Stable dispersions of covalently tethered polymer improved graphene

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Biological and Medical Applications of Materials and Interfaces

Stable dispersions of covalently tethered polymer improved graphene oxide nanoconjugates as an effective vector for siRNA delivery Nisha Yadav, Naveen Kumar, Peeyush Prasad, Shivani Shirbhate, Seema Sehrawat, and Bimlesh Lochab ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03477 • Publication Date (Web): 10 Apr 2018 Downloaded from http://pubs.acs.org on April 10, 2018

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ACS Applied Materials & Interfaces

Stable dispersions of covalently tethered polymer improved graphene oxide nanoconjugates as an effective vector for siRNA delivery Nisha Yadav,1# Naveen Kumar,2# Peeyush Prasad 2, Shivani Shirbate2, Seema Sehrawat,2,3* Bimlesh Lochab1* 1

Materials Chemistry Laboratory, Department of Chemistry, School of Natural Sciences,

Shiv Nadar University, Gautam Buddha Nagar, Uttar Pradesh 201314, India. 2

Brain Metastasis and Neurovascular Disease Modeling Lab, Department of Life Sciences,

School of Natural Sciences, Shiv Nadar University, Gautam Buddha Nagar, Uttar Pradesh 201314, India. 3

Department of Medicine, Harvard Medical School, Boston, Massachusetts, 02115, USA.

*[email protected]; *[email protected]; [email protected] #

Authors contributed equally.

KEYWORDS Graphene oxide, siRNA delivery, gene knockdown, pH dependent release, binding efficiency, cancer.

ABSTRACT Conjugates of poly(amidoamine) (PAMAM) with modified graphene oxide (GO) are attractive nonviral vectors for gene based cancer therapeutics. GO protects siRNA from enzymatic cleavage and showed reasonable transfection efficiency along with simultaneous benefits of low cost and large scale production. PAMAM is highly effective in siRNA

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delivery but suffers from high toxicity with poor in-vivo efficacy. Co-reaction of GO and PAMAM led to aggregation and more importantly, have detrimental effect on stability of dispersion at physiological pH preventing their exploration at clinical level. In the current work, we have designed, synthesized, characterized and explored a new type of hybrid vector (GPD), using GO synthesized via improved method which was covalently tethered with poly(ethylene glycol) (PEG) and PAMAM. The existence of covalent linkage, relative structural changes and properties of GPD is well supported by fourier transform infrared (FTIR), UV-visible (UV-vis.), raman,

X-ray photoelectron (XPS), elemental analysis,

powder X-Ray diffraction (XRD), thermogravimetry analysis (TGA), dynamic light scattering (DLS), and zeta potential. Scanning electron microscopy (SEM), and transmission electron microscopy (TEM) of GPD showed longitudinally aligned columnar self-assembled ~ 10 nm thick polymeric nano-architectures onto the GO surface accounting to an average size reduction to ~ 20 nm. GPD revealed an outstanding stability in both phosphate buffer saline (PBS) and serum containing cell medium. The binding efficiency of EPAC1 siRNA to GPD was supported by gel retardation assay, DLS, zeta potential and photoluminescence (PL) studies. A lower cytotoxicity with enhanced cellular uptake and homogeneous intracellular distribution of GPD/siRNA complex is confirmed by imaging studies. GPD exhibited a higher transfection efficiency with remarkable inhibition of cell migration and lower invasion than PAMAM and Lipofectamine 2000 suggesting its role in prevention of breast cancer progression and metastasis. A significant reduction in the expression of the specific protein against which siRNA was delivered is revealed by western blot assay. Furthermore, a pH-triggered release of siRNA from the GPD/siRNA complex was studied to provide a mechanistic insight towards unloading of siRNA from the vector. Current strategy is a way forward for designing effective therapeutic vectors for gene based antitumor therapy.


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1. INTRODUCTION Recent increase in breast cancer accounted a very high annual mortality rate (0.51 million deaths, WHO 2013) globally.1 In cancerous cells, one of the key signalling, well-known second messenger, cyclic adenosine monophosphate (cAMP) pathway is altered resulting in uncontrolled cell proliferation via inducing specific signals for migration, cycle regulation and ultimately cell death.2-3 The main role of cAMP is to relay hormonal responses via mediating barrier properties and control of cellular processes including activation of exchange protein (EPAC1). The overexpression of EPAC1 especially in breast cancer is considered as one of the reason for cell metastasis.4 Gene therapy is an upcoming promising method for treatment of cancer where conventional remedial measures fails. The approach involved either replacement of mutated gene, or “knocking out,” or introduction of a new gene to arm the cells to fight against a disease via controlling the specific protein expression. Tumor targeting ability of EPAC1 synthetic double-stranded ribonucleotide (siRNA) is worth exploring to regulate the cellular production of cAMP in breast cancer cells. Generally, siRNA consists of 21-25 nucleotides accounting polyanionic nature (40-50 negative phosphate charges) with a molecular weight of ~ 13 kDa. In blood and serum, bare siRNA is unstable and it lacks the gate pass to move across the cell membrane due to both electrostatic and steric repulsion by negatively charged cell membrane.5 As a result, it often undergoes enzymolysis followed by its subsequent elimination without realizing its therapeutic potential. Therefore, an ideal vector is desired to improve siRNA biological stability with efficient delivery into the cells through specific formulations. Gene delivery vectors explored so far, includes cationic lipids, polymers, dendrimers, polysaccharide, and graphene oxide (GO).6-9 Despite numerous efforts, a limited success is achieved due to their prevalent cytotoxicity, solubility, stability, higher gene loading efficiency and low cellular uptake. GO appears as a potential vector due to well expose, larger surface area with an easier 3 ACS Paragon Plus Environment


large scale production. Nano-size GO (< 100 nm) loaded with single stranded DNA via noncovalent adsorption showed effectiveness in gene transfection efficiency with a simultaneous protection against enzymatic cleavage.10 The physical adsorption of genetic material via weak π−π interactions accounts to a lower loading, poor transfection efficiency and genetic material may leach out with time.11 However, problems associated with GO includes poor biocompatibility, generation of reactive oxidant species (ROS), and lower stability at physiological pH.12 The improvement in biocompatibility and stability of GO is widely tackled by functionalization with poly(ethylene glycol) (PEG), chitosan, poly(lactic acid). So far, GO-PEG formulations gained an inspiring attention as PEG being biocompatible, relatively inexpensive, versatile and approved by Food and Drug Administration (FDA).13 Furthermore, it minimize opsonization, with a prolonged blood circulation time14 along with ease of linker chemistries to affect stability of GO dispersions.15 A higher loading efficiency of genetic material via stronger electrostatic interaction is realized by modification of GO with cationic hyperbranched polyethyleneimine (PEI)

16

and dendritic polyamidoamine

(PAMAM).17 An increase in molecular weight of PEI from 10 kDa to 25 kDa led to an enhancement in transfection efficiency with prevalence of cytotoxicity thus limiting its gene loading efficiency.18-19 PEGylation of reduced GO-PEI nanoconjugates showed both enhanced and controlled gene transfection efficiency which is attributed to the co-formation of reduced graphene domains.20 In contrast to hyperbranched PEI, PAMAM dendrimer inherits a nano-topographic structure with symmetrical geometry allowing a higher number of well exposed surface amine functionalities.21-23 Furthermore, PAMAM offers a distinct advantage of biodegradability and generation dependent variable size and amine functionalities enabling a higher gene loading capacity.24-25 Amongst various dendrimer generations, a 4th generation PAMAM revealed an optimum and adaptable structural configuration to accommodate siRNA with an efficient binding and releasing capability in an


