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Dual-functionalized graphene oxide for enhanced siRNA delivery to breast cancer cells
Colloids and Surfaces B: Biointerfaces 147 (2016) 315–325
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Dual-functionalized graphene oxide for enhanced siRNA delivery to breast cancer cells Rana Imani a,c , Wei Shao a , Samira Taherkhani d , Shahriar Hojjati Emami c , Satya Prakash (Professor) a,∗∗ , Shahab Faghihi b,∗ a Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering, Faculty of Medicine, McGill University, Montréal, QC H3A 2B4, Canada b Tissue Engineering and Biomaterials Research Center, National Institute of Genetic Engineering and Biotechnology, Tehran 14965/161, Iran c Department of Biomedical Engineering, Amirkabir University of Technology, Tehran 15875/4413, Iran d Department of Computer and Software Engineering, Institute of Biomedical Engineering, Polytechnique Montréal, Montréal, Québec H3C3A7, Canada
a r t i c l e
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Article history: Received 18 May 2016 Received in revised form 7 August 2016 Accepted 11 August 2016 Available online 12 August 2016 Keywords: Gene delivery Nano-carrier Graphene oxide Amphiphilic polymer Cell penetrating peptide siRNA
a b s t r a c t The aim of this study is to improve hydrocolloid stability and siRNA transfection ability of a reduced graphene oxide (rGO) based nano-carrier using a phospholipid-based amphiphilic polymer (PL-PEG) and cell penetrating peptide (CPPs). The dual functionalized nano-carrier is comprehensively characterized for its chemical structure, size, surface charge and morphology as well as thermal stability. The nano-carrier cytocompatibility, siRNA condensation ability both in the presence and absence of enzyme, endosomal buffering capacity, cellular uptake and intracellular localization are also assessed. The siRNA loaded nano-carrier is used for internalization to MCF-7 cells and its gene silencing ability is compared with AllStars Hs Cell Death siRNA as a model gene. The nano-carrier remains stable in biological solution, exhibits excellent cytocompatibility, retards the siRNA migration and protects it against enzyme degradation. The buffering capacity analysis shows that incorporation of the peptide in nano-carrier structure would increase the resistance to endo/lysosomal like acidic condition (pH 6–4) The functionalized nanocarrier which is loaded with siRNA in an optimal N:P ratio presents superior internalization efficiency (82 ± 5.1% compared to HiPerFect® ), endosomal escape quality and capable of inducing cell death in MCF7 cancer cells (51 ± 3.1% compared to non-treated cells). The success of siRNA-based therapy is largely dependent on the safe and efficient delivery system, therefore; the dual functionalized rGO introduced here could have a great potential to be used as a carrier for siRNA delivery with relevancy in therapeutics and clinical applications. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Gene therapy is a powerful and novel strategy that has a great potential to cure genetic based diseases particularly cancer [1]. Recently, ribonucleic acid interference (RNAi) has been attracted many attention as a promising candidate for the treatment of a variety of diseases that suffer from expression of undesired genes [2]. Small interfering RNA (siRNA) can suppress the expression of target
∗ Corresponding author at: Tissue Engineering and Biomaterials Research Center, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran 14965/161, Iran. Tel.: +98 21 44787386; fax: +98 21 44787386. ∗∗ Corresponding author at: Department of Biomedical Engineering, Faculty of Medicine, 3775 University Street, Montréal, QC H3A 2B4, Canada. E-mail addresses: [email protected] (S. Prakash), [email protected], [email protected] (S. Faghihi). http://dx.doi.org/10.1016/j.colsurfb.2016.08.015 0927-7765/© 2016 Elsevier B.V. All rights reserved.
pathogenic genes using degradation of the target messenger RNA (mRNA). However, duo to the limited delivery of siRNA through cell membrane, various nano-delivery systems have been developed to facilitate siRNA access to the cytoplasm of targeted cells [3–5]. A nano-carrier system is also essential to provide in vitro and in vivo biostability and to ease cellular uptake [6]. Graphene and its derivatives e.g. graphene oxide (GO) and reduced graphene oxide (rGO) have been considered as a biocompatible and conjugable nano-carriers for delivery of targeting compounds and biomolecules [7]. Lately, GO/rGO has been utilized for siRNA delivery and showed a great success in gene knockdown. Tripathi et al. covalently grafted polyethylenimine (PEI) to GO and evaluated delivery of GFP specific siRNA with 70% suppression of the target gene expression [8]. In another study, Cheng et al. have shown that a polyethylene glycol (PEG)-PEI-grafted graphene/Au composites could efficiently load anti-apoptosis Bcl-2 siRNA and
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successfully down-regulated the Bcl-2 expression in HL-60 cells [9]. Zhang et al. have used PEI for covalent functionalization of GO which was followed by loading of hTERT siRNA and targeting HeLa cells [10]. Feng et al. have reported synthesis of ultra-small size dual-polymer-functionalized GO (GO-PEG-PEI) and effective delivery of Polo-like kinase 1 (Plk1) siRNA using laser irradiation [11]. It is also demonstrated that GO-PEG-PEI is an excellent nanocarrier for delivery of Stat3 siRNA which could lead to significant regression in tumor growth [12]. Finally, Zhang et al. were investigated the sequential delivery of siRNA and anticancer drugs via PEI-functionalized GO [13]. Non-covalent functionalization of graphene with hydrophobic interactions or electrostatic is the most effective and nondestructive method as enables the modification of graphene without alteration of its chemical structure [14]. While much research have been devoted to covalent functionalization of graphene sheets using PEI or PEG for siRNA delivery, there has been little investigation of non-covalent functionalization of graphene as a siRNA nano-carrier using amphiphilic polymers. We have previously synthesized GO, optimized the concentration of carboxylic acid on its surface [15], and successfully used it for plasmid delivery [16]. Here we introduced a novel reduced graphene oxide (rGO) based nano-carrier which was double functionalized by a phospholipid-based amphiphilic polymer and cell penetrating peptide (CPPs) for enhanced hydrocolloid stability and high siRNA transfection ability. After a comprehensive characterization, the positively charged nano-carrier was evaluated for delivery of siRNA into the MCF-7 breast cancer cells and the knockdown of the cell survival related genes.
2. Materials and methods 2.1. Materials All the reagents used for synthesis and functionalization of rGO nano-sheets were purchase from Sigma Aldrich, Canada except otherwise indicated specifically. The MCF-7 breast cancer cell line was purchased from American Type Cell Culture (ATCC, Manassas, USA). Fetal bovine serum (FBS) and Dulbecco’s modified eagle’s medium (DMEM) were purchased from Invitrogen (Montreal, CA). The MTS viability assay kit was obtained from Promega (Madison, USA). The FITC-labeled scrambled siRNA and AllStars Hs Cell Death Control siRNA were supplied by Santa Cruz Biotechnology (Ontario, CA) and QIAGEN (Montreal, CA). HiPerFect® , was purchased from QIAGEN. LysoBright® and Hoechst 33342 staining reagents were bought from AAT Bioquest® (Montreal, CA) and GIBCO® -Life Technology (Montreal, CA). RNase A and ethidium bromide (EtBr) were supplied by Sigma (Montreal, CA). Ocataarginine (R8) cell penetrating peptide (MW 1267 g/mol- Supplementary data) was bought from AbbiotecTM (CA, Montreal).
