC. Tel.: þ886 3 3281200x2412; fax: þ886 3 3285818. E-mail addresses: [email protected] (K.-C. Wei), [email protected] (C.-C.M. Ma). 1 Contributed equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2014.04.057 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.
One of the most promising candidates for targeting specific genes is microRNA (miRNA). miRNAs are a class of endogenous, noncoding small RNA molecules that participate in gene regulation and various physiological processes. Altered miRNA expression has been detected in human cancers: tumor suppressive miRNAs are downregulated while oncogenic miRNAs are upregulated. Nine members of the Let-7 miRNA family have been detected in human tissue. Let-7 miRNAs function as tumor suppressors by decreasing expression of the Ras oncogene family [5,6]. Downregulation of Let-7 has been observed in lung cancer, colon cancer, and melanoma and is correlated with poor survival [7e9]. Conversely, Ras proteins (K-Ras, N-Ras and H-Ras) are known to be upregulated in these cancers. Overexpression of Let-7 has also been shown to inhibit cancer cell growth [11]. In human brain tumor cells, overexpressed Let-7 miRNA leads to a decrease in Ras proteins, inhibition of cell proliferation and reduced tumor growth, without harming normal astrocytes [10]. This selective growth inhibition of brain tumor cells suggests Let-7 miRNA as an attractive candidate for gene-targeting therapy to treat malignant brain
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tumors. The development of a safe and effective method for delivery of these miRNAs to cancer cells has therefore emerged as a critical goal of current cancer therapeutic research. Nanomaterials have been attracting increasing interest for their potential use as tumor-selective drug delivery vehicles. Tumor vessels are usually leaky, with large endothelial interstitial gap junctions of about 100e600 nm [12]. Nanomaterials can reach tumors by the enhanced permeability and retention (EPR) effect [13]. Such systems are controlled by a number of parameters including pH, redox environment, light, heat, ultrasound, and magnetic and electrical fields [2,14e16]. Functionalized nanoscale graphene oxide (NGO) is an example of a nanomaterial that is capable of delivering oligonucleotides into cells and protecting them from enzymatic cleavage [17]. In addition, pep stacking between anti-cancer drugs and NGO allows high-efficiency loading and controlled release of anti-cancer drugs [18]. Polyethylene glycol (PEG)-functionalized NGO was initially developed as a nanocarrier and evaluated for its in vitro cellular uptake in the Dai laboratory [19]. Anticancer drugs are loaded into these PEG-NGOs via noncovalent physisorption. Yang et al. investigated the loading and release behaviors of doxorubicin hydrochloride (DOX) on NGO [18]. They found that the weight ratio of loaded drug to NGO carrier could reach 200%. Besides, the NGO also can be used for gene delivery; some cationic polymers such as polyethylenimine (PEI), chitosan, and Polyamidoamine (PAMAM), high molecular weight branched PEI (HMW BPEI) or low molecular weight branched PEI (LMW BPEI) were conjugated to the inorganic materials (i.e. silica, iron oxide, and carbon materials) to enhance cellular uptake and transfection efficiency [20,21]. However, the polymers with high molecular weight would induce cytotoxicity toward the cells [22]. Therefore, we modified the NGO with low molecular weight PAMAM and GdDTPA as drug/gene carriers which can be monitored by MRI to enhance the transfection efficiency with ultralow cytotoxicity. In
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addition to its use for drug and gene delivery, graphene oxide (GO) has also been extensively explored for bioimaging, biosensing, and many other biomedical applications. This versatility results from the excellent physiochemical, optical, and electrical properties of GO [23e26]. In this study, we report the design, synthesis, and evaluation of a new delivery system consisting of PAMAM-dendrimer and gadolinium (Gd)-functionalized NGO (Gd-NGO) loaded with miRNA (Let7g) and Epirubicin (EPI) (Gd-NGO/Let-7g/EPI) and designed for chemo/gene therapy and magnetic resonance imaging (MRI) (Fig. 1). This system could potentially allow safe delivery of other potent and toxic drugs into tumor cells and provide high transfection efficiency for gene delivery (miRNA, siRNA, pDNA) to overcome the therapeutic challenges of some tumors which are currently considered difficult to treat. We suggest that, with these new functionalities, Gd-NGO can be considered important therapeutic and diagnostic targets for the treatment and detection of cancers. 2. Materials and methods 2.1. Materials Graphite platelets (model xGnP; width 100 mm, thickness 5e15 nm) were obtained from XG Sciences Inc. (East Lansing, MI). Acrylic acid, potassium persulfate, sulfuric acid (H2SO4, 98%), sodium sulfate (Na2SO4), potassium permanganate (KMnO4), hydrogen peroxide solution (H2O2), and ammonia were purchased from Showa Chemical Co. (Tokyo, Japan). U87 human glioblastoma cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Hank’s balanced salt solution and RPMI-1640 medium were purchased from Gibco (Grand Island, NY). Fetal bovine serum was purchased from Biological Industries (Beit-Haemek, Israel). Gentamicin, penicillin, and streptomycin were obtained from MDBio. Minimum essential medium was purchased from Invitrogen (Carlsbad, CA). Epirubicin (EPI), glutaraldehyde, 1-ethyl-3-(3dimethylaminepropyl) carbodiimide hydrochloride (EDC), 2-(N-morpholino)ethanesulfonic acid hydrate (MES), sulfanilamide, N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), diethylenetriaminepentaacetic acid gadolinium (III) dihydrogen
Fig. 1. Schematic of the procedure for preparation of Gd-NGO/Let-7g/EPI.
