Materials Science and Engineering C 39 (2014) 288–298

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Graphene and carbon nanotube nanocomposite for gene transfection L.M. Hollanda a, A.O. Lobo b, M. Lancellotti a, E. Berni c, E.J. Corat d, H. Zanin d,⁎ a

Laboratory of Biotechnology, Department of Biochemistry, Institute of Biology at UNICAMP, Rua Monteiro Lobato 255, Campinas, SP CEP 13083-862, Brazil Laboratory of Biomedical Nanotechnology, Institute of Research and Development at the UNIVAP, Av. Shishima Hifumi, 2911, CEP: 12244-000 Sao Jose dos Campos, SP, Brazil c Biological Chemistry Laboratory, Department of Physical Chemistry, Institute of Chemistry at UNICAMP, R. José de Castro, Campinas, SP CEP 13083-970, Brazil d Associated Laboratory of Sensors and Materials of the INPE, Av. dos Astronautas 1758, Sao Jose dos Campos CEP: 12227-010 SP, Brazil b

a r t i c l e

i n f o

Article history: Received 8 April 2013 Received in revised form 26 January 2014 Accepted 1 March 2014 Available online 12 March 2014 Keywords: Graphene oxide Gene transfer Carbon nanotubes Composite Cell viability Plasma

a b s t r a c t Graphene and carbon nanotube nanocomposite (GCN) was synthesised and applied in gene transfection of pIRES plasmid conjugated with green fluorescent protein (GFP) in NIH-3T3 and NG97 cell lines. The tips of the multiwalled carbon nanotubes (MWCNTs) were exfoliated by oxygen plasma etching, which is also known to attach oxygen content groups on the MWCNT surfaces, changing their hydrophobicity. The nanocomposite was characterised by high resolution scanning electron microscopy; energy-dispersive X-ray, Fourier transform infrared and Raman spectroscopies, as well as zeta potential and particle size analyses using dynamic light scattering. BET adsorption isotherms showed the GCN to have an effective surface area of 38.5 m2/g. The GCN and pIRES plasmid conjugated with the GFP gene, forming π-stacking when dispersed in water by magnetic stirring, resulting in a helical wrap. The measured zeta potential confirmed that the plasmid was connected to the nanocomposite. The NIH-3T3 and NG97 cell lines could phagocytize this wrap. The gene transfection was characterised by fluorescent protein produced in the cells and pictured by fluorescent microscopy. Before application, we studied GCN cell viability in NIH-3T3 and NG97 line cells using both MTT and Neutral Red uptake assays. Our results suggest that GCN has moderate stability behaviour as colloid solution and has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity and good transfection efficiency. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The greatest advantage of nanotechnology lies in its potential to create novel structures with enhanced abilities to translocate through cell membranes, as well as increased solution stability, and bioavailability of biomolecules, by improving their delivery efficiency [1]. Gene delivery is an emerging tool to obtain expression products and assign work to a nucleic acid sequence to achieve on/off order of the endogenous genes or repair missing/defective genes for therapeutic intervention [2–4]. Methods of gene transfection for cells and tissues range from bacterial to mammalian. Effective virus-based systems carry risks, and thus, efficient synthetic systems that are non-toxic need to be found. Non-viral vectors such as cationic polymers, lipids, or peptides have several advantages such as low immunogenicity, target-cell specificity, and relative safety profiles. However, their low transfection efficiency compared with virus systems combined with their high toxicity is a big obstacle for their application [2,5]. Therefore, extensive effort has been done to develop new materials for high efficient gene transfection with low cytotoxicity. Carbon-based materials, including graphene oxide (GO) and carbon nanotubes (CNTs) are considered attractive candidates for biomedical applications such as scaffolds in tissue ⁎ Corresponding author. E-mail address: [email protected] (H. Zanin).

http://dx.doi.org/10.1016/j.msec.2014.03.002 0928-4931/© 2014 Elsevier B.V. All rights reserved.

engineering, substrates for stem cell differentiation, and parts of implant devices [6]. GO and CNTs are topic of extensive interest in the nanomaterial world, because of their outstanding properties such as high surface area, lightweight, exceptional mechanical elasticity, large carrier mobility, biocompatibility, facility for functionalisation, and low thermal and electrical resistivity [7]. In addition, GO and CNTs are currently being used in engineered tissues [6,8], implants [6,9], diagnostic tools and chips [6,10,11], biological imaging [6,12], drug delivery carriers [6,13], and antibacterial materials [6,14]. However, one problem that limits their use in nanobiology and nanomedicine is that pristine graphene and raw CNTs are hydrophobic. Those materials precipitate in such solvents as result of aggregation. The surface material functionalisation is the primary key to allow their effective application. In addition, several studies suggest low biocompatibility of CNTs, while others give the opposite conclusion [15–18]. Other studies found an obvious preference of cell growth on a CNT surface. Several studies highlighted CNT and other carbon based materials interfering with cytotoxic dyes, commonly used to study cell integrity, viability, and proliferation [19–24]. The toxicity of CNTs is attributed to their physicochemical properties, including structure, length and aspect ratio, surface area, degree of aggregation, extent of oxidation, surface topology, bound functional group(s), the manufacturing method, concentration, and dose offered to cells or organisms [1,25–28]. CNTs can elicit toxicity through membrane damage, DNA damage, oxidative stress, changes in mitochondrial activities,