2.2. Preparation of functionalized nano-carrier 2.2.1. Preparation of reduced graphene oxide nano-sheets (rGON) Graphene oxide nano-sheets (GON) were synthesized using Hummers’ method as previously described [15]. For more exfoliation and size reduction of GON, 1.2 g of sodium hydroxide (NaOH) was added to 10 mL of GON solution (1 mg/mL) followed by sonication for 3 h (MicrosonTM, XL2000, 100W, USA). After adjusting pH to 1, the solution was filtered and washed. The ultra-small nanoparticles were re-dispersed in DI water at concentration of 1 mg/mL. To reduce GON, 0.05% v/v of hydrazine monohydrate was added to 15 mL of GON (1 mg/mL) and the solution was heated up to 80 ◦ C for 4 h. The black precipitations were washed by DI water 5 times
using centrifugation filtration units (Amicon, 30 kDa) and freeze dried. 2.2.2. Functionalization of rGON To immobilize PL-PEG (Avanti Polar Lipids, Inc. US) on the surface of the rGON, 2 mg of rGON was partially dispersed in 8 mL of DI water under ultrasonication process (5 min). Subsequently, 2 mL of PL-PEG solution (3 mg/mL in DI water) was mixed with dispersed rGON. The mixture was dispersed under the bath sonication for 3 h. The suspension was washed 5 times by filtration (Amicon filters- 30 kDa) to remove excess non-bonded polymer. Finally, the ultracentrifugation (50Ti, Beckman Coulter, USA) at 20000 rpm for 1 h was used to obtain the stable supernatant which was referred as rGON-PLPEG. In the next step, octaarginine (R8) solution was added into the rGON-PLPEG solution at the final concentration of 1 mM and the mixture was sonicated for 1 h, then washed. The final functionalized double layer coated sample was referred as rGONPLPEG-R8. 2.3. Characterization of nano-carrier 2.3.1. Instrumentation FT-IR spectra were recorded by a Nicolet 6700 FT-IR spectrometer within the range of 3500–500 cm−1 at a resolution of 4 cm−1 . UV–vis spectra of specimens suspended in DI water were obtained in the range of 190–600 nm in UV–vis spectrophotometer (Cary100 bio, Varian Inc). The morphology and size of the nano-carriers were evaluated by an atomic force microscope (AFM, Nanoscope III Multimode,VEECO) in tapping mode. The single sheet morphology of GO before and after treatment with base was characterized by transmission electron microscopy (TEM, Philips169 CM200) operated at 200 kV. Thermal gravimeter analysis (TGA) was performed using TGA Q500 (TGA instruments Ltd., UK) under nitrogen atmosphere at a rate of 20 ◦ C/min. Finally, the size and surface charges were evaluated using size and zeta potential analyzer (Brookhaven Instruments Corporation, USA). 2.3.2. Peptide quantification Fluorescamine assay was utilized to measure peptide concentration [17]. Briefly, phosphate buffer (pH 7.4, 80 L) and 20 L of nano-carrier solution were mixed followed by addition of 32 L fluorescamine solutions (3 mg/mL in acetonitrile) to the mixture. The excitation wavelength was set at 400 nm and the fluorescence emission was recorded rapidly after the addition of reagent to the mixture at 480 nm using SpectraMax i3 Multi-Mode Microplate Reader (Molecular devices). To estimate peptide molarity in rGONPLPEG-R8 formulation, standard curve was plotted based on R8 solution in different molar ratio. 2.3.3. Stability and dispersibility assessments The ability of PL-PEG to disperse rGON in aqueous solution after the first layer of coating was assessed by ultracentrifugation (20000 rpm, 1 h) of the samples. The concentration of stable supernatants was determined using UV–vis spectrophotometer. Stability of rGON-PLPEG and rGON-PLPEG-R8 in buffer phosphate saline (PBS) and cell culture media in the presence of 10% serum was observed after 48 h incubation at room temperature. 2.3.4. Cell culture and MTS cell viability assay The MCF-7 breast cancer cells were cultured in DMEM supplemented with 10% FBS in a humidified incubator with 5% CO2 at 37 ◦ C. The cytotoxicity of rGON-PLPEG and rGONPLPEG-R8 was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay kit (Promega, Madison, USA) using the Cell Titer 96® . Briefly,
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triplicates of 1 × 104 cells were seeded in a 96-well plate and cultured for 24 h followed by treatment with samples at 10–250 g/mL (37 ◦ C). The MTS assay was performed based on the manufacturer protocol. The absorbance of formazan in cell culture was measured at 490 nm using Victor3 Multilabel Counter (Perkin Elmer, USA). The viability of cells was calculated as the percentage of viable cells to untreated cells as control.
plate with gentle shaking. After 4 h incubation, the cells were fixed with 0.3% glutaraldehyde in PBS for 5 min and stained with Hoechst® (0.5 g/mL in PBS) for 15 min followed by lysosomes staining using Lysobrigh® (0.1 g/mL in PBS) for 5 min. Confocal images were taken with a Zeiss LSM 510 microscope (Carl Zeiss, Jena, Germany) using × 60 oil-immersion objectives and captured and post-processed using Zeiss LSM 510 software.
2.3.5. Gel retardation assay For gel electrophoresis, 2% agarose gel was used to examine the affinity of siRNA for the nano-carrier. First, 50 ng FITClabeled siRNA were mixed with rNGO-PLPEG-R8 sample in different N/P (5, 10, and 15) ratios by gentle pipeting and incubation at room temperature for 30 min. The complexes were then electrophoresed in the agarose gel containing EtBr (0.5ug/mL) in the presence of 2 L dye (6×) with a TAE buffer at 100 V for 30 min. The gels were detected by an automatic digital gel image analysis system (Hercules, CA). Migration of genetic material condensed with functionalized-rGON was compared to the naked samples and HyPerfect® reagent as a positive control.
2.3.10. Cell death induction by siRNA delivery Cell death siRNA (cd-siRNA) was transfected against MCF-7 breast cancer cells by rGON-PLPEG-R8 in optimized N/P ratios. The cells were seeded into a 96-well plate at a density of 1 × 104 cells/well. After 24 h, an appropriate amount of the samples were mixed with 75 ng cd-siRNA in a 100 L medium without serum and left for 30 min at room temperature. The cells were washed with PBS and the above mixture was added drop-wise during gentle shaking. The HiPerFect® and naked cd-siRNA were used as positive and negative controls, respectively. The cell death caused by siRNA delivery was evaluated using MTS viability assay 72 h of transfection.
2.3.6. Buffering capacity of nano-carrier The capacity of the nano-carrier to buffer endosome in acidic condition were evaluated by acid-base titration and compared to PEI and NaCl solutions [18]. First, 2 mL of rGON-PLPEG-R8 solution (0.1 mg/mL) was adjusted to pH 10 by addition of 0.1N NaOH. The titration was performed with 5 L additions of 0.1 N HCl to reach to pH 3. A graph was plotted based on the pH and the amount of HCl used to reach to pH 3.
2.4. Statistical analysis
2.3.7. RNase protection assay 2 units of RNase A was added to the complex of nano-carrier and siRNA and incubated for 30 min at room temperature (naked siRNA was used as control). Subsequently, EDTA (4 L, 0.25 M) was added to the sample in order to inactivate the RNase A and continued to incubate for another 10 min. Finally, 5 L, 2 unit/L of heparin solution was added to the samples and incubated for 1 h followed by gel electrophoresis (30 min at 100 V) in 2% agarose gel with TBE buffer [8].
3.1. Structural characterization of rGO-based nano-carrier
2.3.8. Cell internalization MCF-7 cells were cultured in DMEM+ 10% FBS medium with density of 1 × 104 cells per well in a 96-well plate. After 24 h, 100 ng FITC-siRNA was mixed with an appropriate amount of sample (30 min, 25 ◦ C). The complexes were added to the cells drop-wise during of a gentle shaking. A commercial cationic lipid transfection agent, HiPerFect® , was used as positive control. After 12 h of incubation, the FITC-labeled siRNA samples were observed by fluorescent microscopy (Nikon, Eclipse, Te2000-4, Japan). The cellular uptake efficiency of the sample was calculated by the measurement of fluorescence intensity using a microplate reader (excitation: 480 emission: 520) and compared to that of positive control. The internalization efficacy of the nano-carrier was expressed as relative fluorescence intensity which was calculated based on Eq. (1): Internalizationefficacy = (Et −Eb )/Ec × 100
(1)
Where Et , Eb and Ec represent the average fluorescence emission of test samples, blank sample and positive control (HiPerFect® ), respectively. 2.3.9. Intracellular localization 100 ng FITC-siRNA was mixed with samples (in an optimized N/P ratio) and left for 30 min at room temperature. The complexes drop-wise were added to 5 × 103 MCF-7 cells in Petri dish
Statistical analysis was carried out using SPSS software (v 17.0; IBM New York, NY, USA) when statistical differences were detected, a t-Student test was performed. Data are reported as mean ± SD at a significance level of p < 0.05. 3. Results and discussion
3.1.1. FTIR spectroscopy The chemical characteristic of as-prepared GON and rGON was confirmed by FT-IR spectroscopy (Fig. 1a). The base-treated GON showed multiple peaks around 1060, 1250, 1365, and 1720 cm−1 which are assigned to vibrational modes of C O, C O C, C OH and C O in carboxylic acid and carbonyl moieties [19]. After the reduction reaction, the peaks of the oxygenated groups e.g. carboxyl in rGON were disappeared and the intensity of peaks between 900 and 1500 cm−1 decreased significantly [20]. In addition, the band at 3500 cm-1 which is assigned to the stretching mode of hydroxyl groups of GON became significantly weaker suggesting the removal of the hydroxyl groups. These observations confirm that most oxygen functionalities of the pristine GO were removed. After the surface coating of rGON by PL-PEG the FT-IR spectrum showed the peaks of PEG segments (-CH2-) at 2850 cm−1 . The Comparison between FTIR spectra of rGON-PLPEG, rGON and PL-PEG suggested that the polymer has been successfully immobilized on the surface of rGON. 3.1.2. Uv-vis spectroscopy analysis Upon reduction of GO nano-sheets, the brownish aqueous solution darkened and the reduced sheets were aggregated and precipitated. As shown in Fig. 1b, GON sheets display a maximum absorption peak at 230 nm (attributed to –* transitions of aromatic C C bonds) and a shoulder peak around 300 nm (attributed to n– * transitions of C O bonds) [17]. After GON reduction, the peak at 230 nm red-shifted to 258 nm and the shoulder peak at 300 nm was disappeared [22]. The UV–vis spectrum of the PL-PEG coated rGON which was dispersed in water presented similar features as that of polymer itself. The rGON characteristic peak around 260 nm was also detectable in rGON-PLPEG. The R8 peptide functionalized rGON-PLPEG spectrum showed a characteristic peak at ∼208 nm which is correlated to R8 guanidine groups. The R8 functionalized
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Fig. 1. The FTIR (a) Uv-vis (b) TGA (c) spectra of specimens.