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salt hydrate (Gd-DTPA), ethylenediamine, transfection reagent (Lipofectamine 2000), and poly(amidoamine) (PAMAM) dendrimer (generation 4.0 solution; Mw: 516.68) were purchased from Sigma (St Louis, MO). Synthetic Let-7g (50 -uga ggu agu agu uug uac agu u-30 ), fluorescein amidite (FAM)-labeled Let-7g, and a scrambled control sequence were purchased from MDBio, Inc (Taiwan). All reagents were of analytical grade and were used without any further purification. Deionized (DI) water was used in all experiments.
2.7. EPI and Let-7g release study Gd-NGO/Let-7g/EPI samples were placed in dialysis bags containing 10 mL of phosphate buffered saline (PBS) at pH 7.4 and 6.0, respectively, after adjusting with HCl. Drug release was assumed to start as soon as the dialysis bags were placed into the reservoir. The release reservoir was constantly stirred, and at various time points, the dialysis bags were taken out for characterization. The concentrations of Let-7g and EPI released from Gd-NGO/Let-7g/EPI into the distilled water of the reservoir were quantified using ELISA and HPLC, respectively.
2.2. Preparation of carboxylic nanographene oxide (NGO-COOH) NGO-COOH was prepared by a modified Hummers’ method. Graphite platelets (250 mg), NaNO3 (0.125 g), and H2SO4 (12 mL 98%) were magnetically mixed in a 100 mL flask in an ice bath, followed by slow addition of 0.75 g KMnO4, while keeping the temperature below 5 C. The flask was then heated to 100 C, followed by slow addition of 12 mL DI. The temperature of the solution was increased to 98 C for a 30 min incubation period. A 50 mL volume of 10% H2O2 was added to the solution until the cessation of gas evolution. The solution was centrifuged at 11,000 rpm and washed several times with DI to remove impurities and obtain large-scale GO (LGO). The precipitate was subjected to ultrasonication for 8 h at 800 W using an Ultrasonic Liquid Processor 2020 from Misonix (Farmingdale, NY) and centrifuged at 14,000 rpm for 0.5 h. The supernatant was filtered three times with Acrodisc 25 mm syringe filters (0.2 mm Supor membrane), and the NGO collected in the filtrate was subjected to further modification. NaOH (10 mL, 12 mg/ mL) was added to an aqueous suspension of the NGO (10 mL, 2 mg/mL) followed by sonication at 800 W for 2 h to convert OH groups to COOH, resulting in NGO-COOH. X-ray photoelectron spectroscopy (XPS) was used to investigate the structure, COOH conversion, and functional groups of NGO.
2.8. In vitro gene transfection studies For Western blot analysis, U87 cells were added to 24-well plates at a density of about 1 105 cells per well, 24 h before transfection. Transfection was carried out by adding 150 mL of Gd-NGO, Gd-NGO/Let-7g, or Gd-NGO/Let-7g/EPI into each well. After incubation for 12 h at 37 C, the media containing different transfection complexes were removed. Cells were washed three times with serum-free DMEM medium before addition of 750 mL formal DMEM growth medium containing 10% FBS. An additional 48 h incubation period was needed for efficient reaction of Let-7g inside the cells. Pure Let-7g without any transfection agent was used as a control. Expression of Pan-ras, K-ras, and N-ras proteins were determined by Western blot. For fluorescence microscopy, U87 cells were added to 8-well chamber slides at a density of about 5 103 cells per well, 24 h before transfection. Transfection was carried out by adding 50 mL of Gd-NGO/Let-7g or Gd-NGO/GFP-pDNA to each well. After incubation for 12 h at 37 C, the media containing different transfection complexes were removed. After another 36 h of incubation, cells were washed three times with serum-free DMEM medium. Fluorescence images of cells were captured using a laser scanning confocal fluorescence microscope (Leica TCS SP2, Germany). All images were collected using the same parameters.