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Fig. 1. Typical SEM images of GCN.

altered intracellular metabolic routes, and protein synthesis [1,29–34]. The most common mechanisms of CNT cytotoxicity also encompass apoptosis and necrosis [1,17,23,35]. However, CNT cytotoxicity is significantly controversial, with a large number of studies reporting altered toxic responses to CNTs both in vitro and in vivo [1,29,36–38]. In addition, the question remains if the cytotoxicity is due to CNT itself, or to its contaminants, such as metallic particles or amorphous carbon structures, generated simultaneously to the CNT production process. The studies that brought good cell viability used some kind of purification or functionalisation of the CNT. Some authors suggest that functionalisation is necessary to increase cell viability [1,39]. On the other hand, others show that functionalisation increases cytotoxicity [1,40]. There are some alternatives to improve their dispersion and remove metallic nanoparticles, but it is inconvenient to manipulate strong chemical oxidants, which are dangerous and require appropriate disposal. However, the oxygen plasma treatment is a simple, low cost, and green methodology to improve dispersibility and wettability. Oxygen plasma etching has been extensively studied for selective dispersion control [41], due to its ability to attach oxygen functional groups on material surfaces. Furthermore, oxygen plasma etching causes the exfoliation of CNTs, opening their walls and exposing their fundamental structure: graphene. Following this methodology, we could produce exfoliated graphene and carbon nanotube nanocomposites (GCN), which are dispersible in water. Furthermore, functionalised CNTs (fCNTs) with DNA have been shown to enhance stability [1,42,43]. DNA can bind to SWCNTs, forming tight helices around them [44], or can form non-covalent conjugates with CNTs [1,45]. CNTs wrapped with flavin mononucleotide and DNA enhances dispersion of these nanotubes [1,46,47]. DNA-functionalised CNTs can be used as biological transporters and as biosensors [1,48]. DNA-encased MWCNTs were more effective than plain MWCNTs against malignant tissues when tested in vivo for their thermal ablation capability [1,49]. DNA-CNTs could penetrate lymphocytes instantaneously with a needle-like mechanism, thus reducing cytotoxic effects [1,50]. fCNTs were found to be similar to cell-penetrating proteins, because they can penetrate cells without endocytosis [51]; however, the internalisation of nanomaterials depends on the type of functionalisation process [1]. Moreover, the interactions between proteins and CNTs could play a key role in the biological effects of CNTs [52,53]. A π–π stacking occurs between CNTs and aromatic residues (Trp, Phe, Tyr) of proteins, enhancing their adsorptivity and biocompatibility, which renders them less toxic compared to pristine CNTs [1,54–56] The CNT–protein

nanoconjugates are very beneficial in biosensor fabrication [57], drug delivery [58], and cancer therapy [1,56]. For the first time, we analysed the cell viability by (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) and Neutral Red assay and demonstrated that GCN is not cytotoxic. In addition, we inserted into the NIH-3T3 and NG97 cell the GCN nanocomposites,

Fig. 2. Typical Raman spectra of MWCNT and GCN.

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Table 1 Evaluation of elements using energy dispersive spectroscopy (EDS). Element

Series

Net

Wt.%

Atom%

Error (wt.%)

87.54 1.58 9.16

94.97 1.29 2.14

10.97 0.70 0.41

Graphene/carbon nanotubes nanocomposite C K 25,407 78.75 O K 1798 18.25 Fe L 139 0.31

83.05 14.45 0.07

9.88 3.57 0.05

As-grown multi-wall carbon nanotubes C K 25,536 O K 152 Fe L 1504

functionalised with pIRES plasmid. The complex plasmid-GCN could penetrate these cell lines instantaneously with a needle-like mechanism, thus reducing cytotoxic effects, and transfecting these lineages with fluorescent protein only expressed in the cell. The GCNs were probably dispersed in water and conjugated with the pIRES plasmid resulting in a helical wrap. As shown in this work, GCNs are linked with the plasmid in the potential zeta assay, probably in the same way that was observed by Tasis et al. [45].

2. Experimental 2.1. Synthesis of multi-walled carbon nanotubes (MWCNT) The MWCNTs were prepared using a mixture of camphor (85% wt) and ferrocene in a thermal chemical vapour deposition (CVD) furnace, as reported elsewhere [59]. The mixture vapourised at 220 °C in an antechamber, and then, the vapour was carried by an argon gas flow at atmospheric pressure to the chamber of the CVD furnace set at 850 °C. The time elapsed during the process used to produce the MWCNTs was only a few minutes.