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energetically favorable fashion.26 It safeguards siRNA towards enzymatic degradation with its effective delivery and subsequent release via proton sponge effect.27 However PAMAM’s inherent cellular toxicity is preventing its exploration and demands strenuous chemical modifications protocol.28-29 Hybrid nanoconjugates of PEG and PAMAM with GO are worth exploring as vectors to overcome above limitations of gene loading efficiency and biocompatibility. Wang et al. reported a 40 nm size of GO-PEG-PAMAM nanoparticles where PAMAM is bound via electrostatic interactions to GO exhibited remarkable inhibition in both cancerous cell migration and invasion.30 A smaller and narrow size distribution of nanoconjugates is preferred to facilitate a better in-vivo distribution, as size > 200 nm are known to clog the microcapillaries with a higher rate of clearance from the blood circulation. Recently, surface modification of graphene with oleic acid and PAMAM led to an improved transfection efficiency, however, a substantial aggregation of nanoparticles is reported.31 The chemistry of linking of PAMAM directly to pristine or modified GO either via electrostatic or covalent conjugation suffers from the problem of aggregation accounting to poor dispersion stability of so-formed nanoparticles at physiological pH. Although, structurally GO-PAMAM may exhibit excellent gene delivery capacity, but the following reasons are responsible to restricts its full potential. First, PAMAM acts as a bridge between GO nanoparticles and led to an enhancement in nanoparticle size.32 Second, earlier studies mainly utilized GO synthesized by modified Hummer’s method33 which consists of a mainly large nanoparticles with a very high polydisperse index, reflecting a non-uniformity in both size and chemical reactivity. This accounts to a heterogeneous nature of modified nanoparticles that is highly detrimental for stability of dispersion and affects pharmacokinetics, circulation and distribution characteristics. Third, prior-modification of GO with PEG-amine led to a substantial consumption of predominantly existing carboxylic functionalities on edges of GO.34



Therefore, leaving a meagre amount of carboxylic functionality to react with PAMAM that accounts to a lower and ineffective loading of genetic material. To counteract the above limitations, additional approaches are employed to reduce the GO size followed by selective size purification techniques. Inopportunely, additional processing steps are not cost-effective, as they involve strenuous purification protocol, which may hamper the yield of desired quality of GO with irreproducible results reducing their scope for clinical applications. This demands designing and exploration of alternative synthetic route to achieve stable GO-polymer dispersions with nearly monodisperse nature to ensure homogeneous physio-chemical and biological properties. Therefore, facilitation of GO synthesis via synthetic protocol that offers surplus or variable reactive functionalities with appreciable control on homogeneity of smaller size nanoparticles is an important consideration, to advance their utility as potential carrier. Recently, GO synthesized by improved method exhibited benefits of higher yield, easy purification strategy and smaller size of nanoparticles as compared to modified Hummer’s method.35-36 To the best of our knowledge, GO synthesized by improved method is not explored as a nanocargo for gene delivery. Herein we report, an efficiently designed new nanovehicle based on improved GO platform which is chemically modified with PEG (6 armed, 15 kDa) and PAMAM (G4, 10 kDa) to enable it as an efficient gene delivery vehicle (Figure 1).


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Figure 1. Synthetic scheme for the preparation of GO based nanocarrier to load siRNA (GP: PEGylated GO; GPD: PAMAM functionalized GP).

The integration of three entities, GO, PEG and PAMAM via covalent functionalization is to enhance the stability of aqueous dispersion with several benefits namely, (i) PEG ensures lower cytotoxicity of GO and PAMAM, protects siRNA from nuclease activity thereby increasing its life time; (ii) PAMAM possess amine groups to link to siRNA and empower its cellular entry with simultaneous release of siRNA; (iii) GO mediates conjugation platform with additional advantages of loading siRNA, lowering toxicity of PAMAM, enhancing stability of dispersion and prosperity to load multiple functionalities for further benefits. 2. EXPERIMENTAL SECTION 2.1 Synthesis of GPD (covalently linked GO-PEG-PAMAM). A mixture of concentrated sulfuric acid: phosphoric acid (134:15 mL, v/v) was added to a mixture of graphite flakes



(Alfa Aesar, 99.8%, natural graphite -325 mesh, 1.0 g) with stirring at room temperature followed by slow addition of potassium permanganate (6.0 g). After addition, the reaction mixture was heated to 50 oC for further 12 h. After cooling to room temperature, and a mixture of ice cold water: H2O2 (250:3 mL) was added slowly. A noticeable color change from dark brown to yellow was observed. The reaction mixture was allowed to settle under gravity and supernatant was decanted. The residue so formed was washed, re-dispersed in water and then the process was repeated till the pH of the dispersion was found to be neutral. The drying of the residue on rotary evaporator at 50 ºC under vacuum yield a light brown solid (2.3 g) of GO.35 GP was synthesized by base mediated ring opening reaction of epoxide ring present on basal plane of GO by nucleophilic attack of 6-arm PEG-amine (Jenkem Technology, 15 kDa). Firstly, GO aqueous suspension (0.5 mg/ mL, 10 mL) was subjected to probe sonication for 2 min. followed by bath sonication for 1.5 h. To this, PEG-amine (30 mg) and KOH (40 mg) were added and reaction was stirred at 80 oC for 24 h. The reaction mixture was subsequently dialyzed against water using dialysis membrane (mol. weight cut off 10 kDa) for 24 h to form GO-PEG (GP). PAMAM (4th generation, ethylene diamine core, 10 wt% in methanol, Sigma Aldrich, 10 kDa) was covalently attached to GP by using a coupling reagent, EDC [1-ethyl-3-(3-dimethylaminopropyl) carbodiimide] and NHS (Nhydroxy succinimide) to activate carboxylic acid to form amide bond. Firstly, EDC (30 mg) and NHS (30 mg) were mixed in aqueous suspension of GP (0.5 mg/mL, 10 mL) and stirred at room temperature for 15 min. After activation, PAMAM (400 µL) was added in the reaction mixture and stirred at room temperature for 24 h. Finally, unreacted PAMAM was removed through dialysis membrane (mol. weight cut off 10 kDa) against water for 24 h with multiple washings to form GO-PEG-PAMAM (GPD). 2.2 Characterizations of GPD and GPD/siRNA complex. Absorbance measurements were carried out on Thermo Scientific Evolution 201 UV-visible (UV-vis.) spectrophotometer in 1


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cm quartz cuvettes over the range of 200-900 nm by preparing the dispersions of respective nanomaterial in deionised water. Fourier transform infrared (FTIR) spectra were recorded on a Nicolet iS5 spectrometer equipped with iD5-ATR (Diamond) accessory, in the range of 4000 to 400 cm−1 with a resolution of 4 cm-1. Raman spectra were obtained using a Jobin Yvon HR800 Raman microscope at λ of 514.5 nm. The integrated intensity ratio, ID/IG for the D and G band was used to determine the relative degree of disorder/defects in graphitic materials. The intensity ratio increases with structural disorder in the graphite network and is inversely proportional to the average size of the sp2 cluster.37 X-Ray diffraction (XRD) studies were performed on Rigaku Smart Lab X-Ray Diffractometer, CuKα radiation (λ = 1.5406 Å). Thermal behaviour was investigated using Perkin Elmer Diamond STG-DTA in the temperature range 50-700 ºC under nitrogen atmosphere at a heating rate of 10 °C/min. The average particle size and zeta potential (ζ) of the particles was measured by the dynamic light scattering (DLS) instrument (Nanosizer, Malvern, UK) with an argon laser wavelength λ = 830 nm, at a detector angle = 90°, and typical sample volume = 10 µL at room temperature. The obtained DLS data represent the average of three runs. X-ray photoelectron spectroscopy (XPS, Omicron Multi-probe Surface Analysis System) measurements were carried out using monochromatized AlKα (1486.7 eV) radiation source to analyze the surface chemistry of nanomaterials. Elemental analysis was performed using FLASH EA1112 series Thermo-Finnigan, Italy to determine C, H, N, and O in samples. The surface morphology of samples was studied using a scanning electron microscope (SEM) (Zeiss EVO MA15) with an acceleration voltage of 20 kV and high resolution transmission electron microscopy (HRTEM, JEOL-2100F) under an acceleration voltage of 200 kV. Photoluminescence (PL) was measured using spectrofluorometer (Horiba Model No. FL3C-2iHR300) at an excitation wavelength of 600 nm and integration time of 0.1 sec at a slit width of 2 for both excitation



