Article pubs.acs.org/Biomac
Toward Chemical Perfection of Graphene-Based Gene Carrier via Ugi Multicomponent Assembly Process Aram Rezaei,*,†,‡,§ Omid Akhavan,*,†,∥ Ehsan Hashemi,§ and Mehdi Shamsara§ †
Department of Physics, Sharif University of Technology, P.O. Box 11155-9161, Tehran, Iran Institute for Nanoscience and Nanotechnology, Sharif University of Technology, P.O. Box 14588-89694, Tehran, Iran ‡ Nano Drug Delivery Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran § National Research Center for Transgenic Mouse and Animal Biotechnology Division, National Institute of Genetic Engineering and Biotechnology, P.O. Box 14965-161, Tehran, Iran ∥
S Supporting Information *
ABSTRACT: The graphene-based materials with unique, versatile, and tunable properties have brought new opportunities for the leading edge of advanced nanobiotechnology. In this regard, the use of graphene in gene delivery applications is still at early stages. In this study, we successfully designed a new complex of carboxylated-graphene (G-COOH) with ethidium bromide (EtBr) and used it as a nanovector for efficient gene delivery into the AGS cells. G-COOH, with carboxyl functions on its surface, in the presence of EtBr, formaldehyde, and cyclohexylisocyanide were participated in Ugi four component reaction to fabricate a stable amphiphilic graphene-EtBr (AG-EtBr) composite. The coupling reaction was confirmed by further analyses with FT-IR, AFM, UV−vis, Raman, photoluminescence, EDS, and XPS. The AG-EtBr nanocomposite was able to interact with a plasmid DNA (pDNA). This nanocomposite has been applied for transfection of cultured mammalian cells successfully. Moreover, the AG-EtBr composites showed a remarkable decreased cytotoxicity in compared to EtBr. Interestingly, the advantages of AG-EtBr in cell transfection are more dramatic (3-fold higher) than Lipofectamine2000 as a commercial nonviral vector. To the best of our knowledge, this is the first report in which EtBr is used as an intercalating agent along with graphene to serve as a new vehicle for gene delivery application.
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INTRODUCTION Gene transfection is a revolutionary therapeutic strategy in recent years, bringing many fascinating ideas and opportunities for treatment of diseases caused by genetic disorders.1−3 Transfection of genetic materials, including DNA, RNA, and oligonucleotides, into a specific cell type permit the selective inhibition or expression of genes for therapeutic purposes.4,5 However, transfer of naked nucleic acids across the cell to the nucleus cannot be successful easily due to several cellular barriers such as cell membranes, endosomal degradation and nucleases activities.6 Consequently, the assistance of suitable vehicles, such as viral and nonviral vectors can help cellular uptake of nucleic acids. Dangerous effects of viral vectors, such as carcinogenesis, pathogenicity, broad tropism, and immunogenicity,7 as well as difficult scaling up, encourage efforts to develop synthetic nonviral vectors, which are suitable to carry larger genetic materials and also easier to synthesize than that of viral vectors with high safety, efficiency, and specificity.3 A diverse collection of synthetic nonviral gene carriers, including dendrimers,8 liposomes,9 polymers,10,11 and nanomaterials12−15 (e.g., gold, silica nanoparticles and carbon nanotubes), has been developed for therapeutic purposes exhibiting remarkable © XXXX American Chemical Society
transfection efficiency for many eukaryotic cells. In this regards, for the unique chemical and physical properties of graphenebased materials, they have wide applications in biomedical fields such as biosensors, tissue engineering, cellular imaging, drug delivery, and cancer treatment.16,17 Graphene and its derivatives possess an ultrahigh surface area that are characterized to hydrophobic and hydrophilic regions, and can be served as potential sites for specific modification to enhance their biological application. Dai and colleagues demonstrated that nanographene oxide (NGO) sheets functionalized by polyethylene glycol (PEG) acquired a great potential to deliver hydrophobic aromatic molecules such as camptothecin analogue to cancer cells.18 Wang and co-workers have founded that fluorescein-labeled aptamers complexed with NGO could be applied for intracellular tracking of biomolecules.19 Lu’s group proved that NGO could bind and protect oligo DNAs from nucleases and deliver them into cells.20 Zhi et al. used GO composed with Received: May 26, 2016 Revised: July 19, 2016
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DOI: 10.1021/acs.biomac.6b00767 Biomacromolecules XXXX, XXX, XXX−XXX