sample showed both main peaks of rGON-PLPEG and R8 around 260 and 200 nm, respectively. 3.1.3. TGA analysis The TGA analysis (Fig. 1c) showed that a major mass loss occurs for the GON at 180 ◦ C presumably due to pyrolysis of the oxygencontaining functional groups while for the rGON no significant mass loss was detected up to 700 ◦ C [23]. This is an indication for successful reduction of GON and significant increase of thermal stability of the rGON [21]. The TGA was also used to estimate the content of stabilized polymer on the surface of rGON. The TGA curve for the polymer-functionalized rGON was shown in Fig. 1c. The PL-PEG had weigh loss of 95% in a nitrogen atmosphere at 700 ◦ C whereas functionalized rGON showed a different pathway which indicates that polymer were immobilized on the rGON. The polymer content in rGON-PLPEG was calculated around 40 wt% which provides evidence for PL-PEG immobilization on the surface of rGON via PL hydrophobic interaction with basal plane of rGON. 3.1.4. AFM and TEM analyses The results of AFM and TEM indicated that as prepared GON had a sheet-like morphology with the average size of 250–400 nm (Fig. 2a,b). The TEM image of GON showed a wrinkled sheet-like structure. The thickness of unmodified GO was estimated around 1–1.5 nm according to the height profile of AFM image which is corresponded to 1–2 layer of graphene sheets. The base treatment of GON was resulted in smaller exfoliated nano-sheets with the mean thickness of ∼0.8 nm that indicates the formation of single-
layered sheets (Fig. 2c). It has been previously shown that the oxidation/base treatment of graphene would form a single-sheet morphology with a size reduction [15]. The reduction reaction of the GON did not affect morphology of nano-sheets significantly (Fig. 2d). However, after immobilization of PL-PEG on the surface of rGON, the height profile of the nano-sheets was significantly changed as rGON-PLPEG showed 1 nm height increase as compared to rGON (Fig. 2e). It was confirmed that the polymer could successfully interact with rGON basal plane. By the addition of R8 to the sample the nano-sheets presented further round-shaped morphology while rGON sheets showed very sharp edges with flat surface (Fig. 2f). The thickness increase of the nano-sheets from 2.6 to 3.8 nm indicated successful integration of peptide on the single sheet. 3.1.5. DLS and zeta potential analyses The size (effective diameter, polydispersity index (PDI)) and surface charges of samples were evaluated by size and zeta analyzer. Based on DLS analysis, rGON (146 ± 4.6 nm, PDI = 0.005) showed smaller size and narrower distribution than base-treated GON (180 ± 6.1 nm, PDI = 0.212). It should be noted that the size analysis of rGON was performed immediately after strong sonication duo to the fast aggregation of rGO nano-sheets. As expected the DLS analysis showed a significant size increase after the coating of rGON with PL-PEG (146 ± 4.6 to 188 ± 3.2 nm) which is corresponded to immobilization of polymer on the nano-sheets surface. After the peptide incorporation, the size of nano-sheets showed further increase to 246.7 ± 2.7 nm. Surface charge screening using
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Fig. 2. The morphological characterization of as-prepared GON; TEM (a) and AFM (b). The AFM images of base treated GON (c), rGON (d), rGON-PLPEG (e) and rGON-PLPEG-R8 (f) specimens.
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zeta analyzer demonstrated that significant increasing of zeta value from −45 to −25 mv and dramatically decreasing solubility also confirm proper reducing GON to rGON. The rGON-PLPEG showed the less negative charge around −20.55 ± 1.73 mv duo to NH2 functionality of PEG chains. The results showed that by introduction of R8 into the stabilized rGON formulation, the zeta potential was significantly increased from −20.55 ± 1.73 to +42.61 ± 2.02 mv which is a confirmation on the addition of positively charged R8 to the final structure. It was revealed that the R8 could significantly interact with rGON-PLPEG structure to cover the PL-PEG layer. 3.1.6. Fluorescamine assay In order to quantify the incorporated R8 peptide molarity in the functionalized rGON, FL-amine assay was used. The FL-amine would react with amino groups of R8 in aqueous solution and form a fluorescent component which can be detected at 480 nm emission (excitation at 400 nm) and quantify using a standard curve. The amount of bonded R8 peptide (mol/mg) was calculated. The molarity of R8 was estimated around 1.2 mol/mg which was much higher than covalently conjugated R8 to GON that has been reported previously [16]. It is expected that the stabilized rGON could present a high ability to be internalized into the cells. 3.1.7. Hydrocolloidal stability assessment For biomedical applications, functionalized rGONs need to be stable in physiological fluids, such as PBS or serum containing media[21,22]. In gene delivery applications agglomeration and precipitation of nano-carriers in serum containing media would harshly affect the transfection efficacy and consequently limit their applications. Therefore, the stability of double layer coated rGON was investigated by incubation in PBS (0.01 M, PH7.4) and growth media containing 10% FBS for 48 h at 37 ◦ C. The stability of rGON-PLPEG-R8 sample was also tested by the size measurements through DLS (Table 1). The sample showed a slight size increase after 1 h incubation in cell culture media compared to DI water which could be duo to the protein absorption from the serum. The DLS results confirmed no significant size alteration after 48 h incubation at room temperature (258 ± 3.7 compared to 266 ± 7.4) which is an indication for a good colloidal stability of the sample in the presence of serum and salts. As mentioned before by the addition of R8 the zeta potential dramatically rose to positive values (-20.55 ± 1.73 to +42.61 ± 2.02 mv), however, it is believed that the stability of rGON-PLPEG in aqueous solution was largely supported by hydrophilic PEG chains that provide enough steric hindrance. The charge repulsion between cationic amine groups of PEG was also a great help for higher dispersability of rGON-PLPEG-R8 sample. 3.2. Functional characterization of nano-carrier 3.2.1. MTS assay To examine cytotoxicity, the MCF-7 cells were treated with different concentrations (200–10 g/mL) of rGON-PLPEG-R8 for 24 and 48 h. The results showed that cell viability remained above 85% even after the addition of 200 g/mL nano-carrier (Fig. 3a). There was a slight cell viability reduction after the addition of 100 and 200 g/mL of the nano-carrier by 5.5, 12%, respectively. However, the reduction in cell viability was not statistically significant. It can be seen that functionalization of rGON with PL-PEG and R8 could significantly improve the relative viability of nano-carrier. In particular peptide incorporated formulation showed superior cell proliferation rather than control (Fig. 3b). 3.2.2. Evaluation of buffering capacity The interaction of DNA or RNA loaded nano-carriers with cell membrane would typically lead to their endocytosis and entrap-
Fig. 3. Histogram represents cell viability based on MTS assay for rGON-PLPEG-R8 at different concentrations (10–200 g/mL) (a) and comparison between different formulations with the same concentration (50 g/mL) (b). All samples compared to the non-treated cells as control * p < 0.05.
ment within the cellular endosomal vesicles [23]. The amino groups of cationic polymers such as PEI act as a proton sponge and cause swelling and eventual rupture of the endosome. This would lead to release of the nano-vector into the cell cytoplasm, a process which is known as endosomal escape [24]. The surface chemistry of nano-carriers could affect their buffering capacity and proton sponge characteristic. It is believed that the charge and composition of the cationic polymer could regulate the endosomal escape capability [25]. To evaluate this, R8 functionalized rGON was titrated with HCl and the pH value was monitored. The PEI which is commonly considered as a golden cationic polymer and has a great buffering capacity was considered as positive and NaCl solution as negative controls. Interestingly, the sample showed a resistance to pH changes in acidic pH (6–4) that indicates an increase in proton absorption that is attributed to the R8 peptide contribution. The buffering capacity of rGON-PLPEG-R8 and PEI was in a similar range 6–4 (Fig. 4 top). However, the amount of HCl which was consumed to reduce the pH value of PEI solution from 5 to 3 was 45 L while rGON-PLPEG-R8 reached to pH 3 after adding 25 L HCl. This could be a reflection for the effective role of R8 as a buffering component in a strong acidic environment.
3.3. siRNA/nano-carrier complex formation 3.3.1. Retardation assay Gene loading and condensation capability of nano-carrier for siRNA was assessed using gel retardation assay (Fig. 4 down, a). The complex of rGON-PLPEG-R8 with siRNA was prepared at three N/P ratios (5, 10, and 15). The rGON-PLPEG-R8 formulation showed significant retardation of siRNA moving for N/P ratio of 10. The positive charge of R8 amine groups were completely neutralized by negative charges of phosphate groups in siRNA structure in N/P ratio of 10.