and emission study in 3 mL quartz cuvettes. Probe sonicator (Sonics USA, 500 Watt, 20 kHz) was used at 40% max. amplitude with a pulse rate of 10 sec on and off. 2.3 Cell culture conditions. The human breast cancer cell line, MDA-MB-231 (NCCS, India) was cultured in 25 cm2 vented culture flasks (Corning, USA) in L-15 medium (HiMedia, India) supplemented with 10% fetal bovine serum (FBS, HiMedia), 1% antibiotics (penicillin: 10,000 units/mL, streptomycin: 10,000 µg/mL, Gibco-Thermofisher, USA). The media was stored at 4 oC. Media was changed when the cells became 80-90% confluent. The cells were sub-cultured twice a week by using 0.25% trypsin and seeded at a density of 40,000-50,000 cells/cm2 area. Cells were grown at 37 oC in a humidified CO2 incubator (Galaxy 170R, Eppendorf, Germany). 2.4 Cytotoxicity analysis. Cytotoxicity of GPD, PAMAM and Lipofectamine 2000 (Invitrogen, USA) was analyzed by using MTT assay (colorimetric assay). MDA-MB-231 cells were seeded at a density of 10,000 cells/ well in a 96 well plate. After 24 h, different concentrations of GPD, PAMAM and Lipofectamine 2000 were added into each well and cells were further incubated for 48 h. After 48 h, cells were incubated with 0.5 mg/mL of 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, HiMEDIA, India) for 3 h. MTT is a yellow colour salt which gets reduced to purple colour insoluble formazon by the enzymes in living cells. To each well, a 100 µL of DMSO (Thermo Fisher Scientific, USA) was added and incubated for 15 min. Absorbance was recorded at 590 nm with microplate reader (Bio-Rad, USA).

2.5 Preparation of GPD/siRNA complex. For preparing GPD/siRNA complex, a dispersion of GPD (0.5 mg/mL in doubly deionised water) at different volumes 3, 4, 5, 6, 8, and 10 µL was incubated with a constant volume of siRNA (3 µL,10 nM) in a 6 separate sterile glass


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vial for 45 min. at room temperature to give different volume ratios of GPD/siRNA (v/v as 3:3, 4:3, 5:3, 6:3, 8:3, 10:3) complex. 2.6 Agarose gel retardation assay. The volume of pre-incubated GPD/siRNA complex at different volume ratios was made to 20 µL using RNase free water (pH 7). A volume of 20 µL of complex diluted complex was loaded onto 1 % agarose gel (HiMedia, India) containing EtBr dye (ethidium bromide, 0.5%, 10 mg/mL, Bio-Rad). The gel was run at 60 V for 2 h. After running the gel, the image was recorded and analyzed by Alpha Imager using UV light. 2.7 Enzymatic treatment of siRNA. Enzymatic treatment was carried out using RNase (Qiagen) with a constant concentration of siRNA (10 nM). During siRNA digestion experiments, a mixture of siRNA (3 µL) with different volumes of GO was incubated for 2 h. The volume of GO (0.5 mg/mL) used was 5, 6, and 7 µL with same dosage of RNase (0.5 µL, 50 µg/mL) and gel loading dye (2 µL, bromophenol blue, HiMedia). The total volume of each well was kept constant (14 µL). RNA digestion was carried out at a temperature of 37 o

C. The digested products were run on an agarose gel electrophoresis (1.0%) containing

ethidium bromide (0.5%, 10 mg/mL, Bio-Rad) at 100 V for different time intervals. After running the gel, the images were recorded at 5 min., 10 min. and 20 min. and analyzed by Alpha Imager using UV light.

2.8 In vitro gene transfection. The transfection analysis was performed with GPD/siRNA complex to determine transfection efficiency of GPD. Approximately, 0.6 million MDA-MB231 cells/well were seeded in a 6 well plate for 24 h. After 24 h, cells were washed twice with Opti-MEM (Gibco-Thermo, USA) to remove FBS and antibiotics in the solution. A 10 nM of Cy5 tagged siRNA (3 µL, pentamethine cyanine dye tagged siRNA, Eurogentec, Belgium) alone and in combination with GPD (4 µl, 6 µl, and 10 µl) was initially diluted in Opti-MEM (75 µL) and incubated for 45 min. Bare siRNA and GPD/siRNA complex were added to cells and incubated for 4 h in Opti-MEM (1.5 mL). After 4 h, cells were washed with Opti-MEM and incubated with L-15 medium at 37 oC for 48 h. Thereafter, culture media was removed and cells were washed with 1 X PBS. Nuclear stain, DAPI (4',611 ACS Paragon Plus Environment


diamidino-2-phenylindole, Life Technologies, USA) was added onto the cells for 30 min. in 1 X PBS and siRNA transfection was quantified by counting number of coloured cells (Cy5 excitation at 647 nm) with respect to total number of cells using immunofluorescence microscope (Leica DM IL LED). 2.9 Flow analysis. To quantify the number of transfected cells, we performed flow based analysis of Cy5-siRNA transfected cells. MDA-MB-231 cells were seeded at a density of 0.3 million cells/well in a 12 well plate and transfected with GPD/siRNA complex. After 48 h, cells were trypsinized and washed with 1 X PBS buffer in dark. Before acquiring the samples, control cells with and without fluorescence labelled siRNA was used to set the voltage and gate for 5000 events. After setting the gate, Cy5-siRNA transfected cells were acquired by fluorescence activated cell sorting of live cells (FACS, BD FACSAria™ III cell sorter). The efficiency of transfection was compared with the control cells. To evaluate the efficiency of GPD nanovehicle, we also used Lipofectamine 2000 (3 µL) to transfect Cy5siRNA (3 µL, 10 nM). Results were analyzed and compared to determine the transfection efficiency. 2.10 Western blot assay. Cellular knockdown efficiency of transfected cells with GPD/siRNA complex was analyzed by western blot. Cells were transfected in same manner as mentioned above. Transfected MDA-MB-231 cells were harvested by trypsinization and lysed with lysis buffer (0.5% SDS, 50 mM Tris-Cl, 1 mM EDTA). Cell lysate were loaded at 20 µg/well and separated by 10% SDS-PAGE at 120 V for 3 h. After running the gel, it was transferred by semi-dry method on to the nitrocellulose membrane (Bio-Rad, USA) for 1.5 h and membrane was blocked with blocking buffer (5 % skim milk in 1X PBST, where 1X PBST is 0.1% Tween20 and 1X phosphate buffer saline) for 1 h. Nitrocellulose membrane was incubated with primary antibody of EPAC1 (anti-mouse EPAC1, Cell Signaling Technology, USA), and GAPDH (anti-mouse glyceraldehyde 3-phosphate dehydrogenase,


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Novus Biologicals, USA) at 1:1000 dilution in 1X PBST overnight (O/N) at 4 oC. Thereafter, the membranes were washed with 1X PBST and incubated with secondary antibody at 1:2000 dilution [secondary anti-mouse horse radish peroxidase (HRP) labelled, BD Biosciences, USA].

After

washing,

the

membranes

were

developed

by

ECL

(enhanced

chemiluminescence, Thermofisher, USA) solution and images were taken by Alpha Imager (Protein Simple, USA). Quantification of protein bands was performed by using the ImageJ software.38 2.11 In vitro wound healing assay. Wound healing assay was performed to analyze migratory properties of cancer cells. MDA-MB-231 cells were seeded and transfected with EPAC1 siRNA via GPD, PAMAM and Lipofectamine 2000 in a 12 well plate as described above. After the transfection, cancer cell monolayer was scratched using a sterile micropipette tip (Eppendorf, 10 µL) through the center to create an artificial and uniform wound. After making the uniform wound, media was changed after 1 h of scratch and cells were incubated for 24 h. To investigate the migratory properties after transfection of EPAC1, images were captured by microscope (Leica DM IL LED) at 0 h and 24 h. Wound closure was calculated using the following equation.