2.2. Preparation of graphene and carbon nanotubes nanocomposite (GCN) The removal of the catalytic particles from the MWCNTs was performed by acid treatment. The samples were subjected to ultrasonication for 1 h in 10 M of HCl at 150 °C, then washed intensively in water, and dried. Incorporating oxygen-containing groups was carried out in a pulsed-direct current plasma reactor with an oxygen flow rate of 1 sccm, at a pressure of 85 mTorr, −700 V and with pulse frequency of 20 kHz [60]. We have developed an apparatus that shakes and deagglomerates the carbon nanotube powder during oxygen plasma etching. The oxygen plasma etching was performed up to 1 h.

Fig. 3. FTIR spectra of as-grown MWCNT and GCN nanocomposites.

Fig. 4. Apparent negative zeta potential (a) and the average particle sizes distribution of the water dispersible GCN (b) using conventional sonication and ultra-power sound irradiation.

2.3. Characterisation of GCN nanocomposites The nanocomposites were characterised by several techniques, including high resolution scanning electron microscopy (HR-SEM), energy-dispersive X-ray (EDS) and Raman spectroscopies. Zeta potential and particle size were analysed by dynamic light scattering (DLS)

Fig. 5. Zeta frequency of plasmid, GCN, and their conjugates.

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(Zetasizer, Malvern — UK). For morphological and Raman characterization the samples were dropped onto titanium substrate. Morphological analyses were performed with JEOL 6330 — operated at 10 kV and coupled with an energy dispersive X-ray spectrometer (EDS) for chemical analysis, operating with a Si(Li) detector with an energy resolution of 126 eV. Raman spectra were recorded at ambient temperature using a Renishaw microprobe system, employing an argon laser for excitation (λ = 514.5 nm) with a laser power of approximately 6 mW. Fourier transform infrared spectroscopy (FTIR: Spotlight 400 — Perkin-Elmer) was used to identify the oxygen content groups on GCN. All measurements were conducted at room temperature. The size distribution and zeta potential of the GCNs were measured by DLS at 150 μg mL−1 in 1 mM of KCl solution (pH 7.4). The solution was deagglomerated in two different ways, i) conventional sonication for up to 60 min, and ii) using an ultra-power irradiation at 200 W for up to 60 min. Then samples were placed in polystyrene cuvette and measures were performed 2 h after re-suspension.

2.4. Biological in vitro assays 2.4.1. MTT and Neutral Red (NR) assay We purchased mouse fibroblasts NIH-3T3 cell lines from ATCC-CCL1658 and obtained human-derived glioma NG97 cell lines, which are from a patient diagnosed with glioblastoma multiform of the right temporal lobe. These cell lines were used to examine the cytotoxicity and gene transfection of plasmid-GCN substrates [63]. Before testing, we dispersed the GCN nanocomposites 1 mg mL− 1 in culture medium. After, we separately plated both cells lines in 96-well plates. We used a density of 1 × 105 cells mL−1 to 1 × 107 cells mL−1 per well, respectively. They were incubated at 37 °C, in a humidified incubator with 5% CO2, for 24 h. Then, the medium was replaced and the test was repeated with different GCN nanocomposite concentrations in the range from 3.9 to 500 μm mL−1, which was added to the wells in triplicate for each concentration. Before the analyses, we removed GCN nanocomposites after 24 h incubation. After this, we washed each well 3 times with 0.1 mL of PBS Buffer (137 mM NaCl, 10 mM phosphate, KCl 2.7 mM, and a pH of 7.4). For MTT assays, 0.2 mL of RPMI1640 medium (without FBS and antibiotic) containing the dye MTT (0.5 mg mL−1) was added. After incubation for 3 h at 37 °C, the medium with dye was removed and 0.2 mL of ethanol was carefully added to solubilise the blue formazan (yielded from MTT reduction by viable cells). The plates were shaken for 10 min, and the absorbance for each well was read in a spectrophotometer ELx800 Absorbance Microplate Reader (BioTek, USA) at λ = 570 nm [64]. For the NR assay, we removed the different medium containing the dye and washed the wells rapidly with 0.2 mL of calciumformaldehyde solution (10 mL of 40% formaldehyde, 10 mL of CaCl2, 10 mL deionised water). The calcium–formaldehyde solution was discarded and 0.2 mL of ethanol–acetic acid was added (1 mL of glacial acetic acid, 100 mL of ethanol 50%). The plate was kept for 15 min at room temperature and the absorbance for each well was read in a spectrophotometer ELx800 Absorbance Microplate Reader (BioTek, USA) at λ = 540 nm [65]. For both assays, we expressed the values as percentages compared to the control and cells not exposed to test agents. For statistical analysis we used GraphPad InStat version 3.06. All experiments of MTT and NR were conducted in triplicate. Data from each assay were analysed statistically by ANOVA. Multiple comparisons among groups were determined with Tukey test. Differences were considered significant when the p value was less than 0.05 [66].