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Table 1 The DLS particle size measurement of the functionalized nano-carrier after incubation in cell culture medium and PBS for 48 h. Sample
rGON-PLPEG-R8
Effective diameter (nm) in DMEM
Effective diameter (nm) in PBS
After 1 h incubation
After 48 h incubation
After 1 h incubation
After 48 h incubation
255.1 ± 7.7
259.4 ± 4.3
248.2 ± 3.1
253.6 ± 6.7
Fig. 4. The buffering capacity of nano-carrier compared to PEI and NaCl solutions (Up). The gel retardation assay of nano-carrier at different N:P ratio (N:P = 0 (lane 1), N:P = 5(lane 2), N:P = 10 (lane 3) and N:P = 15 (lane 4)) (a). The RNase protection assay for nano-carrier at N:P = 10 (lane 1: nacked siRNA, lane 2: siRNA + RNase, lane 3: siRNA + nano-carrier, lane 4: siRNA + nanocarrier + RNase, lane 5: siRNA + nanocarrier + RNase + Heparin, lane 6: siRNA + nanocarrier + Heparin) (b).
3.3.2. Protection of condensed siRNA against nuclease degradation For a successful gene delivery, the carrier not only should effectively condense gene but also protect it against nuclease enzyme degradation [26]. Therefore, the ability of functionalized rGON to protect siRNA from nuclease degradation was examined by agarose gel electrophoresis. As it is shown in Fig. 4b (down), siRNA was completely degraded after incubation with enzymes (lane 2) compared to the nacked siRNA in the absence of enzyme (lane 1). As expected siRNA fully condensed and retarded in interaction
with nano-carrier (lane 3). In contrast, siRNA released from nanocarrier complexes and remained intact after enzyme treatment (lanes 4 and 5). The rGON-PLPEG-R8 formulation at N/P ratio of 10 could entirely protect siRNA from enzyme degradation. It was also revealed that after heparin treatment similar bonds were detected as naked siRNA (lanes 1 and 6) that suggests rGON-PLPEG-R8 nanocarrier could deliver more integral siRNA without degradation by enzymes.
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Fig. 5. The MCF-7 cell internalization of nacked FITC-siRNA (a) FITC- siRNA/nano-carrier complexes (b) and FITC- siRNA/HiPerFect® complexes (c) after 12 h. The internalization efficacy of rGON-PLPEG-R8 nano-carrier (d) compared to the HiPerFect® (* p < 0.05).
3.3.3. Cell internalization A key point for an efficient transfection with siRNA would be the delivery of the carrier across the cell membrane [4]. To study cell internalization ability of siRNA loaded rGON-PLPEG-R8, FITC-
labeled siRNA was complexed with the nano-carrier at N/P ratio of 10 and incubated with MCF-7 cells for 12 h. After incubation plenty of green dots were detected inside the cytoplasm of the cells compared to the treated cells with naked siRNA-FITC (Fig. 5a and b).
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Fig. 6. Confocal images of rGON-PLPEG-R8 nano-sheets uptake by MCF-7 cell after 4 h of incubation (up) and 12 h (down). The images show siRNA labeled-FITC (a), lysosome stained (b), nucleus stained with Hochest3342 (c), co-localization of siRNA and lysosomal region (d-f).
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transfected by the cd-siRNA loaded nano-carrier which resulted in gene silencing and subsequent cell death. 4. Summary and conclusions
Fig. 7. Histogram represents cell viability based on MTS assay for MCF-7 cells treated with nacked cell death siRNA (cd-siRNA) and without cd-siRNA compared to HiPerFect® as control (* p < 0.05).
However, it can be seen that HiPerFect® delivered markedly higher quantity of siRNA to the MCF7 cells rather than rGON-PLPEG-R8 (Fig. 5c). Interestingly, the R8 coated sample showed comparable internalization potential compared to the HiPerFect® treated cells. For further confirmation fluorescence intensity of the samples were also measured by mico-plate reader after 24 h (Fig. 5d). The rGON-PLPEG-R8 sample showed 82 ± 5.1% internalization efficacy in delivery of FITC-siRNA compared to HiPerFect® that considered as control. These results showed an excellent ability of nano-carrier in transporting siRNA into the cells. 3.3.4. Confocal microscopy The MCF-7 cells were treated with FITC-siRNA/nanocarrier complex and imaged with laser confocal microscopy after nucleus and lysosome staining. As it can be seen in Fig. 6, after 4 h, the majority of the complexes (green dots) were detected in the cytoplasm of the cells. It can be seen that the most of the green dots were co-localized with red ones (endo/lysosomal compartments). After 12 h, the density of FITC-siRNA in the endo/lysosomal compartments was significantly declined whereas the green dots could be detectable and more spread in cytoplasmic regions. This could be translated to possible endosomal scape of nano-carrier duo to buffering capacity as it mentioned before. However, some overlapping of green and red dots was still detectable around the nuclease. This is evidence for endosomal scape capability of designed nanocarrier after localization inside endo/lysosomal compartments. This also shows the effective role of R8 peptide in providing buffering capacity and destabilization of lysosomal membrane [27]. 3.3.5. Gene transfection The AllStars Cell death control siRNA is a blend of highly potent siRNAs targeting ubiquitously expressed genes that are vital for cell survival. Knockdown of these genes induces a high degree of cell death which could be detected by light microscopy. In this study we utilized cell death siRNA to assess cell death induction through siRNA delivery using rGON-PLPEG-R8 nano-carrier[28]. The cells were transfected by rGON-PLPEG-R8 nano-carrier and the cell death was quantitatively evaluated by MTS assay. The results showed that designed formulation complexed with cdsiRNA induced cell death after 72 h. The Fig. 7 shows that delivery of bare nano-carrier into the cells did not affect their viability, however, when cd-siRNA was delivered into the cell cytoplasm by nano-carrier there was a significant decrease in cell viability. Even though HiPerFect® showed significant cell death compared to rGON-PLPEG-R8, it decreased cell viability even when it was delivered without cd-siRNA. This indicated that MCF-7 cells can be safely
Gene therapy is based on the introduction of nucleic acids typically either siRNA or pDNA into targeted cells through gene knockdown and expression. However nucleic acids are highly unstable and have little cell penetration ability. In present study, an efficient gene nano-carrier is introduced based on reduced graphene oxide that co-functionalized with an amphiphilic polymer (PL-PEG) and R8 cell penetrating peptide. The rGON-PLPEG-R8 remained stable in biological solution and exhibited excellent cell viability. Incorporation of R8 into the nano-carrier formulation provided highly positive surface charged which facilitate siRNA loading and condensation as well as cell penetration. At the optimal N/P ratio of rGON-PLPEG-R8/siRNA, the transfection efficiency of designed nano-carrier is similar to HiPerFect® , a commercial reagent for the transfection of siRNA into mammalian cells. Furthermore, confocal microscopy and buffering capacity assessments demonstrated that functionalized rGON had a potential for endosomal scape. In summary, the rGON-PLPEG-R8 showed a great potential as efficient and biocompatible siRNA delivery vector for gene therapy purposes. Conflict of interest There is no conflict of interest to declare. Acknowledgments The authors wish to acknowledge scientific help and guidance of Prof. Hojatollah Vali, McGill University, Montreal, Canada. SF also would like to thank partial financial support of this work by National Institute of Genetic Engineering and Biotechnology (940801-I-536). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.08. 015. References [1] S.L. Ginn, I.E. Alexander, M.L. Edelstein, M.R. Abedi, J. Wixon, Gene therapy clinical trials worldwide to 2012-an update, J. Gene Med. 15 (2013) 65–77. [2] B.L. Davidson, P.B. McCray, Current prospects for RNA interference-based therapies, Nat. Rev. Genet. 12 (2011) 329–340. [3] A. de Fougerolles, H.-P. Vornlocher, J. Maraganore, J. Lieberman, Interfering with disease: a progress report on siRNA-based therapeutics, Nat. Rev. Drug Discov. 6 (2007) 443–453. [4] J. Wang, Z. Lu, M.G. Wientjes, J.L.-S. Au, Delivery of siRNA therapeutics: barriers and carriers, AAPS J. 12 (2010) 492–503. [5] R. Molinaro, J. Wolfram, C. Federico, F. Cilurzo, L. Di Marzio, C.A. Ventura, M. Carafa, C. Celia, M. Fresta, Polyethylenimine and chitosan carriers for the delivery of RNA interference effectors, Expert Opin. Drug Deliv. 10 (2013) 1653–1668. [6] V. Labhasetwar, Nanotechnology for drug and gene therapy: the importance of understanding molecular mechanisms of delivery, Curr. Opin. Biotechnol. 16 (2005) 674–680. [7] H.Y. Mao, S. Laurent, W. Chen, O. Akhavan, M. Imani, A.A. Ashkarran, M. Mahmoudi, Graphene: promises, facts, opportunities, and challenges in nanomedicine, Chem. Rev. 113 (2013) 3407–3424. [8] S.K. Tripathi, R. Goyal, K.C. Gupta, P. Kumar, Functionalized graphene oxide mediated nucleic acid delivery, Carbon 51 (2013) 224–235. [9] F.-F. Cheng, W. Chen, L.-H. Hu, G. Chen, H.-T. Miao, C. Li, J.-J. Zhu, Highly dispersible PEGylated graphene/Au composites as gene delivery vector and potential cancer therapeutic agent, J. Mater. Chem. B 1 (2013) 4956–4962. [10] X. Yang, G. Niu, X. Cao, Y. Wen, R. Xiang, H. Duan, Y. Chen, The preparation of functionalized graphene oxide for targeted intracellular delivery of siRNA, J. Mater. Chem. 22 (2012) 6649–6654.