% =

ℎ, 0 ℎ − ℎ − , 24 ℎ × 100 ℎ, 0 ℎ

A total of 5 random areas from each well were selected and the experiment was repeated in triplicates. 2.12 In vitro invasion assay. To analyze the invasive properties of cancer cells, we performed invasion assay with 8 µm pore size culture inserts (Corning, USA). Culture inserts were coated overnight at 37 oC in humidified CO2 incubator with Matrigel (BD Biosciences, USA) at a concentration of 50 µg/mL in each well. Matrigel is a gelatinous protein mixture which resembles the extracellular matrix of many tissues and is utilized as a substrate for



growing cells. MDA-MB-231 cells were transfected with GPD, PAMAM and Lipofectamine 2000. After 48 h, transfected MDA-MB-231 cells were trypsinized and seeded on to the culture insert with incomplete L-15 medium. A total of 2.5 x 105 cells/mL in 200 µL media was seeded to each insert. Lower chamber of insert was filled with complete L-15 media (10% FBS). After 24 h, cells were washed with 1X PBS and processed for imaging. Initially cells were fixed with 4% formaldehyde in 1X PBS for 10 minutes and permeabilized by 100% methanol for 10 minutes. After permeabilization, cells were stained with crystal violet for 10 min. Cells were washed carefully at each step and after staining culture insert was taken out and put on a transparent glass slide (HiMedia, India) and then images of the invaded cells were captured by microscope (Leica, Germany). A total of 5 random areas from each well were selected and the experiment was repeated in triplicates. 2.13 In vitro siRNA release profile. EPAC1 siRNA 3ꞌend tagged with fluorescence dye Cy5siRNA was employed for the fluorescence based analysis. In vitro release kinetics of Cy5siRNA from GPD/Cy5-siRNA nanoconjugate was evaluated by the dialysis method. Dialysis was carried out to evaluate the release of Cy5 labelled siRNA from GPD/siRNA complex at different pH (8, 7.4, 7 and 6.5) buffer solutions separately. A 5 µL of GPD (0.5 mg/mL) was incubated with 3 µL of Cy5-siRNA (10 nM) in an incubator shaker in the dark at room temperature for 2 h to form GPD/siRNA complex. In a typical experiment, the so obtained GPD/siRNA complex was dispersed in PBS buffer solution (1 mL, pH 7.4), transferred to a dialysis bag (molecular weight cut-off: 10 kDa) and allowed to dialyze against PBS buffer (4 mL, pH 7.4) with constant stirring at 100 rpm maintained at 37 °C. Aliquots (1 mL) from dialysate were withdrawn at predetermined time intervals, and the container was replenished with the same amount of fresh PBS (pH 7.4). The fluorescence intensity of detached Cy5siRNA from GPD nanocarrier was estimated by measuring its fluorescence (excitation 600 nm, emission 663 nm). The amount of the released siRNA was calculated using a standard


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curve of Cy5-siRNA in PBS buffer. The same experiment was performed at different buffer pH solution. 2.14 Statistical analysis. Data obtained were analyzed using descriptive statistics, single factor analysis of variance (ANOVA) and presented as a mean value ± standard deviation (SD) from five independent measurements. We analyzed data sets for significance with Student's t test and considered P values of less than 0.05 as statistically significant. 3. RESULTS AND DISCUSSION 3.1 Synthesis of GO, GO-PEG (GP) and GO-PEG-PAMAM (GPD). Graphene based delivery vectors are vastly designed utilizing the GO synthesized by modified Hummer’s method. The procedure involved chemical exfoliation of graphite using potassium permanganate and sodium nitrate in concentrated sulfuric acid.34 To advance the above oxidation process in an industrially viable and sustainable manner demands elimination of both exothermic requirement (98 oC) and control measures to manage the larger volumes of generated toxic NOx gas(es). Moreover, reaction is known to be hazardous and may explode as it produces large exotherm during scale up of the process. In present work, an improved method with slight modifications is utilized to generate GO at milder temperature (50 oC) without the usage of NaNO3. GO synthesized by improved method is obtained in excellent yields (nearly three-fold higher than Hummer’s method) with simpler purification strategy.36 The GO synthesized by improved method is covalently functionalized with PEG and PAMAM to form GPD nanoparticles, as illustrated in Figure 2, to enable its exploration as a nanocargo for gene delivery.



Figure 2. Schematic presentation of covalent conjugation of PEG and PAMAM onto graphene oxide (GO) to form GP and GPD. Probable reaction mechanisms: (Step 1) oxidative cleavage of C=C bond via manganate cyclic ester intermediate resulting in formation of hydroxyl, epoxide, carbonyl and carboxylic functionalities to form improved GO; (Step 2) addition reaction of PEG to form GP; (Step 3) amidation reaction of PAMAM to form GPD.

The first step of synthetic pathway involves introduction of oxygen containing functionalities in graphite using a mixture of sulfuric acid: phosphoric acid in potassium permanganate to form GO. The generated functionalities includes carboxylic groups at the edges of GO while epoxide and carbonyl groups within the GO framework. It is reported that phosphoric acid acts as a better intercalant than sulfuric acid alone thereby mediating potassium permanganate oxidation within the graphene layers.39 In second step, the epoxide and carbonyl 16 ACS Paragon Plus Environment

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functionalities are reacted with amine groups of PEG via base mediated nucleophilic ringopening and addition reaction. This resulted in formation of covalently bound PEG chains in GO to form GP. In third step, the available carboxylic groups present in GP was chemically modified to amide bonds with reaction of primary amino groups of PAMAM using NHS/EDC chemistry to form GPD. It must be noted that the dangling free amino groups of both PAMAM and PEG linked to GO are available to bind with siRNA via electrostatic interactions. In accordance to reported procedures in literature,

32-33

also explored the

potential of the synthesized GO to directly link to PAMAM via electrostatic or covalent chemistry to form GO-PAMAM. However, precipitation of nanoconjugates is observed (Figure S1). This demonstrates PEGylation as a necessary step to stabilize the aqueous dispersion of modified GO NPs. Additionally, the covalently tethered PEG and PAMAM at different reactive sites in GO is expected to provide stronger energetic interactions with respective stability. The physically bound polymers on to GO may leach out with time affecting their stability in different media thereby restricting their applicability.

3.2 Characterization of GO, GP and GPD.

3.2.1 Spectroscopic and thermal analysis of nanoparticles. The conversion of graphite to GPD was studied by FTIR spectroscopy (Figure 3a). The appearance of characteristic vibration modes due to oxidation of graphite to GO is evident from peaks centered at 3200 cm-1 (O-H), 1617 cm-1 (broad peak due to C=O of carboxyl, ketonic, adsorbed water, sp2hybridized C=C), 1415 cm-1 (C-OH) and 1050 cm-1 (epoxide C–O–C). In order to quantify the nature of functionalities in the region 1500-1780 cm-1, the peak at 1617 cm-1 was deconvoluted (Figure S2) into three regions, namely 1718 cm-1 (7 %), 1620 cm-1 (69%) and 1572 cm-1 (24%). The deconvoluted peak at 1718 cm-1 is suggestive of meagre amount of carboxylic functionalities. Furthermore, the predominance of epoxide, ketone, and alkene rich



regions in GO framework is indicated from the FTIR studies. The epoxide, and ketone groups in GO may undergo nucleophilic attack by the amine groups of PEG to form GP. The covalent conjugation of PEG to GO to form GP is indicated by the development of a noticeable stretching vibration due to C-N, C-O-C (symmetric and asymmetric), C-H (methylene groups) and N-H at 1105 cm-1, 1050 cm-1, 1250 cm-1, 2877 cm-1, and 3400 cm-1 respectively. PAMAM was covalently tethered successfully at the edges of GP via amide bonds as confirmed by the existence of amide (NH−CO) bonds stretching vibrations at 1645 cm-1 and 1555 cm-1 (Figure 3a).40 Besides appearance of amide stretch, an apparent shift of ~ 73 cm-1 due to carbonyl of carboxylic groups (1718 cm-1) in GO to a lower wavenumber with a noticeable intensity is suggestive of carboxylic group transformation to amide linkages in GPD. The above results indicated successful chemical conjugation of PEG and PAMAM to the GO sheets and is expected to be robust due to covalent nature. The atomic percentage and chemical states of functionalities in GO, GP and GPD was determined by XPS. The wide-survey spectra of GO, GP and GPD (Figure S3) showed the presence of heteroatoms O and N along with C. The C(1s) spectra of the graphene derivatives (Figure 3b-d) were deconvoluted and fitted according to the literature.20 In GO, the existence of distinct Gaussian peaks due to oxygen rich functionalities, namely C-OH/epoxy, C=O, and COOH are observed at binding energy 286.2, 288.1, and 289.1 eV respectively along with C=C/C-C/C-H rich domains at ~ 284.6 eV. The relative percentages of C=C/C-C/C-H, epoxy, C=O, and COOH groups calculated from deconvoluted peaks, indicated nearly 19%, 53%, 24% and 4% respectively, which is also in good agreement with the FTIR studies (Figure 3a and S2). Deconvoluted C(1s) spectrum in GP (Figure 3c) showed a significant increase in % of C-C/C=C to 40% and development of new C-N peak at 287.3 eV confirmed successful attachment of carbon-rich PEG polymer chains via PEGylation reaction. In addition, carbonyl region in GO subsequently vanished in GP supporting nucleophilic addition reaction of amine