2.3. Dendrimer-functionalization and Gd-DTPA conjugation to NGO-COOH Twenty-four milligrams of EDC and 27 mg of sulfo-NHS were dissolved in 2 mL of 0.5 M MES buffer (pH ¼ 6.3) in the dark. A 0.2 mL aliquot of this solution was mixed with 0.2 mL of Gd-DTPA (5 mg/mL) at 25 C and reacted for 60 min in the dark to allow the formation of amide bonds between activated carboxyl groups. The activated Gd-DTPA was reacted with ethylenediamine at 25 C for 3 h in the dark to form amine-functionalized Gd-DTPA. A 0.2 mL aliquot of the EDC/sulfo-NHS solution was mixed with 0.2 mL of NGOCOOH (3 mg/mL) at 25 C and reacted for 30 min in the dark to activate the carboxylic groups of NGO-COOH. Activated NGO-COOH was washed with 0.8 mL 0.1 M MES buffer, resuspended in 0.2 mL of MES buffer, and then mixed with 0.05 mL of amine-functionalized Gd-DTPA (2 mg/mL) and 0.15 mL of dendrimer (2 mg/mL) at 25 C by vortexing for 12 h. The product was then separated from the solution, washed with DI water and centrifuged at 14,000 rpm for 0.5 h to remove both MES buffer and unbound amine-functionalize Gd-DTPA and dendrimer, and finally dispersed in DI water to obtain Gd-conjugated NGO (Gd-NGO). 2.4. MRI phantom studies with Gd-DTPA and Gd-NGO For in vitro measurements, Gd-DTPA and Gd-NGO were diluted with physiological saline to 0.000625, 0.00125, 0.0025, 0.005, 0.01, and 0.02 mM. Circular wells (inner diameter ¼ 5 mm) were filled with 200 mL of contrast agent sample or physiological saline as control and were placed in the MR scanner (Clinscan, Bruker, Germany; 7 T). Spin-lattice relaxivity maps were calculated from two T1-weighted images with different flip angles (gradient recalled echo sequence, TR/ TE ¼ 2.3 ms/0.76 ms, slide thickness ¼ 0.8 mm, matrix ¼ 132 192, and flip angle ¼ 5 /20 ). The correlation between R1 (¼1/T1) mapping and Gd-DTPA concentration was determined [27].
2.9. Western blots Treated U87 glioma cells were washed twice in ice-cold PBS and lysed on ice in ice-cold T-PER tissue protein extraction reagent (Pierce, Rockford, IL, USA) containing protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Lysates were cleared by centrifugation, and total protein concentrations were determined by Bradford assay (Bio-Rad, Hercules, CA, USA). Protein samples (20 mg/lane) were separated on 12% polyacrylamide gels by SDSePAGE and transferred to polyvinylidene difluoride membranes (Millipore). Blots were blocked overnight in TBS (20 mM TriseHCl, 150 mM NaCl, 0.1% Tween-20, 0.5 mM EDTA, pH 7.4) containing 5% nonfat dry milk. Blots were then incubated for 2 h with primary antibodies (K-Ras 1:1000, Pan-Ras 1:1000, K-Ras 1:500; Santa Cruz), followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG (1:20,000; PerkinElmer) for 1 h. Ras proteins were detected using the Western Lightning kit (PerkinElmer) according to the manufacturer’s instructions. Beta-actin antibody purchased from Cell Signal was used as an internal control. 2.10. In vitro cytotoxicity assay U87 cells were cultured in DMEM supplemented with 2.2 mg/mL sodium carbonate, 10% fetal bovine serum, 50 mg/mL gentamicin, 50 mg/mL penicillin, and 50 mg/mL streptomycin at 37 C and 5% CO2. Approximately 10,000 cells (i.e., 150 mL of a suspension of 6.67 104 cells/mL) were placed in each well of a 96-well culture plate and incubated in a humidified chamber at 37 C and 5% CO2 for 24 h. Different concentrations of samples (50 mL each) were added to the medium, and incubation was continued for 48 h. Before counting, the culture medium was removed, and the cells were incubated in 120 mL of XTT solution for 3 h. Subsequently, 100 mL of the XTT solution was removed from each well and transferred to a 96-well counting dish. The cytotoxicity toward U87 cells in vitro was evaluated by measuring the OD at 490 nm using an ELISA reader.