2.4.2. Gene transfection We compared the efficiency gene transfection between Effectene Transfection Reagent® (Qiagen, USA) and GCN nanocomposites. For this, we incubated NIH-3T3 and NG97 cell lines at density of 8 × 104 cells/mL in 24 well plates, with 350 μL of culture medium for 24 h at 37 °C in 5% CO2. The gene delivery was done with Effectene Transfection Reagent® (control), GCN nanocomposite, and Negative Control. We did not performed the Statistical analysis due to the specific procedure of this assay. For Effectene Transfection Reagent®, we added 1 μg of plasmid DNA (Clontech, USA) into 8 μL of Enhancer belonging to Effectene kit. For GCN nanocomposites, 1 mg of plasmid DNA (Clontech) was added to nanocomposite suspension 10 or 100 mg mL− 1 diluted in RPMI1640 containing 10% FBS and 1% penicillin/streptomycin. For both assays, the solution was vigorously stirred with the aid of vortex for 1 s (Effectene kit) and 10 s (GCN nanocomposites) and incubated at room temperature for 5 min (Effectene kit) and 30 min (GCN nanocomposites), respectively. Then, 10 μL of Effectene was added and again the solution was vigorously stirred for 10 s and incubated for 10 min at room temperature. Next, 1 mL of RPMI1640 medium containing 10% FBS and 1% penicillin/streptomycin was added. Finally, the solution was homogenised using a micropipette five times and added in triplicate into a 24 well plate with no coverslip. We used an optical microscope with and without fluorescence for gene delivery characterisation. The plate was incubated in a humidified cell culture incubator for 24 h at 37 °C in 5% CO2. Then, it was evaluated using a fluorescence inverted Nikon Eclipse TS100 microscope (Nikon, USA) and photographed with a Nikon DS-RI1 (Nikon, USA) coupled to the microscope. The images were taken with the software NIS Elemente AR 3.1 (Nikon, USA).

3. Results and discussion Fig. 1(a–h) shows typical morphology of GCN. Fig. 1(a & e) is top views of two different regions of GCN, which are magnified at higher resolutions to better visualization of the effect of the oxygen plasma etching at MWCNT. Fig. 1(b) shows higher resolution image of a delimited region in Fig. 1(a), revealing that the oxygen plasma etching is able to exfoliate MWCNT tips. In Fig. 1(b), there are other two inserted boxes (α & β, from the left to right), which are magnified and presented in Fig. 1(c & d), respectively. In the same way, Fig. 1(f) shows higher resolution image of a region in Fig. 1(e) and it is a pre-visualization of another two regions (ϕ & Δ, from the left to the right), which are shown in details in Fig. 1(g & h). From all those SEM micrographs, we could observe that oxygen plasma exfoliated the carbon nanotube tips, opening its walls and exposing its fundamental structure: graphene. It is well known that carbon nanotubes are graphene sheets rolled into cylinders. Moreover, graphene sheet rolling up into a tube is a standard picture to illustrate carbon nanotube formation. Nanotube tips are defective and interact directly with plasma, causing tip exfoliation. Further, the nanotube sidewalls were not significantly affected by both hydrochloric acid treatment and oxygen plasma etching that we performed. However, depending on plasma conditions we could etch the whole nanotubes or simply attach oxygen groups to them without exfoliation. At higher plasma pressures (higher than 180 m Torr) or for longer process times, the erosion was completed. With diluted plasma exfoliation do not show up but wettability increased significantly because of oxygen group attachment. The GCN composites were achieved at quite specific conditions. In addition, those micrographs point out GCN as porous structure, which the real surface area and specific density were

Fig. 6 Evaluation of cytotoxicity of GCN nanocomposites using NIH-3T3 cell. NHI-3T3 cells were exposed to different concentrations of GCN nanocomposites for 24 h and 48 h. The values are expressed as percentage of MTT reduction (a) and NR uptake assay (b) compared to the control, where cells were not exposed to the GCN nanocomposites. The experiment was conducted in 96-well microplates, and the results represent the means and standard deviations of the experiment. The statistical analysis for MTT, using the software GraphPad InStat 3.06, showed that *p b 0.05 or ***p b 0.01. The statistical analysis for NR was ***p b 0.01.