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functionalities in PEG chains,15 along with epoxide ring opening reaction. Upon conjugation of PAMAM in GP, GPD C(1s) spectrum (Figure 3d) showed nearly absent carboxylic region at ~ 289 eV with an enhancement in relative percentage of C-N peak to 8.8%.41 Compared to GP, a substantial increase in percentage of N is indicated in GPD due to binding of nitrogen rich PAMAM (Figure 1, Step 3). A successful introduction of PAMAM is further confirmed by deconvolution of N(1s) XPS spectrum of GPD (Figure 3e). The presence of N in two different environments is indicated by two values of binding energy in N(1s) deconvoluted spectrum at 399.3 eV and 401.3 eV which are attributed to the primary amine (-NH2) and amide (NH−CO) bonds respectively.41-42 According to the percentage of N, the amount of N in GP and GPD (Table S2) can be calculated as about 0.57 mmolg-1 and 3.90 mmolg-1 respectively. As expected, the nitrogen contents increases proportionally to the number of nitrogen atoms per functional group: addition of PAMAM (large excess of N-atoms per molecule) increases the surface concentration of nitrogen in GO samples to a large extent as compared with PEG (6 N-atoms per molecule).



Figure 3. (a) FTIR spectra of GO, GP and GPD. The characteristic peak of different chemical bonds formed is shown, suggesting successful covalent linking of PEG and PAMAM onto GO sheets; High resolution deconvoluted XPS: (b-d) C(1s) spectra of GO, GP and GPD respectively; (e) N(1s) spectrum of GPD; (f) TGA curve of GO, GP and GPD at a heating rate of 10 °C /min in nitrogen atmosphere. Elemental analysis of GP and GPD (Table S1) showed the presence of nitrogen confirming the incorporation of nitrogen containing functionalities onto the GO structure. The percentage of N determined in GPD both by bulk (7.0%) and surface (7.5%) studies are in good agreement. A relatively higher percentage of amine groups is essential to facilitate a substantial loading of siRNA. In addition, elemental analysis of GPD revealed a concomitant increase in the C/O ratio with functionalization which is accounted to the tethering of carbonrich PEG and PAMAM chains. The amount of PEG and PAMAM loaded in GO was estimated by thermogravimetry analysis (TGA), Figure 3f. Bare GO showed a significant mass loss in the temperature range of 50-650 oC, the mass loss < 250 oC is attributed to the presence of adsorbed waster, labile and oxygen rich functionalities. The higher percentage of oxygen containing functionalities in GO corroborated well with XPS and FTIR studies. PEG and PAMAM modified GO showed a substantial changes in TGA traces accounted to the considerable consumption of oxygen functionalities in GO to form a covalently linked, more thermally stable GP and GPD nanoarchitecture. A smaller mass loss (~ 4%) in GP between 150-210 ºC is attributed to the removal of oxygen containing functional groups and volatilization of hydrogen bonded water molecules from the graphene sheets.43 Unlike GP, thermal degradation of GPD showed a multistep degradation again confirming structural changes in GP due to modification with PAMAM dendrimer. GPD showed a higher char yield than GP which is accounted to the amount of grafted dendrimer. GO undergoes complete decomposition and showed a zero char


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yield at 650 ºC. Therefore, the relative amount of grafted PEG and PAMAM chains in GO was calculated from the char yields at 650 ºC as 13% and 25% respectively, supporting successful covalent and effective polymer tethered GO nanoparticles.

Figure 4. Characterization of GO, GP and GPD: a) UV-vis. spectra (inset shows the photomicrograph of aqueous dispersion of nanoparticles (0.5 mg/mL); b) Raman spectra; c) XRD pattern.

UV-visible spectrum of GO, Figure 4a, showed absorption due to π → π* transition of carbon double bonds and n → π* transition of carbonyl groups at ~ 228 nm and ~ 300 nm (shoulder) respectively.44-45 Both GP and GPD showed a red shift in π → π* transition from 228 to 238 nm with an unnoticeable shoulder at 300 nm suggesting enhancement in conjugation and reaction at carbonyl groups in GO, which is also confirmed by XPS studies (Figure 3b-d). The color of aqueous dispersion and synthesized NPs changed from yellowishbrown to dark-black (Figure 4a, inset) is also an indication for formation of more reduced conjugated structures after the reaction of amine rich PEG units with GO at a higher temperature.46 As compared to GO, both GP and GPD showed a nearly ∼ 87.5% increment in absorbance value in near IR region, 800- 900 nm. This further confirms the existence of more reduced domains advancing their applicability as photothermal agents for cancer therapy.20, 33



Raman spectra of GO, GP and GPD and its analysis are shown in Figure 4b and Table S3. The characteristic absorption due to graphene structure are observed at 1350 cm-1 (D band) and 1586 cm-1 (G band) respectively. The D band is associated to the defects and disorders while the G band is associated with bond stretching of sp2 hybridized carbon in graphene sheets.47 The ratio of the intensities of D and G band (ID/IG) can be correlated to the degree of chemical functionalization, i.e., a higher ID/IG ratio indicates a higher degree of disorder in graphene nanomaterials.48 The conversion of a highly crystalline graphite to nanocrystalline form accounts to the broadening of D and G bands.49 Both GP and GPD led to an increase in ID/IG ratio to ~ 1.0 along with a subsequent broadening of bands. This can be visualized as intercalation and wrapping up of GO sheets by covalently tethered PEG and PAMAM chains led to the formation of nano-size crystalline structures. Moreover, the distance between defects (LD) in GO (~ 10 nm) was found to be similar in GP and GPD which further supports chemical functionalization occurred on the initial generated, preexisting defects in GO, as proposed in Figure 1. The in-plane crystallite size (La) further supports reduction in size after chemical functionalization suggestive effectiveness of PEG and PAMAM to former smaller GO domains.20 XRD pattern of GO (Figure 4c) indicated the appearance of a typical diffraction peak of graphite oxide at 2θ = 9.7o corresponding to a d001 planes.35 The interlayer spacing (d) evaluated as 9.0 Å using Bragg’s equation (nλ = 2d.sinθ) which is ascribed to the formation of oxygenated groups on graphene. As evident from Figure 4c, both GP and GPD exhibited no XRD peaks suggesting formation of non-diffracting nano-conjugates due to wrapping of polymer around GO nanoparticles. The disappearance of GO diffraction peak indicates an excellent dispersion and exfoliation of GO particulates, as the interlayer spacing is enhanced with the formation of nanostructure.


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3.2.2 Morphology and stability studies of nanoparticles. The morphology of GPD is analyzed using SEM (Figure 5a-d) and TEM (Figure 5e-f) images.

Figure 5. Representative FESEM (a-d) and TEM (e-f) images of GPD showing the surface morphology at different resolutions. FESEM of GPD displays flowery structures with longitudinally aligned self-assembled nano-architectures onto the GO surface. TEM images showed smaller aggregates with nano-columnar assembly.