2.5. Adsorption of Let-7g or GFP-pDNA on Gd-NGO Gd-NGO/Let-7g and Gd-NGO/GFP-pDNA complexes were freshly prepared prior to use. Briefly, 0.1 mL of Gd-NGO aqueous solution (1 mg/mL) was mixed with 0.1 mL of Let-7g (2 nmol/mL) or 0.1 mL of green fluorescent protein-expressing plasmid DNA (GFP-pDNA, 1 mg/mL) by gentle vortexing, and incubated at room temperature for 30 min. Complexes were separated from the solution, washed with DI water, centrifuged at 14,000 rpm for 0.5 h to remove free Let-7g or GFP-pDNA, and finally dispersed in DI water to obtain Gd-NGO/Let-7g or Gd-NGO/GFP-pDNA. 2.6. Drug loading onto Gd-NGO/Let-7g Epirubicin (EPI) loading onto Gd-NGO/Let-7g was accomplished by simply mixing EPI (0.5 mL in 0.5% dendrimer solution, 1 mg/mL) with a solution of Gd-NGO/ Let-7g (0.5 mL, 1 mg/mL) at pH 8 and 4 C overnight. Unbound EPI was removed by repeated washing with DI water. The resulting Gd-NGO/Let-7g/EPI complexes were resuspended and stored at 4 C. The concentration of EPI loaded onto Gd-NGO/Let7g was measured by high-performance liquid chromatography (HPLC) on a SUPELCOSILTM LC-18 column (4.6 250 mm) using an L-2130 pump and an L-2400 UV-detector (Hitachi). The mobile phase of the HPLC was a 50/15/35 (v/v/v) mixture of DI water, acetonitrile and methanol with a flow rate of 1.5 mL/min and data were collected at 256 nm.
2.11. In vivo MRI of Gd-DTPA and Gd-NGO In the animal experimental group, focused ultrasound (FUS)-induced BBB opening was monitored by MRI with a 7-T magnetic resonance scanner and a 4channel surface coil. The mouse was placed in an acrylic holder, positioned in the center of the magnet, and anesthetized with isoflurane gas (1e2%) at 50e70 breaths/ min during the entire MRI procedure. The distribution of Gd-DTPA (n ¼ 3) or GdNGO (n ¼ 3) leakage was investigated immediately after conducting FUS-BBB opening. After BBB opening, the Gd-DTPA and Gd-NGO were injected into animals then the animals were relocated into the MRI scanning room for imaging after 10 min of injection. Contrast-enhanced T1-weighted images with different flip angles were acquired to calculate spin-lattice relaxivity maps by transferring two images with different flip angles (gradient recalled echo sequence, TR/TE ¼ 2.3 ms/ 0.76 ms, slice thickness ¼ 0.8 mm, slice number ¼ 14, matrix ¼ 132 192, and flip angle ¼ 5 /20 ). 2.12. Histology and microscopy Animals were sacrificed 12 h after post-injection of Gd-NGO/FAM-labeled Let7g. Slides were stained with DAPI (Sigma) to detect nuclei. Briefly, brain sections mounted on slides were stained in a 1:1 mixture of 2% potassium ferrocyanide and
H.-W. Yang et al. / Biomaterials 35 (2014) 6534e6542 2% hydrochloric acid for 30 min at room temperature. The slides were rinsed with distilled water, dehydrated, and photographed. Nuclei were stained with the fluorescent dye DAPI. Fluorescent microscopic observations were performed using TCS SP2 confocal spectral microscopy using Leica QWin software (Leica).