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Contents lists available at ScienceDirect
Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Dual-functionalized graphene oxide for enhanced siRNA delivery to breast cancer cells Rana Imani a,c , Wei Shao a , Samira Taherkhani d , Shahriar Hojjati Emami c , Satya Prakash (Professor) a,∗∗ , Shahab Faghihi b,∗ a Biomedical Technology and Cell Therapy Research Laboratory, Department of Biomedical Engineering, Faculty of Medicine, McGill University, Montréal, QC H3A 2B4, Canada b Tissue Engineering and Biomaterials Research Center, National Institute of Genetic Engineering and Biotechnology, Tehran 14965/161, Iran c Department of Biomedical Engineering, Amirkabir University of Technology, Tehran 15875/4413, Iran d Department of Computer and Software Engineering, Institute of Biomedical Engineering, Polytechnique Montréal, Montréal, Québec H3C3A7, Canada
a r t i c l e
i n f o
Article history: Received 18 May 2016 Received in revised form 7 August 2016 Accepted 11 August 2016 Available online 12 August 2016 Keywords: Gene delivery Nano-carrier Graphene oxide Amphiphilic polymer Cell penetrating peptide siRNA
a b s t r a c t The aim of this study is to improve hydrocolloid stability and siRNA transfection ability of a reduced graphene oxide (rGO) based nano-carrier using a phospholipid-based amphiphilic polymer (PL-PEG) and cell penetrating peptide (CPPs). The dual functionalized nano-carrier is comprehensively characterized for its chemical structure, size, surface charge and morphology as well as thermal stability. The nano-carrier cytocompatibility, siRNA condensation ability both in the presence and absence of enzyme, endosomal buffering capacity, cellular uptake and intracellular localization are also assessed. The siRNA loaded nano-carrier is used for internalization to MCF-7 cells and its gene silencing ability is compared with AllStars Hs Cell Death siRNA as a model gene. The nano-carrier remains stable in biological solution, exhibits excellent cytocompatibility, retards the siRNA migration and protects it against enzyme degradation. The buffering capacity analysis shows that incorporation of the peptide in nano-carrier structure would increase the resistance to endo/lysosomal like acidic condition (pH 6–4) The functionalized nanocarrier which is loaded with siRNA in an optimal N:P ratio presents superior internalization efficiency (82 ± 5.1% compared to HiPerFect® ), endosomal escape quality and capable of inducing cell death in MCF7 cancer cells (51 ± 3.1% compared to non-treated cells). The success of siRNA-based therapy is largely dependent on the safe and efficient delivery system, therefore; the dual functionalized rGO introduced here could have a great potential to be used as a carrier for siRNA delivery with relevancy in therapeutics and clinical applications. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Gene therapy is a powerful and novel strategy that has a great potential to cure genetic based diseases particularly cancer [1]. Recently, ribonucleic acid interference (RNAi) has been attracted many attention as a promising candidate for the treatment of a variety of diseases that suffer from expression of undesired genes [2]. Small interfering RNA (siRNA) can suppress the expression of target
∗ Corresponding author at: Tissue Engineering and Biomaterials Research Center, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran 14965/161, Iran. Tel.: +98 21 44787386; fax: +98 21 44787386. ∗∗ Corresponding author at: Department of Biomedical Engineering, Faculty of Medicine, 3775 University Street, Montréal, QC H3A 2B4, Canada. E-mail addresses: [email protected] (S. Prakash), [email protected], [email protected] (S. Faghihi). http://dx.doi.org/10.1016/j.colsurfb.2016.08.015 0927-7765/© 2016 Elsevier B.V. All rights reserved.
pathogenic genes using degradation of the target messenger RNA (mRNA). However, duo to the limited delivery of siRNA through cell membrane, various nano-delivery systems have been developed to facilitate siRNA access to the cytoplasm of targeted cells [3–5]. A nano-carrier system is also essential to provide in vitro and in vivo biostability and to ease cellular uptake [6]. Graphene and its derivatives e.g. graphene oxide (GO) and reduced graphene oxide (rGO) have been considered as a biocompatible and conjugable nano-carriers for delivery of targeting compounds and biomolecules [7]. Lately, GO/rGO has been utilized for siRNA delivery and showed a great success in gene knockdown. Tripathi et al. covalently grafted polyethylenimine (PEI) to GO and evaluated delivery of GFP specific siRNA with 70% suppression of the target gene expression [8]. In another study, Cheng et al. have shown that a polyethylene glycol (PEG)-PEI-grafted graphene/Au composites could efficiently load anti-apoptosis Bcl-2 siRNA and
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successfully down-regulated the Bcl-2 expression in HL-60 cells [9]. Zhang et al. have used PEI for covalent functionalization of GO which was followed by loading of hTERT siRNA and targeting HeLa cells [10]. Feng et al. have reported synthesis of ultra-small size dual-polymer-functionalized GO (GO-PEG-PEI) and effective delivery of Polo-like kinase 1 (Plk1) siRNA using laser irradiation [11]. It is also demonstrated that GO-PEG-PEI is an excellent nanocarrier for delivery of Stat3 siRNA which could lead to significant regression in tumor growth [12]. Finally, Zhang et al. were investigated the sequential delivery of siRNA and anticancer drugs via PEI-functionalized GO [13]. Non-covalent functionalization of graphene with hydrophobic interactions or electrostatic is the most effective and nondestructive method as enables the modification of graphene without alteration of its chemical structure [14]. While much research have been devoted to covalent functionalization of graphene sheets using PEI or PEG for siRNA delivery, there has been little investigation of non-covalent functionalization of graphene as a siRNA nano-carrier using amphiphilic polymers. We have previously synthesized GO, optimized the concentration of carboxylic acid on its surface [15], and successfully used it for plasmid delivery [16]. Here we introduced a novel reduced graphene oxide (rGO) based nano-carrier which was double functionalized by a phospholipid-based amphiphilic polymer and cell penetrating peptide (CPPs) for enhanced hydrocolloid stability and high siRNA transfection ability. After a comprehensive characterization, the positively charged nano-carrier was evaluated for delivery of siRNA into the MCF-7 breast cancer cells and the knockdown of the cell survival related genes.
2. Materials and methods 2.1. Materials All the reagents used for synthesis and functionalization of rGO nano-sheets were purchase from Sigma Aldrich, Canada except otherwise indicated specifically. The MCF-7 breast cancer cell line was purchased from American Type Cell Culture (ATCC, Manassas, USA). Fetal bovine serum (FBS) and Dulbecco’s modified eagle’s medium (DMEM) were purchased from Invitrogen (Montreal, CA). The MTS viability assay kit was obtained from Promega (Madison, USA). The FITC-labeled scrambled siRNA and AllStars Hs Cell Death Control siRNA were supplied by Santa Cruz Biotechnology (Ontario, CA) and QIAGEN (Montreal, CA). HiPerFect® , was purchased from QIAGEN. LysoBright® and Hoechst 33342 staining reagents were bought from AAT Bioquest® (Montreal, CA) and GIBCO® -Life Technology (Montreal, CA). RNase A and ethidium bromide (EtBr) were supplied by Sigma (Montreal, CA). Ocataarginine (R8) cell penetrating peptide (MW 1267 g/mol- Supplementary data) was bought from AbbiotecTM (CA, Montreal).