The existence of mainly smooth surface morphology with intermittent rough surface is observed in SEM (Figure 5a) image of GPD. Further analysis of GPD, showed widely spaced flowery structures (Figure 5b-c) which consists of longitudinally aligned columnar architectures (Figure 5d). TEM images illustrated the formation of multiple smaller nanoaggregates along with self-assembled dendritic wrapping on graphene structure. The diameter of 4th generation dendrimer is 4.5 nm.50 We observed a nearly ~ 10 nm wrapping of organic layer on GO nanoparticles. This can be visualized as formation of inter- and intra-molecularly linked polymer and GO frameworks via electrostatic and H-bonding interactions between polar groups.31 23 ACS Paragon Plus Environment


Both particle size and surface charge (zeta potential, ζ) of nanoparticles are crucial parameters to dictate their cellular uptake efficiency along with pertinent pharmacodynamic profiles mainly biodistribution, elimination and delivery of payload to the intended target sites. In addition, they also empower the stability to nanoparticle dispersion to influence their stability in media and designing drug formulations.51-52 A highly positively or negatively charged smaller size NPs (~10 nm) accounts to an undesirable uptake by liver with a simultaneous issues of hemolysis and cytotoxicity.53 On the contrary, a value of ζ > + 30 mV and < – 30 mV are considered as stable dispersions due to a higher inter-particle electrostatic repulsions. There is no generalization of size and charge of NPs but surely a very large size (> 200 nm) and highly positively charged NPs may face inhibition towards cellular uptake. However, lowering of charge on GO NPs may affect their stability at physiological pH but it can be resolved by tethering polymers.12 Figure 6a revealed an average hydrodynamic size of GO, GP and GPD as 220 nm, 54 nm and 130 nm respectively. The smaller size of both GP and GPD promotes their applicability as nano-cargo for cellular delivery. PEGylation induces a size reduction suggesting wrapping of polymer chains on to the GO sheets thereby preventing their aggregation.9 Modification of PAMAM to form GPD led to an increment in hydrodynamic size, due to formation of additional coating on GP surface.30 The ζ of GO, GP and GPD was determined as – 30, – 10, + 10 mV (Figure S4, Table S4) respectively.


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Figure 6. (a) DLS measurement of GO, GP, and GPD; (b) Digital images of GO, GP and GPD in different media at a concentration of 0.5 mg/mL, recorded after centrifugation at 10,000 rpm for 5 min. The settling of GO is observed in all media while both GP and GPD was found to form stable dispersions in water, PBS, cell medium and saline (10%).

The variation in ζ towards the positive value is attributed to the amino functionalities of covalently decorated PEG and PAMAM chains.54 A higher amine functionalities form positively charged ammonium surface groups which counteract the initially present negatively charged carboxylate ions from carboxylic groups in GO. Both hydrodynamic size and ζ measurement further confirms a successful attachment of PEG and PAMAM to GO. In comparison to GO, as-prepared GP and GPD exhibited excellent dispersion stability in variety of media including complete Dulbecco's modified eagle's medium (cDMEM) and sodium chloride solution (Figure 6b). This behaviour is attributed to the hydrophilic nature of PEG and PAMAM, presence of charge and reduction in their size. Surprisingly, GPD showed excellent dispersion stability as GP confirming existence of stable, covalent binding of PEG



and PAMAM. While non-covalent binding of cationic polymers such as PEI and PAMAM to graphene is associated with their dissociation due to relatively weaker interactions.20, 40, 55

3.3 Characterization of GPD-siRNA nanoconjugates 3.3.1 Cell viability and binding of siRNA with GPD nanoparticles. The biocompatibility of newly designed and synthesized nanocargo, GPD vs. neat PAMAM dendrimer was investigated in breast cancer cells, MDA-MB-231 cells, Figure 7a. Both GPD and PAMAM at different concentrations were incubated with cells for 48 h before determination of cell viability using MTT assay. Figure 7a revealed PAMAM is more toxic to the cells as compared to GPD with the half maximal inhibitory concentration (IC50) of 10 µg/mL. While GPD showed an IC50 value of 20 µg/mL. Even at high concentrations of up to 10 µg/mL, the viabilities of GPD treated cells was nearly 73%, which is approximately 1.5 fold higher than PAMAM treated cells. The lowering in cytotoxicity of GPD is attributed to the beneficial and excellent biocompatibility effect provided by PEG, enabling GPD to acts as a relatively safer and promising gene delivery agent.13 To gain further insight into the properties of GPD, we evaluated its binding ability with EPAC1 siRNA. This conjugate can be used for translational approach for oncotherapy. Binding analysis was performed by gel retardation assay (electrophoretic mobility shift assay, EMSA or band shift assay). A constant volume of EPAC1 siRNA was mixed with different volumes of GPD to give different predetermined volume ratios (GPD:siRNA as 0:3, 1:3, 2:3, 3:3, 4:3 and 5:3). The unbound siRNA moved swiftly across the gel as an intense band (Figure 7b). While GPD/siRNA complex at a lower volume of GPD (1-3 µL) retarded the movement of siRNA in an exponential manner with a reduction in intensity. This suggests probably further addition of GPD is required to effectively inhibit the movement of siRNA in


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agarose gel.

A further increase in volume of GPD (4-10 µL) effectively inhibit the

movement of siRNA with no detectable free siRNA in the gel. The bands appeared at the base line indicating excellent binding efficacy of GPD to siRNA. The volume ratios of GPD: siRNA as 4:3, 6:3 and 10:3 were used in subsequent studies.

Figure 7. (a) Relative cell viability of MDA-MB-231 cells analyzed with GPD and PAMAM. GPD exhibited lower toxicity. Binding strength of siRNA (3 µL, 10 nM) with GPD at different volumes: (b) Gel retardation assay of GPD/siRNA; (c) Zeta potential (inset shows the hydrodynamic diameter of GPD, siRNA and the resultant complex prepared under same conditions); (d) Quenching of fluorescence of Cy5 tagged siRNA due to the complex formation with GPD. To further understand the interaction between GPD and siRNA, variation in surface charge was monitored at same volume ratios by zeta potential measurements (Figure 7c).



Bare siRNA showed a ζ of – 12 mV due to presence of negatively charged phosphate groups. Addition of GPD (ζ of + 10 mV) shifted the ζ values towards positive values suggesting binding of siRNA on to GPD via electrostatic interactions with effective lowering of charge due to neutralization. As expected from gel retardation assay, ζ measurement further confirms nearly 4-6 µL of GPD was sufficient to completely pack siRNA elaborating their potentials as nucleic acid carriers. In addition to energetically favorable ionic binding, it is possible that, siRNA can also bind to surface of GO through hydrophobic and π-π stacking interactions.56-58 The bases present in siRNA contain aromatic rings which are known to facilitate such shortrange interactions. A ~ 100 nm decrease in hydrodynamic size of neat siRNA from ~ 345 nm is observed due to binding of GPD supporting a better packing and efficient interactions of siRNA units with GPD (inset, Figure 7c). The presence of negative surface charge on siRNA hinders their passive diffusion across the negatively charged cell membrane. Simultaneously, an excess of positive charge on cationic PAMAM dendrimer is known to induce cytotoxicity.59 Moreover, the GPD at 4-6 µL attains the plateau of charge of ~ 5 mV, indicating effective loading of siRNA. The development of positive charge acquired by GPDsiRNA nanoconjugates may enable their swift entry across the membrane. Fluorescence emission spectra (Figure 7d) further confirms the interaction of GPD with siRNA tagged with near infrared fluorescent cyanine dye, Cy5. Fluorescence of free Cy5-siRNA was quenched significantly at same concentration as compared to GPD/Cy5siRNA complex suggesting close proximity of Cy5-siRNA to the GO sheets to allow fluorescence resonance energy transfer (FRET). A concentration dependent quenching in fluorescence is observed which increases with increasing concentrations of GPD. A complete quenching of fluorescence in GPD/ Cy5-siRNA is not observed due to non-covalent immobilization of Cy5-siRNA at the periphery of linked PAMAM. In addition, further wrapping of PEG and PAMAM to GO accounts to blocked interactions between GO and