3. Results and discussion 3.1. Synthesis and characterization of Gd-NGO To avoid serious obstructions in major organs and blood vessels, the original GO-COOH was shattered by sonication to produce nanometer-scale GO-COOH (NGO-COOH), following a previously reported protocol [15,16]. Positively charged Gd-functionalized NGO was synthesized by carbodiimide-catalyzed amide formation, by sonicating an aqueous solution of NGO-COOH in the presence of amine-functionalized Gd-DTPA and dendrimer solution. The concentration of Gd3þ ions conjugated on the surface of NGOCOOH was 168 29 mg/mg NGO-COOH. Dendrimer drugs are emerging as ideal candidates for gene delivery with reasonable transfection efficiency and minimal cytotoxicity. Gd-NGO was then used for association with Let-7g and EPI (Fig. 1). The sheet sizes of NGO-COOH and Gd-NGO were determined to be between 140 and 150 nm by atomic force microscopy (AFM) imaging (Fig. 2A, B). AFM imaging also showed that the thicknesses (T) of NGO-COOH and Gd-NGO were 1.77 0.21 nm and 2.95 0.39 nm, respectively. The
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average roughness (Ra) of Gd-NGO was 0.36 0.06 nm, which was higher than that of NGO-COOH (0.18 0.05 nm), likely owing to covalent conjugation of the Gd-DTPA and dendrimer on the rougher surface of NGO-COOH sheets (Fig. 2C). Synthesized compounds were characterized by X-ray photoelectron spectroscopy (XPS) (Fig. 3A). The NGO-COOH sample showed C1s and O1s peaks at 285 eV and 532 eV, respectively. After conjugation of Gd-DTPA and dendrimer, N1s and Gd peaks appeared at 399 eV and 147 eV, respectively [28], confirming covalent binding of Gd-DTPA and dendrimer to NGO-COOH. The surface composition, structure and functional groups present on NGO-COOH and Gd-NGO were also investigated. The NGO-COOH C1s XPS spectrum revealed a significant degree of oxidation indicated by numerous oxygen-containing groups; peaks at 284.2, 284.8, 285.6, 286.5, and 288.5 eV were assigned to carbon atoms in C]C, CeC, CeOH, CeOeC, and OeC]O, respectively (Fig. 3B). After modification, the C1s XPS spectrum of the resulting Gd-NGO showed significantly decreased carboxyl acid groups (288.5 eV). Additional peaks at 287.4 eV for Gd-NGO originated from CeN bonds [29]. We concluded that Gd-DTPA and dendrimer had been successfully grafted onto NGO carboxyls and the conjugation ratio was about 22.8% (Fig. 3C). Thermogravimetric analysis (TGA) was used to investigate the thermal stability of the graphene based-materials and determine the quantity of grafted organic substances, based on thermal
Fig. 2. (A) AFM image of NGO-COOH deposited on mica substrate. (B) AFM image of Gd-NGO deposited on mica substrate. (C) Thickness distribution of NGO-COOH and Gd-NGO. T ¼ thickness, Ra ¼ average roughness.
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Fig. 3. (A) The XPS survey spectra of NGO-COOH and Gd-NGO. The C1s peaks in the XPS spectra of (B) NGO-COOH and (C) Gd-NGO.
stripping of the covalent bonds that link the graphene sheet and its substituents in the 200e500 C temperature range [30]. Typical TGA thermograms indicated weight loss, demonstrating thermal stability of surface-functionalized NGO-COOH materials (Fig. 4A). Unlike NGO-COOH, the thermal degradation of Gd-NGO occurred in a single stage between 300 and 500 C, which we attributed to the thermal degradation of Gd-DTPA and dendrimer at the NGO-COOH surface. Gd-DTPA and dendrimer altered the surface properties of NGO-COOH, preventing weight loss at low temperatures (below 200 C). The results showed that amine-functionalized Gd-DTPA
and dendrimer were covalently conjugated on the surface of NGOCOOH. R1 relaxivities of Gd-DTPA and Gd-NGO were calibrated in vitro. The R1-signal increased in a highly linear manner with increased concentrations of Gd-DTPA or Gd-NGO (input concentrations of 0.000625, 0.00125, 0.0025, 0.005, 0.01, and 0.02 mM) as shown by the calibration curve (r2 ¼ 0.977 for Gd-DTPA, and r2 ¼ 0.998 for Gd-NGO). Both Gd-DTPA and Gd-NGO thus acted as dosedependent positive contrast agents. The relaxivity of Gd-NGO (7.59 mM1 s1) was higher than that of Gd-DTPA (2.65 mM1 s1)
Fig. 4. (A) TGA curves of NGO-COOH and Gd-NGO with a heating rate of 10 C/min in N2. (B) Dependence of R1 on Gd-DTPA and Gd-NGO concentrations; relaxivities were estimated as about 2.65 mM1 s1and 7.59 mM1 s1, respectively.