2.2. Preparation of functionalized nano-carrier 2.2.1. Preparation of reduced graphene oxide nano-sheets (rGON) Graphene oxide nano-sheets (GON) were synthesized using Hummers’ method as previously described [15]. For more exfoliation and size reduction of GON, 1.2 g of sodium hydroxide (NaOH) was added to 10 mL of GON solution (1 mg/mL) followed by sonication for 3 h (MicrosonTM, XL2000, 100W, USA). After adjusting pH to 1, the solution was filtered and washed. The ultra-small nanoparticles were re-dispersed in DI water at concentration of 1 mg/mL. To reduce GON, 0.05% v/v of hydrazine monohydrate was added to 15 mL of GON (1 mg/mL) and the solution was heated up to 80 ◦ C for 4 h. The black precipitations were washed by DI water 5 times
using centrifugation filtration units (Amicon, 30 kDa) and freeze dried. 2.2.2. Functionalization of rGON To immobilize PL-PEG (Avanti Polar Lipids, Inc. US) on the surface of the rGON, 2 mg of rGON was partially dispersed in 8 mL of DI water under ultrasonication process (5 min). Subsequently, 2 mL of PL-PEG solution (3 mg/mL in DI water) was mixed with dispersed rGON. The mixture was dispersed under the bath sonication for 3 h. The suspension was washed 5 times by filtration (Amicon filters- 30 kDa) to remove excess non-bonded polymer. Finally, the ultracentrifugation (50Ti, Beckman Coulter, USA) at 20000 rpm for 1 h was used to obtain the stable supernatant which was referred as rGON-PLPEG. In the next step, octaarginine (R8) solution was added into the rGON-PLPEG solution at the final concentration of 1 mM and the mixture was sonicated for 1 h, then washed. The final functionalized double layer coated sample was referred as rGONPLPEG-R8. 2.3. Characterization of nano-carrier 2.3.1. Instrumentation FT-IR spectra were recorded by a Nicolet 6700 FT-IR spectrometer within the range of 3500–500 cm−1 at a resolution of 4 cm−1 . UV–vis spectra of specimens suspended in DI water were obtained in the range of 190–600 nm in UV–vis spectrophotometer (Cary100 bio, Varian Inc). The morphology and size of the nano-carriers were evaluated by an atomic force microscope (AFM, Nanoscope III Multimode,VEECO) in tapping mode. The single sheet morphology of GO before and after treatment with base was characterized by transmission electron microscopy (TEM, Philips169 CM200) operated at 200 kV. Thermal gravimeter analysis (TGA) was performed using TGA Q500 (TGA instruments Ltd., UK) under nitrogen atmosphere at a rate of 20 ◦ C/min. Finally, the size and surface charges were evaluated using size and zeta potential analyzer (Brookhaven Instruments Corporation, USA). 2.3.2. Peptide quantification Fluorescamine assay was utilized to measure peptide concentration [17]. Briefly, phosphate buffer (pH 7.4, 80 L) and 20 L of nano-carrier solution were mixed followed by addition of 32 L fluorescamine solutions (3 mg/mL in acetonitrile) to the mixture. The excitation wavelength was set at 400 nm and the fluorescence emission was recorded rapidly after the addition of reagent to the mixture at 480 nm using SpectraMax i3 Multi-Mode Microplate Reader (Molecular devices). To estimate peptide molarity in rGONPLPEG-R8 formulation, standard curve was plotted based on R8 solution in different molar ratio. 2.3.3. Stability and dispersibility assessments The ability of PL-PEG to disperse rGON in aqueous solution after the first layer of coating was assessed by ultracentrifugation (20000 rpm, 1 h) of the samples. The concentration of stable supernatants was determined using UV–vis spectrophotometer. Stability of rGON-PLPEG and rGON-PLPEG-R8 in buffer phosphate saline (PBS) and cell culture media in the presence of 10% serum was observed after 48 h incubation at room temperature. 2.3.4. Cell culture and MTS cell viability assay The MCF-7 breast cancer cells were cultured in DMEM supplemented with 10% FBS in a humidified incubator with 5% CO2 at 37 ◦ C. The cytotoxicity of rGON-PLPEG and rGONPLPEG-R8 was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay kit (Promega, Madison, USA) using the Cell Titer 96® . Briefly,
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triplicates of 1 × 104 cells were seeded in a 96-well plate and cultured for 24 h followed by treatment with samples at 10–250 g/mL (37 ◦ C). The MTS assay was performed based on the manufacturer protocol. The absorbance of formazan in cell culture was measured at 490 nm using Victor3 Multilabel Counter (Perkin Elmer, USA). The viability of cells was calculated as the percentage of viable cells to untreated cells as control.
plate with gentle shaking. After 4 h incubation, the cells were fixed with 0.3% glutaraldehyde in PBS for 5 min and stained with Hoechst® (0.5 g/mL in PBS) for 15 min followed by lysosomes staining using Lysobrigh® (0.1 g/mL in PBS) for 5 min. Confocal images were taken with a Zeiss LSM 510 microscope (Carl Zeiss, Jena, Germany) using × 60 oil-immersion objectives and captured and post-processed using Zeiss LSM 510 software.
2.3.5. Gel retardation assay For gel electrophoresis, 2% agarose gel was used to examine the affinity of siRNA for the nano-carrier. First, 50 ng FITClabeled siRNA were mixed with rNGO-PLPEG-R8 sample in different N/P (5, 10, and 15) ratios by gentle pipeting and incubation at room temperature for 30 min. The complexes were then electrophoresed in the agarose gel containing EtBr (0.5ug/mL) in the presence of 2 L dye (6×) with a TAE buffer at 100 V for 30 min. The gels were detected by an automatic digital gel image analysis system (Hercules, CA). Migration of genetic material condensed with functionalized-rGON was compared to the naked samples and HyPerfect® reagent as a positive control.
2.3.10. Cell death induction by siRNA delivery Cell death siRNA (cd-siRNA) was transfected against MCF-7 breast cancer cells by rGON-PLPEG-R8 in optimized N/P ratios. The cells were seeded into a 96-well plate at a density of 1 × 104 cells/well. After 24 h, an appropriate amount of the samples were mixed with 75 ng cd-siRNA in a 100 L medium without serum and left for 30 min at room temperature. The cells were washed with PBS and the above mixture was added drop-wise during gentle shaking. The HiPerFect® and naked cd-siRNA were used as positive and negative controls, respectively. The cell death caused by siRNA delivery was evaluated using MTS viability assay 72 h of transfection.
2.3.6. Buffering capacity of nano-carrier The capacity of the nano-carrier to buffer endosome in acidic condition were evaluated by acid-base titration and compared to PEI and NaCl solutions [18]. First, 2 mL of rGON-PLPEG-R8 solution (0.1 mg/mL) was adjusted to pH 10 by addition of 0.1N NaOH. The titration was performed with 5 L additions of 0.1 N HCl to reach to pH 3. A graph was plotted based on the pH and the amount of HCl used to reach to pH 3.
2.4. Statistical analysis
2.3.7. RNase protection assay 2 units of RNase A was added to the complex of nano-carrier and siRNA and incubated for 30 min at room temperature (naked siRNA was used as control). Subsequently, EDTA (4 L, 0.25 M) was added to the sample in order to inactivate the RNase A and continued to incubate for another 10 min. Finally, 5 L, 2 unit/L of heparin solution was added to the samples and incubated for 1 h followed by gel electrophoresis (30 min at 100 V) in 2% agarose gel with TBE buffer [8].
3.1. Structural characterization of rGO-based nano-carrier
2.3.8. Cell internalization MCF-7 cells were cultured in DMEM+ 10% FBS medium with density of 1 × 104 cells per well in a 96-well plate. After 24 h, 100 ng FITC-siRNA was mixed with an appropriate amount of sample (30 min, 25 ◦ C). The complexes were added to the cells drop-wise during of a gentle shaking. A commercial cationic lipid transfection agent, HiPerFect® , was used as positive control. After 12 h of incubation, the FITC-labeled siRNA samples were observed by fluorescent microscopy (Nikon, Eclipse, Te2000-4, Japan). The cellular uptake efficiency of the sample was calculated by the measurement of fluorescence intensity using a microplate reader (excitation: 480 emission: 520) and compared to that of positive control. The internalization efficacy of the nano-carrier was expressed as relative fluorescence intensity which was calculated based on Eq. (1): Internalizationefficacy = (Et −Eb )/Ec × 100
(1)
Where Et , Eb and Ec represent the average fluorescence emission of test samples, blank sample and positive control (HiPerFect® ), respectively. 2.3.9. Intracellular localization 100 ng FITC-siRNA was mixed with samples (in an optimized N/P ratio) and left for 30 min at room temperature. The complexes drop-wise were added to 5 × 103 MCF-7 cells in Petri dish
Statistical analysis was carried out using SPSS software (v 17.0; IBM New York, NY, USA) when statistical differences were detected, a t-Student test was performed. Data are reported as mean ± SD at a significance level of p < 0.05. 3. Results and discussion
3.1.1. FTIR spectroscopy The chemical characteristic of as-prepared GON and rGON was confirmed by FT-IR spectroscopy (Fig. 1a). The base-treated GON showed multiple peaks around 1060, 1250, 1365, and 1720 cm−1 which are assigned to vibrational modes of C O, C O C, C OH and C O in carboxylic acid and carbonyl moieties [19]. After the reduction reaction, the peaks of the oxygenated groups e.g. carboxyl in rGON were disappeared and the intensity of peaks between 900 and 1500 cm−1 decreased significantly [20]. In addition, the band at 3500 cm-1 which is assigned to the stretching mode of hydroxyl groups of GON became significantly weaker suggesting the removal of the hydroxyl groups. These observations confirm that most oxygen functionalities of the pristine GO were removed. After the surface coating of rGON by PL-PEG the FT-IR spectrum showed the peaks of PEG segments (-CH2-) at 2850 cm−1 . The Comparison between FTIR spectra of rGON-PLPEG, rGON and PL-PEG suggested that the polymer has been successfully immobilized on the surface of rGON. 3.1.2. Uv-vis spectroscopy analysis Upon reduction of GO nano-sheets, the brownish aqueous solution darkened and the reduced sheets were aggregated and precipitated. As shown in Fig. 1b, GON sheets display a maximum absorption peak at 230 nm (attributed to –* transitions of aromatic C C bonds) and a shoulder peak around 300 nm (attributed to n– * transitions of C O bonds) [17]. After GON reduction, the peak at 230 nm red-shifted to 258 nm and the shoulder peak at 300 nm was disappeared [22]. The UV–vis spectrum of the PL-PEG coated rGON which was dispersed in water presented similar features as that of polymer itself. The rGON characteristic peak around 260 nm was also detectable in rGON-PLPEG. The R8 peptide functionalized rGON-PLPEG spectrum showed a characteristic peak at ∼208 nm which is correlated to R8 guanidine groups. The R8 functionalized
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Fig. 1. The FTIR (a) Uv-vis (b) TGA (c) spectra of specimens.