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measured by BET and helium pycnometry to be ~ 38.5 m2/g and ~2.3 g/cm3, respectively. Fig. 2(a & b) shows first- and second-order Raman scattering spectra of MWCNT and GCN. The deconvolutions were performed using Lorentzian shapes for the D, G and G' bands, and Gaussian shape for bands around 1250 (#), 1480 (*) and 1611 cm−1 (D' shoulder) [60, 67–70]. The D band is usually attributed to the disorder and imperfection of the carbon crystallites. The G band is assigned to one of the two E2g modes corresponding to stretching vibrations in the basal plane (sp2 domains) of single crystal graphene [70]. The high intensity G' band reveals that these materials present high structural quality [67]. In the GCN first order Raman, for appropriate deconvolution fitting, two Gaussian peaks centred at around 1250 and 1480 cm− 1 were added necessarily. Probably the shoulder has its origin in double resonance process, because its Raman shift (∼1200 cm−1 #) is a point on graphene phonon dispersion curves [69]. The origin of the 1480 cm− 1 (*) band is probably correlated with the polar groups grafting onto GCN surfaces [67,70]. The energy dispersive spectroscopy identified the chemical elements of as-grown MWCNT and GCN. The results are presented in Table 1. After hydrochloric acid treatment, the percentages of the iron atoms decreased from 9 to 0.3. After oxygen plasma etching, the percentage of oxygen atoms increased from 1.58 to 18.25 [60]. Fig. 3 shows the FTIR spectrum of GCN nanocomposite. The presence of carboxyl \C_O and \C\O (υ = 1741 and 1398 cm−1, respectively), aromatic \C_C (υ = 1623 cm − 1 ), C\O (υ = 1288 cm − 1 ), and alkoxy \C\O (υ = 1076 cm − 1 ) indicates the existent of oxygencontaining functional groups attached in the sample [71-73]. In addition, a higher intensity of the bands localized in the range of 2910–2990 cm− 1 is assigned to C–H methyl groups. Size and zeta potential of nanoparticles are recognised as being very important parameters for the success of transfection process [74]. Fig. 4 shows (a) apparent negative zeta potential and (b) the average particle size distribution of the dispersible water in the GCN nanocomposite. The negative zeta potential ranged from − 37.4 mV to − 39.1 mV, which means moderate stability behaviour of the colloid solution. GCN processed for long periods in plasma etching has zeta potential slightly higher than those processed for short periods of time. The zeta potential of GCN in water is −37.4 after 10 min and increased to −38.6 mV after 60 min of oxygen plasma etching. The dispersion method seems to have a strong influence on the zeta potential. For GCN submitted to 10 min etching and dispersed in water by conventional sonication, the zeta potential was −37.4 and −39.6 mV for dispersion by ultra-power irradiation, respectively. We concluded that there is a slight tendency to increase the zeta potential for a more negative value by applying both dispersive method and long periods of plasma etching. Fig. 4b shows that the average particle sizes ranged from 50 to 2000 nm. The larger sizes are related to nanoparticle agglomeration, and the smaller sizes may have derived from both nanotube diameter and graphene sheet sizes. This considerable difference in measured sizes ranging from 50 to 2000 nm, which shows bi-modal behaviour presented by zeta potential, is mainly due to the higher ratio aspect of the CNT. During the zeta potential measurement, the laser absorption does not change the diameter and length of the CNTs. Although the ultrasound contributed to disperse and break down of the agglomeration, Fig. 4(b-inset) shows that after re-suspension the nanoparticles tend to agglomeration again. The re-agglomeration of the particles may be due to van der Waals forces, which in the long-term (days) will make them decant. The zeta potential dependence with the frequencies is presented in Fig. 5 for GCN, plasmid, and mixtures of them. The well-defined zeta potential frequency for GCN, plasmid, and mixtures of them can be observed. These results are evidence that plasmid is conjugated with GCN in these two concentrations of nanocomposites (0.01 and 0.1 mg/mL). These differences confirm that the carboxyl and carboxylate group attached in GCN are simultaneous efficient to dispersion and