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at 7 T (Fig. 4B). This difference is most likely due to the strong interaction of a significant portion of Gd(III) ions with the entrapped NGO, which limits the water accessibility of the Gd(III) ions [31]. This calibration curve could be used for precise quantitation of Gd-NGO deposition in the brain. 3.2. Let-7g and EPI loading To detect loading of Let-7 and EPI onto Gd-NGO, we determined the zeta potential as a measure of the change in surface charge. GdNGO particles had a positive charge of 48.4 mV 5.8 mV. The charge changed to 18.7 mV 6.1 mV by adsorption of negatively charged Let-7g onto the surface of Gd-NGO. After coating with EPI and dendrimer, the charge returned to 33.4 mV 8.4 mV (Fig. 5A). These findings were consistent with successful loading of Let-7g and EPI onto the Gd-NGO, since Let-7g and EPI are negatively and positively charged, respectively. Drug delivery formulations with relatively small particles and positively charged surfaces have been shown to exhibit high cell penetration and accumulation rates [32]. We used FAM-labeled Let-7g to quantify the loading efficiency of Let-7g onto the Gd-NGO surface by fluorescence intensity. The excitation and emission wavelengths were 485 nm and 528 nm, respectively. The amount of loaded Let-7g reached saturation (0.15 nmole Let-7g/mg Gd-NGO) at 0.2 nmole of added Let-7g (up to 75% adsorption) (Fig. 5B). The loading efficiency was as high as 75% due to the high positive surface charge (48.4 mV) of Gd-NGO which favored adsorption of high quantities of negatively charged Let-7g. Loading of EPI onto Gd-NGO/Let-7g was investigated next, based on the characteristic UVevis absorbance peak of EPI at
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495 nm superimposed on the Gd-NGO/Let-7g/EPI absorption spectrum (Fig. 5C). The readout at 480 nm was compared with a standard curve generated from free EPI at different concentrations to calculate the unbound EPI concentration and thus the loading efficiency. The addition of 800 mg of EPI resulted in a saturating concentration of 606 mg EPI/mg Gd-NGO/Let-7g (up to 76%) (Fig. 5D). Efficient EPI loading onto NGO-COOH could be attributed to simple pep stacking under controlled pH conditions [18]. 3.3. Let-7g and EPI release study Another advantage of using Gd-NGO is pH-dependent drug release, based on weakening of hydrogen bonds at low pH. EPI was located on the outer layer, and was therefore expected to be released earlier than Let-7g. We found that about 15% of EPI was rapidly released from Gd-NGO/Let-7g during the first 18 h of incubation at pH 7.4, with essentially no further release over time (Fig. 6A). However, at pH 6.0 the amine groups of EPI were protonated, resulting in increased solubility and accelerated release from Gd-NGO/Let-7g [33]. The amount of EPI released reached nearly 41% within 24 h, and increased to 57% after another 60 h of incubation. Let-7g release from Gd-NGO at pH 7.4 was delayed until 10 h of incubation and only about 13% was released after 84 h. However, 39% was released at pH 6.0. Presumably Let-7g had to traverse the additional outer shell of EPI and dendrimer, which may have prevented contact with the surrounding acidic medium [34]. These data suggested that Let-7g would not be released from GdNGO/Let-7g/EPI too early, before reaching the cell. pH-dependent drug release by Gd-NGO/Let-7g/EPI could be exploited for
Fig. 5. (A) Zeta potential measurements. (B) Amount of Let-7g bound per 1 mg of Gd-NGO vs. amount of Let-7g added. Values are means SD (n ¼ 8). Inset, standard curve of fluorescence intensity of FAM-labeled Let-7g. (C) UVevis absorption spectra of NGO-COOH, Gd-NGO, Gd-NGO/Let-7g, and Gd-NGO/Let-7g/EPI. (D) Amount of EPI bound per 1 mg of Gd-NGO/Let-7g vs. amount of EPI added. Values are means SD (n ¼ 8).