sample showed both main peaks of rGON-PLPEG and R8 around 260 and 200 nm, respectively. 3.1.3. TGA analysis The TGA analysis (Fig. 1c) showed that a major mass loss occurs for the GON at 180 ◦ C presumably due to pyrolysis of the oxygencontaining functional groups while for the rGON no significant mass loss was detected up to 700 ◦ C [23]. This is an indication for successful reduction of GON and significant increase of thermal stability of the rGON [21]. The TGA was also used to estimate the content of stabilized polymer on the surface of rGON. The TGA curve for the polymer-functionalized rGON was shown in Fig. 1c. The PL-PEG had weigh loss of 95% in a nitrogen atmosphere at 700 ◦ C whereas functionalized rGON showed a different pathway which indicates that polymer were immobilized on the rGON. The polymer content in rGON-PLPEG was calculated around 40 wt% which provides evidence for PL-PEG immobilization on the surface of rGON via PL hydrophobic interaction with basal plane of rGON. 3.1.4. AFM and TEM analyses The results of AFM and TEM indicated that as prepared GON had a sheet-like morphology with the average size of 250–400 nm (Fig. 2a,b). The TEM image of GON showed a wrinkled sheet-like structure. The thickness of unmodified GO was estimated around 1–1.5 nm according to the height profile of AFM image which is corresponded to 1–2 layer of graphene sheets. The base treatment of GON was resulted in smaller exfoliated nano-sheets with the mean thickness of ∼0.8 nm that indicates the formation of single-
layered sheets (Fig. 2c). It has been previously shown that the oxidation/base treatment of graphene would form a single-sheet morphology with a size reduction [15]. The reduction reaction of the GON did not affect morphology of nano-sheets significantly (Fig. 2d). However, after immobilization of PL-PEG on the surface of rGON, the height profile of the nano-sheets was significantly changed as rGON-PLPEG showed 1 nm height increase as compared to rGON (Fig. 2e). It was confirmed that the polymer could successfully interact with rGON basal plane. By the addition of R8 to the sample the nano-sheets presented further round-shaped morphology while rGON sheets showed very sharp edges with flat surface (Fig. 2f). The thickness increase of the nano-sheets from 2.6 to 3.8 nm indicated successful integration of peptide on the single sheet. 3.1.5. DLS and zeta potential analyses The size (effective diameter, polydispersity index (PDI)) and surface charges of samples were evaluated by size and zeta analyzer. Based on DLS analysis, rGON (146 ± 4.6 nm, PDI = 0.005) showed smaller size and narrower distribution than base-treated GON (180 ± 6.1 nm, PDI = 0.212). It should be noted that the size analysis of rGON was performed immediately after strong sonication duo to the fast aggregation of rGO nano-sheets. As expected the DLS analysis showed a significant size increase after the coating of rGON with PL-PEG (146 ± 4.6 to 188 ± 3.2 nm) which is corresponded to immobilization of polymer on the nano-sheets surface. After the peptide incorporation, the size of nano-sheets showed further increase to 246.7 ± 2.7 nm. Surface charge screening using
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Fig. 2. The morphological characterization of as-prepared GON; TEM (a) and AFM (b). The AFM images of base treated GON (c), rGON (d), rGON-PLPEG (e) and rGON-PLPEG-R8 (f) specimens.
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zeta analyzer demonstrated that significant increasing of zeta value from −45 to −25 mv and dramatically decreasing solubility also confirm proper reducing GON to rGON. The rGON-PLPEG showed the less negative charge around −20.55 ± 1.73 mv duo to NH2 functionality of PEG chains. The results showed that by introduction of R8 into the stabilized rGON formulation, the zeta potential was significantly increased from −20.55 ± 1.73 to +42.61 ± 2.02 mv which is a confirmation on the addition of positively charged R8 to the final structure. It was revealed that the R8 could significantly interact with rGON-PLPEG structure to cover the PL-PEG layer. 3.1.6. Fluorescamine assay In order to quantify the incorporated R8 peptide molarity in the functionalized rGON, FL-amine assay was used. The FL-amine would react with amino groups of R8 in aqueous solution and form a fluorescent component which can be detected at 480 nm emission (excitation at 400 nm) and quantify using a standard curve. The amount of bonded R8 peptide (mol/mg) was calculated. The molarity of R8 was estimated around 1.2 mol/mg which was much higher than covalently conjugated R8 to GON that has been reported previously [16]. It is expected that the stabilized rGON could present a high ability to be internalized into the cells. 3.1.7. Hydrocolloidal stability assessment For biomedical applications, functionalized rGONs need to be stable in physiological fluids, such as PBS or serum containing media[21,22]. In gene delivery applications agglomeration and precipitation of nano-carriers in serum containing media would harshly affect the transfection efficacy and consequently limit their applications. Therefore, the stability of double layer coated rGON was investigated by incubation in PBS (0.01 M, PH7.4) and growth media containing 10% FBS for 48 h at 37 ◦ C. The stability of rGON-PLPEG-R8 sample was also tested by the size measurements through DLS (Table 1). The sample showed a slight size increase after 1 h incubation in cell culture media compared to DI water which could be duo to the protein absorption from the serum. The DLS results confirmed no significant size alteration after 48 h incubation at room temperature (258 ± 3.7 compared to 266 ± 7.4) which is an indication for a good colloidal stability of the sample in the presence of serum and salts. As mentioned before by the addition of R8 the zeta potential dramatically rose to positive values (-20.55 ± 1.73 to +42.61 ± 2.02 mv), however, it is believed that the stability of rGON-PLPEG in aqueous solution was largely supported by hydrophilic PEG chains that provide enough steric hindrance. The charge repulsion between cationic amine groups of PEG was also a great help for higher dispersability of rGON-PLPEG-R8 sample. 3.2. Functional characterization of nano-carrier 3.2.1. MTS assay To examine cytotoxicity, the MCF-7 cells were treated with different concentrations (200–10 g/mL) of rGON-PLPEG-R8 for 24 and 48 h. The results showed that cell viability remained above 85% even after the addition of 200 g/mL nano-carrier (Fig. 3a). There was a slight cell viability reduction after the addition of 100 and 200 g/mL of the nano-carrier by 5.5, 12%, respectively. However, the reduction in cell viability was not statistically significant. It can be seen that functionalization of rGON with PL-PEG and R8 could significantly improve the relative viability of nano-carrier. In particular peptide incorporated formulation showed superior cell proliferation rather than control (Fig. 3b). 3.2.2. Evaluation of buffering capacity The interaction of DNA or RNA loaded nano-carriers with cell membrane would typically lead to their endocytosis and entrap-
Fig. 3. Histogram represents cell viability based on MTS assay for rGON-PLPEG-R8 at different concentrations (10–200 g/mL) (a) and comparison between different formulations with the same concentration (50 g/mL) (b). All samples compared to the non-treated cells as control * p < 0.05.
ment within the cellular endosomal vesicles [23]. The amino groups of cationic polymers such as PEI act as a proton sponge and cause swelling and eventual rupture of the endosome. This would lead to release of the nano-vector into the cell cytoplasm, a process which is known as endosomal escape [24]. The surface chemistry of nano-carriers could affect their buffering capacity and proton sponge characteristic. It is believed that the charge and composition of the cationic polymer could regulate the endosomal escape capability [25]. To evaluate this, R8 functionalized rGON was titrated with HCl and the pH value was monitored. The PEI which is commonly considered as a golden cationic polymer and has a great buffering capacity was considered as positive and NaCl solution as negative controls. Interestingly, the sample showed a resistance to pH changes in acidic pH (6–4) that indicates an increase in proton absorption that is attributed to the R8 peptide contribution. The buffering capacity of rGON-PLPEG-R8 and PEI was in a similar range 6–4 (Fig. 4 top). However, the amount of HCl which was consumed to reduce the pH value of PEI solution from 5 to 3 was 45 L while rGON-PLPEG-R8 reached to pH 3 after adding 25 L HCl. This could be a reflection for the effective role of R8 as a buffering component in a strong acidic environment.
3.3. siRNA/nano-carrier complex formation 3.3.1. Retardation assay Gene loading and condensation capability of nano-carrier for siRNA was assessed using gel retardation assay (Fig. 4 down, a). The complex of rGON-PLPEG-R8 with siRNA was prepared at three N/P ratios (5, 10, and 15). The rGON-PLPEG-R8 formulation showed significant retardation of siRNA moving for N/P ratio of 10. The positive charge of R8 amine groups were completely neutralized by negative charges of phosphate groups in siRNA structure in N/P ratio of 10.
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Table 1 The DLS particle size measurement of the functionalized nano-carrier after incubation in cell culture medium and PBS for 48 h. Sample
rGON-PLPEG-R8
Effective diameter (nm) in DMEM
Effective diameter (nm) in PBS
After 1 h incubation
After 48 h incubation
After 1 h incubation
After 48 h incubation
255.1 ± 7.7
259.4 ± 4.3
248.2 ± 3.1
253.6 ± 6.7
Fig. 4. The buffering capacity of nano-carrier compared to PEI and NaCl solutions (Up). The gel retardation assay of nano-carrier at different N:P ratio (N:P = 0 (lane 1), N:P = 5(lane 2), N:P = 10 (lane 3) and N:P = 15 (lane 4)) (a). The RNase protection assay for nano-carrier at N:P = 10 (lane 1: nacked siRNA, lane 2: siRNA + RNase, lane 3: siRNA + nano-carrier, lane 4: siRNA + nanocarrier + RNase, lane 5: siRNA + nanocarrier + RNase + Heparin, lane 6: siRNA + nanocarrier + Heparin) (b).