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conjugation of the plasmid. The dispensability in aqueous media is a key requirement for applying GCN nanocomposite to gene transfection. On the other hand, the van der Waals electrostatic forces between those carboxyl and carboxylate groups on GCN and plasmid may induce the adsorption mechanism between them. After the structural, morphological, and water solubility characterisation of GCN, we presented for the first time a consistent cytotoxicity study, comparing two different cell lines known as NIH-3T3 and NG97. NIH-3T3 cells were chosen because they are frequently used in studies of cell functions such as cell shape change, adhesion, movement, and proliferation. They have also been used to demonstrate the key roles of cytoskeletal components in cell adhesion, division, growth [6, 61] and as control in transfection assay [6]. NG97 is an excellent and permanent material for studying the biology of malignant gliomas [62]. Therefore, if plasmid can be transfected into this cell, in the future, genes could be transfected to fight this disease. Colorimetric assays have largely been used to evaluate cell viability of CNT in various concentrations in aqueous solution, using several cell lines. In this way, two different colorimetric assays (MTT and NR) were used. Fig. 6 shows the (a) MTT and (b) NR results of NIH-3T3 cell line after 24 and 48 h exposed to GCN nanocomposite. Clearly, there is no cytotoxicity detected in any of the GCN concentrations. This is a sign of low cytotoxicity of GCN, evidencing the GCN cell viability for NIH-3T3 cells. The MTT statistical analysis showed that the results are extremely significant with p b 0.05 and p b 0.001 between control and GCN 24 h and 48 h, respectively. The statistical analysis for NR (Fig. 6b) confirmed the previous (Fig. 6a) results with p b 0.001 for all GCN concentrations. Fig. 7 demonstrates MTT and NR assay results of the GCN nanocomposites incubated with NG97 cell line. Fig. 7a shows that the GCN nanocomposites were selectively cytotoxic (DL ≥ 50%) for concentration of 0.500 mg/mL, at 24 h, for glioblastoma, because the same concentration did not affect the NIH-3T3 cell line. In the MTT assay, the same concentration did not affect the NIH-3T3 cell lineage, and Fig. 7b shows that no concentration was cytotoxic. The difference between the results of MTT and NR is due to differences in signalling pathways for apoptosis presented by these two techniques. The statistical analysis for MTT, using the software GraphPad InStat 3.06, showed that the results are extremely significant with p b 0.01 between control and GCN (24 h). Statistical analysis between control and GCN (24 h) and between GCN (24 h) and GCN (48 h) is extremely significant with p b 0.001 (Fig. 7a). However, for NR, the statistical difference was not significant (Fig. 7b). Colorimetric assays also have largely been used to evaluate cell viability of CNT dispersed (various concentrations) or on structures, using several cell lines. De Nicola et al. evaluated the cell viability using MWCNT dispersed in cell culture, produced by electric arc discharge and CVD process. Independent of the CNT type, apoptosis was not induced over the basal level up to 48 h of exposure [75]. Mwenifumbo et al. showed that osteoblastic cell cultures have a high metabolic activity on MWCNT constructs produced by the CVD method, using MTT and LDH assays [76]. Kalbacova et al. found a decrease of about 15% in osteoblastic metabolic activity by MTT assay for SWCNT dispersed in culture, compared to the control, but inferior to the Ti6Al4V (20%). This decrease was associated with the impurities of CNT synthesis [77]. Zhang et al. showed a reduction of the primary osteoblast cell viability (60%) using MTT assays with CNT dispersed in culture (concentration of 50 μg/mL) [78]. However, several studies have highlighted the interference of CNTs and other carbon based materials with cytotoxicity dyes, including MTT, NR, WST-1, CB, and AB [18–20,24]. These observations indicate that CNT agglomerates, interfering with reaction products (specifically enzymes) or cell culture medium, which causes erroneous reading of these colorimetric assays. Woörle-Knirsch et al. used three assays to evaluate the cytotoxicity of dispersed SWCNTs (MTT, WST-1 and LDH) with A549, ECV, and NR-8383 cells. The MTT assay produced a false

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Fig. 7. Evaluation of cytotoxicity of GCN nanocomposites exposed to NG97 cell line. NG97 cells were exposed to different concentrations of GCN nanocomposites for 24 h and 48 h. The percentage of (a) MTT reduction and (b) NR uptake assays are compared to the control, where cells were not exposed to the nanocomposite. The experiment was conducted in 96well microplates, and the results represent the means and standard deviations of the experiment. The statistical analyses for MTT were carried out using the software GraphPad InStat 3.06. The results showed **p b 0.01 or ***p b 0.01.

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positive to toxicity, while WST-1 and LDH provided high viability. The problem with MTT assay is that single-walled CNTs (SWCNT) bind to the MTT formazan crystal and stabilise their chemical structure, and as a consequence, these crystals cannot be solubilised [18]. Casey et al. checked the interference of dispersed SWCNT with CB, AB, NR, MTT, and WST-1 dyes with A549 cells. For checking of CB interference with SWCNTs, two sets of experiments varying the concentration of the dyes were performed. The absorbance was observed (1) only with the dye (control) and (2) with the dye and SWCNTs in a 1:1 mass ratio, and expressed as per cent of control. CB, AB, MTT, and WST-1 assays found reduction in the associated absorbance at all concentrations of set 2, with significant toxicity at different SWCNT concentrations: above 0.25 mg/mL for CB, 0.4 mg/mL for AB, 0.003 mg/mL for MTT, and 0.4 mg/ml for WST-1. However, an increase of absorbance was verified with NR dyes, with SWCNT concentration above 0.08 mg/ml [20]. This indicates that the interaction of CNT with the dye produces different responses, depending upon the nature of the dye. However, from the results presented here, any influences were due to higher water solubility of the GCN nanocomposites in all the exposed concentrations of the GCN. Thus, it appears that the GCN nanocomposites act differently in the compartments of the cells and in cell lines, because they affect more in the mitochondria and lysosomes of NG97 cells than the NIH-3 T3 cells. Thus not all the results of cell viability studied by different types of assays present the same result. This behaviour indicates a possible centre of action of the compound in each cell line. Moreover, the difference in the results of the cytotoxicity assays may be due to the variation in the death-signalling pathway activated by the GCN nanocomposites in these two cell lines. Therefore, cellular transfection assays were conducted using GCN concentrations that do not affect cell viability, which were 0.01 mg/mL and 0.1 mg/mL. Our in vitro experiments showed that the GCN nanocomposite were as functional as the Effectene kit for transfection assay. Transfection of plasmid pIRES to NIH-3T3 cells was compared between the commercial Effectene kit and two concentrations of GCN nanocomposites (0.01 and 0.1 mg/mL). Fig. 8 presents the optical microscopy with or without fluorescence images of plasmid pIRES transfected to NIH-3T3 cells using (a,e) no carrier agent; (b,f) commercial Effectene kit; and (c,g) 0.01 mg/mL and (d,h) and 0.1 mg/mL of GCN nanocomposites. The fluorescence in the images is an indication of the cell transfection, because the pIRES plasmid only synthesizes the green fluorescent protein in the cell. Therefore, GCN is as efficient as the Effectene kit for