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Cy5, lowering the quenching efficiency.60-61 The above results confirmed an interaction of siRNA with GPD to form GPD/siRNA complex. 3.3.2 Intracellular uptake of GPD/siRNA complex. A nanovehicle, used in either in vitro or

in-vivo studies, must be efficient to deliver the desired molecules inside the cells for better outcomes. To investigate the gene delivery properties of nanovehicle, we performed in vitro transfection analysis. As a preliminary step, we have determined that whether GO has any effect in providing the protection of siRNA from enzymatic cleavage. We monitored the digestion of siRNA by RNase. From Figure S5, siRNA without GO was digested within 5 min. However, when present with GO, even after 20 min., a significant stability towards enzymatic hydrolysis was observed. This suggested GO has a role in protection of siRNA which is essential for GO-siRNA based therapeutic applications. The cellular uptake of Cy5labeled siRNA (red fluorescence) either bare or complexed with different GPD/Cy5-siRNA volume ratios (4:3, 6:3 and 10:3) was determined by using confocal fluorescence microscopy (Figure 8a). The fluorescence images of cells clearly confirms inability of bare siRNA to penetrate the cells as indicated by the absence of red fluorescence. A measurable red fluorescence was observed when cells were incubated with GPD/Cy5-siRNA complexes as evident from Figure 8a. A uniform distribution of siRNA inside the cytoplasm and around nuclei was observed confirming successful internalization by the cells. Unexpectedly, a further increase in GPD volume to 10 µL lowered the transfection efficiency as indicated by a lower fluorescence intensity (Figure 8a, c). The morphology of cells was found to be deformed at higher volumes of GPD (> 6 µL). This could be attributed to the increase in positive charge > 5 mV for GPD/ siRNA complex (as siRNA volume used is constant, 3 µL, 10 nM). A higher positive surface charge renders cytotoxicity to the cationic PAMAM dendrimer/ linked GPD. A weak positive charge of < +5 mV of cargo-bound with siRNA has been suggested as ideal to effectively induce necessary cell surface interactions and permits 29 ACS Paragon Plus Environment


its endocytosis.62 Moreover our complexes are quite stable despite carrying nearly neutral charge lower charge, which is beneficial to counterbalance cytotoxicity issues. A nearly neutral surface is desired which can be achieved by modification of peripheral amine groups of PAMAM accounted both lowering in cytotoxicity with an enhanced cellular


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Figure 8. Cellular uptake and localization of bare siRNA, GPD/siRNA and Lipofectamine 2000. Representative fluorescence microscopy images of MDA-MB-231 cells (a) incubated with the Cy5-siRNA and GPD/siRNA, nucleus was stained with DAPI (blue) and siRNA was labelled with Cy5 dye (Red); (b) Comparison between transfection capability of Lipofectamine 2000 with GPD nanocarrier on HUVEC cells using fluorescence microscopy; (c-d) Quantitative analysis of transfection in terms of relative intensity of Cy5 labelled dye.

internalization siRNA oligonucleotides. Furthermore, we also analyzed the transfection efficiency of commercially available transfecting agent, Lipofectamine 2000 in primary Human Umbilical Vein Endothelial Cells (HUVEC) using the same protocol (Figure 8b). Primary cells are more sensitive and difficult to transfect comparatively to epithelial cells. A nearly 2-fold higher transfection efficiency in primary cells was observed by GPD as compared to Lipofectamine 2000 transfected cells (Figure 8d) confirming GPD as an effective nanocarrier for delivery of siRNA. After knowing the localization of GPD/siRNA nanoconjugates in cancerous cells, we further quantified the number of transfected cells using FACS analysis, Figure S6 and 9a. A variation in fluorescence intensity in a concentration dependent manner was observed suggesting a substantial influence of GPD to transfect siRNA into the cells. A maximum



transfection efficiency of 56.5% was achieved with loading ratio of 2:1 (6:3 v/v of GPD/siRNA complex). However, a further increase in volume of GPD in GPD/ siRNA complex i.e. 10:3 v/v led to a lowering in transfection efficiency (29.1%), which corroborates well with fluorescence microscopy imaging studies (Figure 8a). The transfection efficiency of Lipofectamine 2000 was found to be 45.6% (Figure S7b). A further increase in volume to 10 µL led to a significant reduction in transfection which may be possibly attributed to its relative serious cytotoxicity (Figure S7a). Our results clearly suggests that a better transfection efficiency of GPD may be because of its better binding efficiency and biocompatibility in cellular microenvironment. Bio-functional aspect of GPD can also be one of the major factor for better transfection efficiency as it can easily get inside the cell without loss of siRNA which confirms its translational potential for oncotherapy. 3.3.3 Influence of serum in transfection efficiency. Various investigations such as cell viability, stability of cationic polymers based nanocargo and their ability to permeate and effectively deliver nucleic acid inside the cells under physiological conditions are truly essential to enable their clinical applications. Such physiological conditions are usually mimicked in FBS. It is reported that there is no significant role of FBS in evaluation of effect of PAMAM on cell viability as determined by MTT assay.63 Only higher concentrations of PAMAM requires higher percentage of FBS to mitigate dendrimer induced cytotoxicity as it supplements and stimulate cells to proliferate. Recent studies reported bare cationic PEI polymers showed a decreased gene transfection ability due to adsorptions of positively charged cationic polymers with negatively charged serum proteins. 20 The presence of FBS is known to affect the dispersion stability leading to aggregation and precipitations of graphene functionalized cationic polymers. A much higher dispersion stability is achieved in physiological media by GPD, despite the negative nature of serum, Figure 6b. This provided us the impetus to explore transfection efficiency of GPD by varying FBS concentration from


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5% to 20%. As evident from Figure S8, a significant impact of FBS concentration is revealed in transfection efficiencies. The change in concentration from 5%, 10%, 15% and 20% revealed transfection efficiency as 0.7%, 56.5%, 41.4% and 29.3% respectively. Interestingly, 5% FBS showed an ineffective behaviour in transfecting siRNA. This is attributed to lower amount of FBS which led to a decrease in cell viability with subsequent poor transfection ability. A 10% FBS concentration was found to be optimum, a further increase in FBS percentage lowered the transfection ability. There is presumably accounted to a competitive preferential interactions of constituents present in FBS (oppositely charged cationic polymers and largely present serum proteins) than with nucleic acid materials as latter is present in extremely inconspicuous amounts. It is further supported by reduced ζ values in presence of FBS (Figure S8b) and with an effect on hydrodynamic size of GPD (Figure S8c). Surprisingly, an extreme size reduction of GPD is observed in 10% FBS accounts to a higher transfection efficiency. Both FACS and fluorescence imaging studies confirmed excellent efficacy of GPD to deliver siRNA inside the cells. 3.3.4 Efficacy of GPD/siRNA against target expression. Gene delivery is a tool to knockdown the desired gene inside the cells to manipulate their functional properties. cAMP signalling pathway participates in many cellular processes and cAMP-EPAC1 axis is one of the key player that affects many cellular mechanism such as intracellular interaction and metastasis in different types of cancer. We previously explored the role of EPAC1 in breast cancer progression, so present aim is to investigate the nanovehicle based delivery of EPAC1 siRNA for translational capability.3

EPAC1 gene is targeted which is one of the major

controlling factor of cAMP pathway. The mechanism of down-regulation of a specific gene by siRNA is mediated by two factors. Firstly, effective cellular internalization of siRNA and secondly, release of siRNA by endosomal escape of the payload to perform the task. However, a higher transfection efficiency of siRNA not necessarily infers effective silencing 33 ACS Paragon Plus Environment


of its targeted mRNA. Several reports discussed excellent cellular uptake of siRNA into the cells, rather unsuccessful in gene knocking down or silencing due to their inefficient unloading of bound siRNA inside the cells.30 To investigate the efficacy of GPD based transfection in cancer cells, western blot of EPAC1 was performed. Antibody probing to the nitrocellulose membrane was developed by ECL method, showed a reduced expression of EPAC1 as compared to control cell lysates. Reduction in protein expression of EPAC1 suggests that GPD nanovehicle efficiently delivers the siRNA into the cells and releases the siRNA for inhibiting the cellular process of target gene. As siRNA targets the gene expression, western blot result indicates significant down regulation of EPAC1 protein (Figure 9b).