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PEI/poly(sodium 4-styrenesulfonates) (PSS) to deliver anticancer agents to overcome multidrug resistance in breast cancer cell line.21 PEGylated reduced GO (PEG-RGO) was used for RNA delivery into HeLa cells.22 In order to further improvement of gene transfection, Feng et al. explored a novel complex of GO with PEI and PEG (NGO-PEG-PEI) to enhance DNA loading capacity and physiological stability.23 This composite showed superior gene delivery efficiency, and reduced cytotoxicity compared to free PEI and GO-PEI. Wang et al. reported encapsulation of gold nanomaterial in GO shell could improve gene delivery compared to gold nanoparticle or nanorods and PEI in HeLa cells.24 More recently, an injectable gelatin hydrogel-based gene delivery system was developed using PEI-GO complexed with VEGF gene for angiogenesis and cardiac repair.25 These studies show that graphene-based materials with a wide range of physicochemical characteristics are potential carriers for efficient delivery of genetic materials to desired cells.26 Ethidium bromide (EtBr) is the most commonly used fluorescent dye for detecting nucleic acids after electrophoresis. This charged flat compound is an intercalating agent that acts by inserting itself into the spaces between the nucleotide base pairs of a DNA duplex.27 The nature of the DNA/EtBr interaction and subsequent inhibition of DNA replication or further protein synthesis make EtBr as a biohazard material.28,29 Despite this serious toxicity, EtBr is still applied in biological studies because it is less expensive than other similar compounds like SYBR-based dyes. In addition, EtBr is widely used as a probe for topological and dynamic studies of DNA due to high fluorescent quantum yield.30 Glazer and co-workers reported a new class of ethidium homodimer that was used successfully to detect and quantify DNA fragments with picogram sensitivity.31 Recent applications of bisintercalators are included in simultaneously detecting live and dead cells,32 sizing of individual double-stranded DNA fragments by flow cytometry,33 fluorescent labeling of DNA to automated gel electrophoresis apparatus,34 and the study of DNA−protein interactions.35 Considering the tremendous intrinsic tendency of EtBr to interact with DNA and also its strong penetration ability into cell membrane might increase EtBr biological application by discovering strategies to decrease cytotoxicity of this component. In the present study, we attempted to develop a novel graphene-EtBr/DNA complex as a nanovector for gene delivery purpose. For this application, EtBr was covalently grafted on carboxylated-graphene oxide (G-COOH) via Ugi four component assembly process (Ugi 4-CAP) between formaldehyde, EtBr, cyclohexylisocyanide, and G-COOH in water under mild conditions to fabricate a novel 2-D nanovector for gene transfection. In this way, we found that G-COOH is able to participate in an isocyanide four-component reaction in the presence of EtBr to form a stable amphiphilic graphene-EtBr (AG-EtBr) composite. This nanocomposite has been applied for transfection of cultured mammalian cells successfully. Moreover, the AG-EtBr composites show a remarkable decreased cytotoxicity in comparison to EtBr solution. To the best of the authors’ knowledge, this study is the first report on the application of a nanocomposite material consisting of intercalating agent and graphene as a new vehicle in gene delivery system.
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EXPERIMENTAL SECTION
Materials and Characterization. All chemical materials for GO and AG-EtBr syntheses were purchased from Aldrich and Merck. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotics (penicillin and streptomycin), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and Lipofectamine2000 were purchased from Invitrogen. Agarose (molecular biology grade) was purchased from Sigma. The pEGFP-C1 plasmidexpressing enhanced green fluorescent protein (EGFP) was obtained from Clontech Laboratories, Inc. AGS cell line was obtained from National Cell Bank of Iran. The samples were characterized by scanning electron microscope (TESCAN Vega Model) equipped with an energy dispersive X-ray spectroscopy measurement system. The Fourier transform infrared (FT-IR) spectra of dried materials were recorded using the KBr pellet method by Bruker Vertex 70/70v FT-IR spectrometers. The hydrodynamic size distribution and Zeta potentials of the samples were evaluated by using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, U.K.). The UV−vis absorption spectra were recorded by a spectrometer PerkinElmer UV−vis (Model Lambda 950). Raman spectra were achieved on Almega Thermo Nicolet Dispersive Raman Spectrometer equipped with an Nd:YLF excitation source operating at wavelength of 532 nm. The XPS were performed by a hemispherical analyzer having an Al Kα X-ray source (hν = 1486.6 eV). pEGFP reporter gene expression was observed under Nikon Eclipse Ti−U inverted microscope. Ethidium Bromide Functionalized Graphene Oxide (AGEtBr) via Ugi Multicomponent Assembly Process (Ugi 4-CAP). The mixture of ethidium bromide (1.1 mmol), 