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transfection of pIRES plasmid in NIH-3T3 cells independent of GCN concentrations tested. However, Fig. 9 shows a different behaviour. Fig. 9 presents the optical microscopy with or without fluorescence images of plasmid pIRES transfected to NG97 cells using (a,e) no carrier agent; (b,f) commercial Effectene kit; and (c,g) 0.01 mg/mL and (d,h) and 0.1 mg/mL of GCN nanocomposites. The higher concentration of GCN was less effective than the lower concentration for plasmid transfection in NG97 cells. While at 0.01 mg/mL, the transfection was as efficient as the Effectene kit, but at higher concentration, we did not observed good transfection activity. We conclude that the GCN nanocomposites at higher concentration can act as a selective antitumor agent due to its cytotoxicity. The higher aspect ratio can cause the agglomeration of them and induce apoptosis and necrosis. However, lower concentrations below 0.062 mg/mL, which are non-cytotoxic for NG97 cells, can be used as a gene carrier agent. Besides this, GCN will be easily an endocyte by the tumoral cells releasing interference RNA (RNAi). RNAi is a natural phenomenon that silences mRNA, inhibiting its gene expression during the translation or transcription [79]. Since, they are expressed from plasmid DNA containing a promoter of RNA polymerase III, we can use this mechanism to decrease the expression of mRNA targets that are carcinogenic or have an important role in tumour development. GCN emerges as a novel nanomaterial for various biomedical applications and can be used to deliver a variety of therapeutic agents. Furthermore, functionalised GCN nanocomposites are promising novel materials for a variety of biomedical applications. These potential applications are particularly enhanced by their ability to penetrate biological membranes with relatively low cytotoxicity [1]. Several functionalisation procedures should be also performed to improve their effectiveness in carrying exogenous genomic material as shown in Figs. 8 and 9. As shown by Lundqvist et al. and Cedervall et al., interactions between proteins and CNTs could play a key role in the biological effects of CNTs. A π–π stacking occurs between CNTs and aromatic residues (Trp, Phe, Tyr) of proteins, enhancing their adsorptivity and biocompatibility, which renders them less toxic compared to pristine CNTs [1,52,53]. Besides proteins, the CNTs can also combine with the plasmid DNA, thus increasing the stability of CNTs, as demonstrated by Sing et al. [42] and Liu et al. [43]. Contrasting what occurs with proteins, the DNA can bind to SWCNTs, forming tight helices around them [44] or can form noncovalent conjugates with CNTs [1,45]. DNA-functionalised CNTs can be used as biological transporters and biosensors [1,48]. For

Fig. 8. Optical microscopy with or without fluorescence images of plasmid pIRES transfected to NIH-3T3 cells using (a,e) no carrier agent; (b,f) commercial Effectene kit; (c,g) 10 μg/mL and (d,h) 100 μg/mL of GCN nanocomposites.

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Fig. 9. Optical microscopy with or without fluorescence images of plasmid pIRES transfected to NG97 cells using (a,e) no carrier agent; (b,f) commercial Effectene kit; (c,g) 10 μg/mL and (d,h) 100 μg/mL of GCN nanocomposites.