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Fig. 6. (A) Cumulative Let-7g and EPI release (%) from Gd-NGO/Let-7g/EPI at 37 C in PBS (pH ¼ 6.0 or 7.4). Values are expressed as means SD (n ¼ 5). (B) Fluorescence microscopic images of U87 cells treated with GFP-pDNA (left) and Gd-NGO/GFP-pDNA (right) at 37 C for 12 h. (C) Fluorescence microscopic images of U87 cells treated with FAM-labeled Let-7g (left) and Gd-NGO/FAM-labeled Let-7g (right) 37 C for 48 h (Blue: nuclei; Green: GFP; Red: FAM). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
controlled drug and gene delivery since microenvironments in both the extracellular tissues of tumors and intracellular lysosomes and endosomes are acidic [35]. 3.4. In vitro transfection study Next we examined the ability of Gd-NGO to deliver pDNA or Let7g into brain tumor cells in vitro. U87 cells were transfected with free GFP-pDNA, free FAM-labeled Let-7g, Gd-NGO/GFP-pDNA, or Gd-NGO/Let-7g complexes. After 48 h of incubation, the cells were observed by fluorescence microscopy. The Gd-NGO/GFP-pDNA complexes showed a significantly higher transfection efficacy (12.7-fold increase) than free GFP-pDNA in U87 cells (Fig. 6B). Thus GFP-pDNA could be successfully carried into the cytoplasm by GdNGO, and then released from Gd-NGO to express GFP. FAM-labeled Let-7g was also delivered into the cells by Gd-NGO, and the miRNA was clearly visible in the nuclei, indicating the miRNA was successfully released from the carrier (Fig. 6C). The efficient transfection results were most likely due to increased electrostatic interactions between the many amino groups of dendrimers and negatively charged pDNA or miRNA, resulting in high adsorption onto the surface of Gd-NGO. In addition, Gd-NGO appeared to be taken up by an endocytotic pathway so the pDNA or miRNA could be delivered into the cells by any transfection protocol. 3.5. In vitro cytotoxicity study We investigated the effect of Let-7g carried into U87 cells by GdNGO after incubation for 48 h. Since the highly adsorptive surface of Gd-NGOs severely restricted RNA extraction, we were unable to use expression profiling to analyze the effect of miRNA. We therefore analyzed downstream protein targets of Let-7g to investigate its bioavailability and function when delivered by Gd-NGO. Since Let7g miRNA is known to reduce cell proliferation and invasion via
inhibition of the oncogenic Ras signaling pathway [5], the expression levels of Ras protein family members were analyzed (Fig. 7A). The expression levels of Pan-Ras proteins were unaltered by GdNGO alone, but were significantly reduced by Gd-NGO/Let-7g. The expression level was the same when EPI was co-incorporated (Gd-NGO/Let-7g/EPI), indicating that EPI was not involved in Ras protein inhibition. Incubation of U87 cells with naked Let-7g miRNA in the presence of transfection reagent had no effect on expression compared to the dramatic decrease of target protein expression with Gd-NGO-carried Let-7g, most likely the concentration of transfection reagent (Lipofectamine 2000) we used was too low to sufficiently transfect the Let-7g into U87 cells to inhibit the expression of Ras family proteins due to the overdose of Lipofectamine 2000 would cause cytotoxicity toward to the cells. We also investigated the viability of U87 cells treated with different materials. Gd-NGO and pure Let-7g were not cytotoxic toward U87 cells (Fig. 7B). Thus pure Let-7g was unable to enter the cells by itself to induce apoptosis. The concentration required for 50% inhibition of cellular growth (IC50) was higher for free EPI (greater than 6.4 mg/mL) than for Gd-NGO/EPI (equivalent of 3.4 mg/ mL EPI). The IC50 of Gd-NGO/Let-7g/EPI was significantly lower (equivalent of 1.3 mg/mL EPI and 16.3 nM Let-7g) than Gd-NGO/EPI (equivalent of 3.4 mg/mL EPI) and Gd-NGO/Let-7g (equivalent of 23.8 nM Let-7g), presumably because of inhibition of the oncogenic Ras signaling pathway by Let-7g. The combination of chemotherapy and gene-targeting therapy thus resulted in better inhibition of cancer cell growth at a lower dose, indicating that this delivery system could reduce the dose of chemotherapeutics and the accompanying serious side-effects. 3.6. In vivo MRI The bloodebrain barrier (BBB) prevents larger molecules from entering the brain parenchyma, thus protecting the brain from toxic
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Fig. 7. (A) Analysis of protein expression downstream of Let-7g miRNA. U87 human glioma cells were co-cultured with control (no substance added), Lipofectamine 2000 þ Let-7g (9.375 nM), Lipofectamine 2000 þ scrambled sequence, Gd-NGO, Gd-NGO/Let-7g (equivalent to 9.375 nM Let-7g), Gd-NGO/scrambled sequence, Gd-NGO/Let-7g/EPI (equivalent to 9.375 nM Let-7g and 1.5625 mg/mL EPI). Gd-NGO/Let-7g and Gd-NGO/Let-7g/EPI significantly reduced the expression of oncogenic Pan-Ras protein. (B) Relative cell viability of U87 cells for 24 h after drug treatment. Values are expressed as means SD (n ¼ 8).