126 μL of cyclohexylisocyanide, G-COOH (2 mL, 0.5 mg/mL), and formaldehyde (1 mmol) was stirred at 25 °C for 2 h, passed from a 0.22 μm filter (Millipore), and then the solid was washed thoroughly with HCl (100 mL, 5%), water (200 mL), and ethanol (50 mL, 70%). The solid was dispersed in water by sonication slightly. A series of AG-EtBr nanocarriers was obtained using various EtBr/G-COOH ratios (mmol/mg) of 0.1, 0.5, and 1, and the resulting complexes were named as AG-EtBr0.1, AG-EtBr0.5, and AG-EtBr1, respectively. Determination of Bromide Ion Concentration by Titration (Mohr’s Method). The amount of bromide ions per gram of graphene was measured in 0.5 mL solutions of AG-EtBr0.1, AGEtBr0.5, and AG-EtBr1 composites in water by the quantitative Mohr’s titration. In this method, silver nitrate was used as titrant and precipitated the bromide ions in the solution. The end point of the titration occurs When all bromide ions precipitated. Subsequently, additional silver ions reacted with potassium chromate, to form a redbrown precipitate of silver chromate. The Mohr’s titration is an argentometric titration that reveals quantitatively the presence of bromide ions, and therefore, the content of functional groups attached to graphene after functionalization.36 Cell Culture. The AGS cells were grown in DMEM, supplemented with 10% FBS and antibiotics (0.1 mg/mL streptomycin and 100 U/ mL penicillin). The cells were grown at 37 °C in a humidified incubator supplied with 5% CO2. Cytotoxicity Assay. Cell toxicity of the series of AG-EtBr nanocomposites were compared with G-COOH and EtBr by standard MTT assay as the manufacture’s protocol. Briefly, AGS cells were seeded onto 96-well plates at a density of 105 cells/well and cultured for 24 h in 100 μL of DMEM. The cells were treated with different concentrations of G-COOH, EtBr, and AG-EtBr complexes for 24 h. The medium was discarded and replaced by MTT solution in PBS (20 μL) and incubated for 4 h at 37 °C and 5% CO2. The absorbance was measured at 570 nm, and the cell viability was estimated as the percentage of alive cells in the treated wells to the control (nontreated wells). Also, cell morphology was visualized in treated samples after 24 h incubation with G-COOH, EtBr, and AG-EtBr1 on a Nikon fluorescence microscope. Ability of Nanocarrier for DNA Extraction. The complex formation capability of the synthesized nanocarriers with pDNA was evaluated on agarose gel electrophoresis. The complexes of pDNA with GO, G-COOH, AG-EtBr0.1, AG-EtBr0.5, and AG-EtBr1 were prepared by addition of the nanoparticle suspensions (20 μL, 300 μg/ B
DOI: 10.1021/acs.biomac.6b00767 Biomacromolecules XXXX, XXX, XXX−XXX
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Biomacromolecules mL) to the pDNA (5 μL, 500 ng/μL) and incubated for 20 min at 25 °C. The nanoplatelets were precipitated by centrifuging at 14000 rpm for 15 min. The supernatant was decanted away and the bonded DNAs were dissociated from nanoplatelets by adding elution buffer (NaCl 100 mM, Tris-HCl 10 mM, pH ∼ 9). Subsequently, 10 μL of the eluted sample were electrophoresed onto a 1% agarose gel at 110 V for 1 h in 0.5X TAE (Tris−acetate−EDTA) buffer. Then the gel was stained with EtBr and visualized on a UV illuminator. In Vitro Gene Transfection Assay. For transfection, the AGS cells were added to 24-well plates at a density of 105 cells/well and incubated overnight. The cells were treated with the complexes of DNA with GO, G-COOH, and AG-EtBr nanocomposites prepared at w/w ratios of 5 and 10 in serum-free DMEM for 8 h. The medium was supported with 10% FBS for another 16 h. The cells were washed with PBS thoroughly and cultured in the fresh medium with 10% FBS for a further 24 h. The naked pDNA, GO, and G-COOH were used as negative controls. Transfection with Lipofectamine2000 was carried out using manufacture’s protocol. The expression of EGFP was observed by a fluorescence inverted microscope under 490 nm excitation wavelength 48 h post-transfection. Total RNA Isolation and Real-Time RT-PCR. Total RNA was extracted from the treated and control samples using High Pure RNA Isolation Kit (Roche, Germany) in accordance with the manufacturer’s protocol. cDNA synthesis was performed by AccuPower RocketScript RT PreMix kit (Bioneer, Korea, K-2101) using random hexamer primers. The PCR reaction was prepared at the final volume of 15 μL by mixing 8 μL of 2× SYBR Green PCR Master Mix (Intron, Korea, 25344), 3 μL of RT product, and 0.2 μM of each primer. Real-Time PCR was carried out with an ABI System (Applied Biosystems StepOne, U.S.A.) under the following thermal conditions: 95 °C for 2 min, and 35 cycles of 95 °C for 10 s and 60 °C for 30 s. Statistical Analysis. All experiments were repeated at least three times. The student t-test and analysis of variance (ANOVA) were used for analysis of the experimental data. The SPSS 16.0 software was used for this analysis and p-value of