example, Gosh et al. demonstrated that the DNA-encased MWCNTs were more effective than plain MWCNTs against malignant tissues when tested in vivo for their thermal ablation capability [1,49]. While Cheung et al. showed that DNA-CNTs could penetrate lymphocytes instantaneously with a needle-like mechanism, thus reducing cytotoxic effects [50]. In Firme et al., the functionalised CNTs were found to be similar to cell-penetrating proteins, because they can penetrate cells without endocytosis [51]; however, the internalisation of nanomaterials depends on the type of functionalisation process [1]. In this paper, we described the preparation of dispersible GCN nanocomposites and presented its characterisation. This material was able to carry plasmid DNA into the analysed cells of NIH-3T3 and NG97. As with other nonviral gene delivery vectors, the charge ratio between the carboxylic and carboxylate groups at the surface of the GCN tips and the phosphate groups of the DNA backbone seems to be a determining factor in the level of gene expression. This probably occurs because the plasmid is wound on GCN nanocomposites forming by tight helices or by establishing noncovalent conjugates with them as previously demonstrated by Nel et al. [55] and Kolosnjaj-Tabi et al. [1,45]. Also, we proposed that the GCN enhancement of cell membrane interactions due to electrostatic forces, increased cellular uptake by endocytosis, and improved trafficking to the nucleus. For this, the different sizes do not influenced in the gene transfection if consider that the GCN is a simple vector to deliver the plasmid DNA for the cells. GOs have been investigated as a drug delivery vehicle for various kinds of drugs including hydrophobic small molecule drugs and siRNAs [79,80]. Although cellular responses of GO have not been intensively investigated yet, a paper reported that GO induced oxidative stress and the decrease of viability of A549 cells, and the effect of GO were dependent on the dose and size [81]. Another report, suggested that small GO exhibited higher hemolytic activity than large GO [82]. However, we have shown, in addition to the zeta potential results, that the plasmid is conjugated with GCN nanocomposites. Thus, the plasmid-GCN can enter the cell by endocytosis instantaneously with a needle-like mechanism, reducing cytotoxicity effects in lower concentrations such as 0.1 mg/mL and 0.01 mg/mL. The study reported herein constitutes the first example of using GCN nanocomposites as components for engineering a novel nanotube-based gene delivery vector system. The GCN nanocomposites formed supramolecular complexes with plasmid DNA through ionic interactions. These complexes are able to bind and penetrate within cells by what seems to be an endosome-independent mechanism. GCN with plasmid DNA were able to facilitate higher DNA uptake and gene expression in vitro than could be achieved with DNA

alone. In view of these interesting properties, the delivery of other types of therapeutic agents by GCN through noncovalent interactions of the nanotubes with the agent can be envisaged. 4. Conclusion We presented, for the first time, a new graphene and carbon nanotube nanocomposite (GCN), which is water dispersible; presents nonor low cytotoxicity for NIH-3T3 and NG97 line cells, and has good efficiency for gene transfection at low GCN concentration. More specifically, all the transfection results are comparable to commercial Effectene kit. This GCN nanocomposite was selectively cytotoxic, because at high doses it reduced the cell viability of NG97 cells while it had no effect in NIH-3T3 cells. For nanocomposite production, the oxygen plasma etching causes a selective exfoliation of carbon nanotube tips, exposing graphene there. The GCN has scalable production. Acknowledgements The authors would like to thank Brazilians agencies CNPq (202439/ 2012-7); FAPESP (2011/17195-3) and (2011/17877-7) for financial support. We also thank the researchers Dr. Silvia Mika Shishido and Dr. Daisy Machado for constructive discussion. Special thanks for Maria Helena Andrade Santana and Daniela Araujo for the ZetaSizerNano analysis, Elaine Minatel for the help in transfection assay, and Alene AlderRangel for reviewing the English language of this manuscript. References [1] S. Vardharajula, S.Z. Ali, P.M. Tiwari, E. Eroglu, K. Vig, V.A. Dennis, et al., Functionalized carbon nanotubes: biomedical applications, Int. J. Nanomedicine 7 (2012) 5361–5374. [2] X. Zhou, F. Laroche, G.E.M. Lamers, V. Torraca, P. Voskamp, T. Lu, et al., Ultra-small graphene oxide functionalized with polyethylenimine (PEI) for very efficient gene delivery in cell and zebrafish embryos, Nano Res. 5 (2012) 703–709. [3] E. Mastrobattista, M.A. van der Aa, W.E. Hennink, D.J. Crommelin, Artificial viruses: a nanotechnological approach to gene delivery, Nat. Rev. Drug Discov. 5 (2006) 115–121. [4] A.O. Lobo, M.A.F. Corat, E.F. Antunes, S.C. Ramos, C. Pacheco-Soares, E.J. Corat, Cytocompatibility studies of vertically-aligned multi-walled carbon nanotubes: raw material and functionalized by oxygen plasma, Mater. Sci. Eng. C-Mater. 32 (2012) 648–652. [5] D. Putnam, Polymers for gene delivery across length scales, Nat. Mater. 5 (2006) 439–451. [6] S.R. Ryoo, Y.K. Kim, M.H. Kim, D.H. Min, Behaviors of NIH-3T3 fibroblasts on graphene and carbon nanotubes: proliferation, focal adhesion, and gene transfection studies, ACS Nano. 4 (2010) 6587–6598.

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