foreign substances. However, it also prohibits delivery of many potentially effective diagnostic or therapeutic agents or nanoparticles. Focused ultrasound (FUS) in the presence of circulating microbubbles can temporarily open the BBB of capillaries in the central nervous system (CNS) parenchyma. This FUS-induced BBB opening can be verified by MRI. Gd-NGO can be used as an effective MRI contrast agent to monitor the location and extent of FUS-induced BBB opening and to
quantify the concentration of EPI/Let-7g delivered into the brain. The Gd-NGO still can be significantly accumulated in the brain, although the Gd-NGO did not be conjugated specific targeting ligands (i.e. EGFR), likely because we used the FUS to open the BBB and allow the Gd-NGO can flow into the brain. Therefore, the BBBopened area was clearly visible at increased contrast intensity in T1-weighted images and R1 maps (Fig. 8A). Gd-NGO could also be observed in the same way as Gd-DTPA in the FUS BBB-opened areas
Fig. 8. (A) Typical MRI contrast-enhanced T1 images of Gd-DTPA and Gd-NGO before and after FUS exposure and R1 maps (after FUS exposure) in normal animals. (Yellow arrow: BBB-opened area). (B) Fluorescence images of tumor tissue: control group (without any treatment; top) and Gd-NGO/FAM-labeled Let-7g treated group. (green: FAM-labeled Let-7g; blue: nuclei). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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in subtracted T1 images. In addition, R1 maps could be used to quantify the concentration and determine the distribution of either Gd-NGO or Gd-DTPA. The signal of Gd-NGO was in fact stronger than that of Gd-DTPA, presumably because of the 2.9-fold higher contrast ability of Gd-NGO compared to Gd-DTPA, and because of the Gd-NGO would be easily accumulated in the brain. We next investigated the transfection of Let-7g in tumor tissue; the GdNGO/FAM-labeled Let-7g was injected in mice via tail vein after FUS treatment. The mice were sacrificed after 12 h of post-injection, the fluorescence microscopy of tumor tissue slides also confirmed that the FAM-labeled Let-7g (green color) could not only be delivered into brain tumor tissues but also successfully transfected in the tumor cells (Fig. 8B). Gd-NGO thus not only provides an important means to simultaneously deliver chemotherapeutics (EPI) and miRNA (Let-7g) for cancer treatment, but can also be monitored by MRI, permitting quantification of drug/miRNA delivery in real time in vivo. 4. Conclusion An MRI-detectable, drug and miRNA carrying vector (Gd-NGO) was developed to effectively load EPI and Let-7g miRNA (Gd-NGO/ Let-7g/EPI) and transfer them into cancer cells. The transfer process was monitored by fluorescence confocal microscopic imaging in glioma U87 cells. The net positive charge density of Gd-NGO resulted in concentrated binding and efficient delivery of GFPpDNA into cells. Gd-NGO was also capable of efficient and simultaneous delivery of EPI and Let-7g miRNA into cells to destroy the DNA and knockdown expression of Ras family proteins, respectively. Moreover, Gd-NGO could be detected by MRI to identify the tumor area and quantify the concentration of therapeutics within the tumor. Taken together, these promising results suggest the potential use of Gd-NGO complexes as efficient non-viral drug and gene delivery vectors for future chemogene therapy applications. Acknowledgements We thank the National Science Council of the Republic of China, National Health Research Institutes of Taiwan, Chang Gung Memorial Hospital, Industrial Technology Research Institute (103A0045J2), and National Tsing Hua University (103N2753E1) for financial support (NSC 102-2221-E-007-003-(102), NSC 1022622-E-007-024-CC3, NSC 102-2314-B-182A-068-MY3, NSC 1022120-M-182A-002-CC1, NHRI-EX103-10004NI, CMRPG3D0571, CMRPG3B1012, CMRPG392103, CORPG3C0041, CMRPG390071, CMRPG380571, 103A0045J2, 103N2753E1). The authors thank the Boost Program from Low Carbon Energy Research Center of National Tsing Hua University; Chang Gung Memorial Hospital Animal Molecular Imaging Center for assistance with 7T-MRI and Microscopy Core Laboratory. References [1] Niidome T, Huang L. Gene therapy progress and prospects: nonviral vectors. Gene Ther 2002;9:1647e52. [2] Wells DJ. Gene therapy progress and prospects: electroporation and other physical methods. Gene Ther 2004;11:1363e9. [3] Dong H, Ding L, Yan F, Ji H, Ju H. The use of polyethylenimine-grafted graphene nanoribbon for cellular delivery of locked nucleic acid modified molecular beacon for recognition of microRNA. Biomaterials 2011;32:3875e82. [4] Karlsen TA, Brinchmann JE. Liposome delivery of microRNA-145 to mesenchymal stem cells leads to immunological off-target effects mediated by Rig-I. Mol Ther 2013;21:1169e81. [5] Johnson SM, Grosshans H, Shingara J, Byrom M, Jarvis R, Cheng A, et al. RAS is regulated by the let-7 microRNA family. Cell 2005;120:635e47. [6] Roush S, Slack FJ. The let-7 family of microRNAs. Trends Cell Biol 2008;18: 505e16.
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