3.3.2. Protection of condensed siRNA against nuclease degradation For a successful gene delivery, the carrier not only should effectively condense gene but also protect it against nuclease enzyme degradation [26]. Therefore, the ability of functionalized rGON to protect siRNA from nuclease degradation was examined by agarose gel electrophoresis. As it is shown in Fig. 4b (down), siRNA was completely degraded after incubation with enzymes (lane 2) compared to the nacked siRNA in the absence of enzyme (lane 1). As expected siRNA fully condensed and retarded in interaction
with nano-carrier (lane 3). In contrast, siRNA released from nanocarrier complexes and remained intact after enzyme treatment (lanes 4 and 5). The rGON-PLPEG-R8 formulation at N/P ratio of 10 could entirely protect siRNA from enzyme degradation. It was also revealed that after heparin treatment similar bonds were detected as naked siRNA (lanes 1 and 6) that suggests rGON-PLPEG-R8 nanocarrier could deliver more integral siRNA without degradation by enzymes.
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Fig. 5. The MCF-7 cell internalization of nacked FITC-siRNA (a) FITC- siRNA/nano-carrier complexes (b) and FITC- siRNA/HiPerFect® complexes (c) after 12 h. The internalization efficacy of rGON-PLPEG-R8 nano-carrier (d) compared to the HiPerFect® (* p < 0.05).
3.3.3. Cell internalization A key point for an efficient transfection with siRNA would be the delivery of the carrier across the cell membrane [4]. To study cell internalization ability of siRNA loaded rGON-PLPEG-R8, FITC-
labeled siRNA was complexed with the nano-carrier at N/P ratio of 10 and incubated with MCF-7 cells for 12 h. After incubation plenty of green dots were detected inside the cytoplasm of the cells compared to the treated cells with naked siRNA-FITC (Fig. 5a and b).
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Fig. 6. Confocal images of rGON-PLPEG-R8 nano-sheets uptake by MCF-7 cell after 4 h of incubation (up) and 12 h (down). The images show siRNA labeled-FITC (a), lysosome stained (b), nucleus stained with Hochest3342 (c), co-localization of siRNA and lysosomal region (d-f).
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transfected by the cd-siRNA loaded nano-carrier which resulted in gene silencing and subsequent cell death. 4. Summary and conclusions
Fig. 7. Histogram represents cell viability based on MTS assay for MCF-7 cells treated with nacked cell death siRNA (cd-siRNA) and without cd-siRNA compared to HiPerFect® as control (* p < 0.05).
However, it can be seen that HiPerFect® delivered markedly higher quantity of siRNA to the MCF7 cells rather than rGON-PLPEG-R8 (Fig. 5c). Interestingly, the R8 coated sample showed comparable internalization potential compared to the HiPerFect® treated cells. For further confirmation fluorescence intensity of the samples were also measured by mico-plate reader after 24 h (Fig. 5d). The rGON-PLPEG-R8 sample showed 82 ± 5.1% internalization efficacy in delivery of FITC-siRNA compared to HiPerFect® that considered as control. These results showed an excellent ability of nano-carrier in transporting siRNA into the cells. 3.3.4. Confocal microscopy The MCF-7 cells were treated with FITC-siRNA/nanocarrier complex and imaged with laser confocal microscopy after nucleus and lysosome staining. As it can be seen in Fig. 6, after 4 h, the majority of the complexes (green dots) were detected in the cytoplasm of the cells. It can be seen that the most of the green dots were co-localized with red ones (endo/lysosomal compartments). After 12 h, the density of FITC-siRNA in the endo/lysosomal compartments was significantly declined whereas the green dots could be detectable and more spread in cytoplasmic regions. This could be translated to possible endosomal scape of nano-carrier duo to buffering capacity as it mentioned before. However, some overlapping of green and red dots was still detectable around the nuclease. This is evidence for endosomal scape capability of designed nanocarrier after localization inside endo/lysosomal compartments. This also shows the effective role of R8 peptide in providing buffering capacity and destabilization of lysosomal membrane [27]. 3.3.5. Gene transfection The AllStars Cell death control siRNA is a blend of highly potent siRNAs targeting ubiquitously expressed genes that are vital for cell survival. Knockdown of these genes induces a high degree of cell death which could be detected by light microscopy. In this study we utilized cell death siRNA to assess cell death induction through siRNA delivery using rGON-PLPEG-R8 nano-carrier[28]. The cells were transfected by rGON-PLPEG-R8 nano-carrier and the cell death was quantitatively evaluated by MTS assay. The results showed that designed formulation complexed with cdsiRNA induced cell death after 72 h. The Fig. 7 shows that delivery of bare nano-carrier into the cells did not affect their viability, however, when cd-siRNA was delivered into the cell cytoplasm by nano-carrier there was a significant decrease in cell viability. Even though HiPerFect® showed significant cell death compared to rGON-PLPEG-R8, it decreased cell viability even when it was delivered without cd-siRNA. This indicated that MCF-7 cells can be safely
Gene therapy is based on the introduction of nucleic acids typically either siRNA or pDNA into targeted cells through gene knockdown and expression. However nucleic acids are highly unstable and have little cell penetration ability. In present study, an efficient gene nano-carrier is introduced based on reduced graphene oxide that co-functionalized with an amphiphilic polymer (PL-PEG) and R8 cell penetrating peptide. The rGON-PLPEG-R8 remained stable in biological solution and exhibited excellent cell viability. Incorporation of R8 into the nano-carrier formulation provided highly positive surface charged which facilitate siRNA loading and condensation as well as cell penetration. At the optimal N/P ratio of rGON-PLPEG-R8/siRNA, the transfection efficiency of designed nano-carrier is similar to HiPerFect® , a commercial reagent for the transfection of siRNA into mammalian cells. Furthermore, confocal microscopy and buffering capacity assessments demonstrated that functionalized rGON had a potential for endosomal scape. In summary, the rGON-PLPEG-R8 showed a great potential as efficient and biocompatible siRNA delivery vector for gene therapy purposes. Conflict of interest There is no conflict of interest to declare. Acknowledgments The authors wish to acknowledge scientific help and guidance of Prof. Hojatollah Vali, McGill University, Montreal, Canada. SF also would like to thank partial financial support of this work by National Institute of Genetic Engineering and Biotechnology (940801-I-536). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.08. 015. References [1] S.L. Ginn, I.E. Alexander, M.L. Edelstein, M.R. Abedi, J. Wixon, Gene therapy clinical trials worldwide to 2012-an update, J. Gene Med. 15 (2013) 65–77. [2] B.L. Davidson, P.B. McCray, Current prospects for RNA interference-based therapies, Nat. Rev. Genet. 12 (2011) 329–340. [3] A. de Fougerolles, H.-P. Vornlocher, J. Maraganore, J. Lieberman, Interfering with disease: a progress report on siRNA-based therapeutics, Nat. Rev. Drug Discov. 6 (2007) 443–453. [4] J. Wang, Z. Lu, M.G. Wientjes, J.L.-S. Au, Delivery of siRNA therapeutics: barriers and carriers, AAPS J. 12 (2010) 492–503. [5] R. Molinaro, J. Wolfram, C. Federico, F. Cilurzo, L. Di Marzio, C.A. Ventura, M. Carafa, C. Celia, M. Fresta, Polyethylenimine and chitosan carriers for the delivery of RNA interference effectors, Expert Opin. Drug Deliv. 10 (2013) 1653–1668. [6] V. Labhasetwar, Nanotechnology for drug and gene therapy: the importance of understanding molecular mechanisms of delivery, Curr. Opin. Biotechnol. 16 (2005) 674–680. [7] H.Y. Mao, S. Laurent, W. Chen, O. Akhavan, M. Imani, A.A. Ashkarran, M. Mahmoudi, Graphene: promises, facts, opportunities, and challenges in nanomedicine, Chem. Rev. 113 (2013) 3407–3424. [8] S.K. Tripathi, R. Goyal, K.C. Gupta, P. Kumar, Functionalized graphene oxide mediated nucleic acid delivery, Carbon 51 (2013) 224–235. [9] F.-F. Cheng, W. Chen, L.-H. Hu, G. Chen, H.-T. Miao, C. Li, J.-J. Zhu, Highly dispersible PEGylated graphene/Au composites as gene delivery vector and potential cancer therapeutic agent, J. Mater. Chem. B 1 (2013) 4956–4962. [10] X. Yang, G. Niu, X. Cao, Y. Wen, R. Xiang, H. Duan, Y. Chen, The preparation of functionalized graphene oxide for targeted intracellular delivery of siRNA, J. Mater. Chem. 22 (2012) 6649–6654.
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