Figure 9. (a) Quantification of flow cytometry analysis on the transfection efficiency of GPD/siRNA complex in MDA-MB-231 cells; (b) Western blot assay was performed for probing EPAC1 protein to check inhibition of EPAC1 mRNA by EPAC1 siRNA. MDA-MB231 cells were transfected by Cy5-siRNA without any vehicle and Cy5-siRNA transfected through GPD. Relative expression of EPAC1 expression is checked by western blot assay after harvesting and lysing the cells. Amount of protein was quantified by nanodrop. Approximately, 20 µg protein was loaded in each well. Relative intensity quantification of western blot band is done by ImageJ software by taking GAPDH as an internal control. The 34 ACS Paragon Plus Environment

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error bars indicated the standard deviation of three individual measurements. Data are mean measurements in triplicate. *Significantly different from control, *p < 0.05, **p < 0.01, ***p < 0.001.

3.3.5 In vitro cell-migration and invasion studies. Cancer cells have the tendency to invade and migrate from surrounding tissue to another part of the body. Targeting in vitro migration and invasion studies could provide an insight into the role of the EPAC1 siRNA against metastatic properties of the MDA-MB-231 cancer cells.To investigate the functional aspect of EPAC1 siRNA delivered through GPD at an optimal and effective volume ratio (6:3 v/v, GPD/siRNA), a scratch wound migration assay was performed (Figure 10a, b).

Figure 10. Cell migration assay, MDA-MB-231 cells were wounded and then transfected with the EPAC1 siRNA via PAMAM, Lipofectamine 2000 and GPD: (a) The migration distance at 0 h and 24 h was imaged and (b) quantified after transfection at 24 h. (c-d) Cy5siRNA transfection was performed to analyze cell invasion properties of cancer cells. Invasion of cancer cells were measured after 48 h using a Boyden chamber assay and number



of invaded cells were counted after staining with crystal violet and quantified. Data are mean ± SE of n = 3 in triplicate. *Significantly different from control, *p < 0.05, **p < 0.005. GPD/siRNA showed a higher inhibitory effect for migration of cells as compared to control cells suggesting the role of EPAC1 in cancer cell migration properties. The migratory properties are linked to a sequential process known as metastasis in which cells migrate and invade surrounding tissues to develop secondary tumour. Later, the invasive properties of cancer cells are analyzed by targeting the expression of EPAC1 while transfection with nanovehicle and Lipofectamine 2000 (Figure 10 c-d). Interestingly, a lower cell invasion was observed in GPD/siRNA transfected cells as compared to siRNA delivered using Lipofectamine 2000 and PAMAM in the cells. This additionally supports effectiveness and compatibility of GPD as a nanovehicle for gene therapy. Above studies provide an indirect clue about the role of GPD/siRNA conjugate for translational oncotherapy.

3.3.6. Effect of pH to unload siRNA from the GPD/siRNA complex. The complex of Cy5siRNA and GPD was prepared by adding Cy5 labelled EPAC1 siRNA to GPD. The release kinetics of siRNA from GPD/ Cy5 -siRNA was studied at variable pH buffers i.e. 6, 7, 7.5, and 8 using dialysis method. The presence of dialysis membrane is necessary to prevent reversible binding of released Cy5-siRNA to GPD.64 Interestingly, a pH dependant release of siRNA from GPD/siRNA complex is observed.65 The fluorescence intensity of release Cy5siRNA follows the order 7 .5 > 7 > 8 > 6 (Figure 11a). From the graph, it is evident a faster siRNA release was observed initially followed by a prolonged sustained release rate. PAMAM possess variable pKa values for external primary (~ 7-9) and internal tertiary (~ 36) amines, 66 which can be tuned with pH of media. The estimated percentage of positively charged surface primary amine groups is 22% and the interior tertiary amine groups are almost uncharged (~ 0.03%) at pH 7.4. The variation in charges on dendrimer with pH is illustrated in Figure 11b. The stability of GPD/ siRNA complex depends on the exposition to 36 ACS Paragon Plus Environment

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the pH. A direct alteration in total charge of the PAMAM dendrimer is anticipated to affect electrostatic forces of attractions with siRNA and therefore, mediate siRNA release from the GPD/siRNA complex.

Figure 11. (a) Effect of pH on Cy5-siRNA. Graph showing variation in fluorescence intensity to determine in vitro siRNA release kinetics of Cy5-siRNA release from GPD nanocarrier at different pH (6.5, 7, 7.4 and 8) and time (0-72 h) using dialysis assay. (b) Pictorial presentation of PAMAM dendrimer at different pH. Blue circle represents the PAMAM structure and one of the branches (out of six) is shown to indicate the presence of tertiary amine (pKa 3-6) groups (inner periphery) and primary amine (pKa 7-9) groups (at the surface). The variation in composition and structural features with increase in pH is shown to



indicate the extent of deprotonation. (c) Fluorescence intensity measurements of the Cy5tagged siRNA suspended in different pH medium at different time intervals. No significant change in the fluorescence intensity of the Cy5-tagged siRNA is observed, and thus ensure its stable behaviour in the medium (inset shows the pH sensitivity of cyanine dye and complementary anhydronium base); (d) Absorbance curves of the Cy5-siRNA at different concentrations at pH 6 (inset shows the correlation between Cy5-siRNA absorbance value at 663 nm and its concentration).

At pH 7.5-8, the percentage of primary amine as ammonium ions is expected to be lower, and below i.e. pH 6-7, the percentage of ammonium ions will be high.67-68At lower pH, a higher net dendrimer charge accounts to more tightly packed siRNA due to higher electrostatic interactions, therefore accounts to a slower release rate. A higher and faster release from the GPD/ Cy5-siRNA complex is observed at higher pH i.e. 7.5. Surprisingly, at pH 8 a slower release rate and reduced fluorescence intensity (Figure 11a) signal is observed. This anomalous behaviour can be explained due to reduced fluorescent nature of Cy5 dyes in an alkaline medium. The fluorescent pentamethine cyanine dye (Cy5™) is sensitive to proton concentration and intensity of the fluorescent emission (at 663 nm) decreases with decrease in concentration of protons (i.e. higher pH). This is further confirmed by time dependant variation in fluorescence intensity of Cy5-siRNA (3 µL, 10 nM) as a function of pH (6.5, 7, 7.5 and 8), Figure 11c. It can be observed that the fluorescent characteristics of the Cy5siRNA are greatly reduced with the increase in pH of buffer. This is accounted to the formation of larger population of the non-fluorescent base species due to increased deprotonation of the cyanine dye (inset, Figure 11c), corroborates well with siRNA release assay (Figure 11a). The percentage release of siRNA at 72 h was estimated as 11.8, 38.9, 60.9 and 38.4% at pH 6.5, 7.0, 7.5 and 8.0 respectively. Alternatively, the amount of siRNA released was determined as 8.3% at pH 6 (using regression equation, Figure 11d), which 38 ACS Paragon Plus Environment

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again is in good agreement with above analysis. A slower and sustained release of siRNA from designed GPD carrier is observed which is desired to ensure its optimal pharmacological effect. Besides pH, under in-vivo conditions intracellular release of siRNA from GPD/siRNA nanocomplex is controlled by several cellular factors and processes, which demands further analysis. One rationale in designing vectors is to achieve higher transfection efficiency, on other side, an understanding of several cellular pathways mainly endosomal escape mechanism, release of gene material and its internalization needs to be developed to realize the therapeutic potential of gene therapy.

69

It is proposed, that the lower pKa of

internal tertiary amine groups in PAMAM induces high buffer capacity to assist the rupture of endosome to release the bound siRNA by increasing osmotic pressure via “proton sponge effect”. In our case, the electrostatic forces of interaction between positively charge, PAMAM and negative charge, siRNA is indicative of dynamics of both loading and unloading of siRNA. Any factor which can weaken such interactions intracellularly may be responsible for release of siRNA. A rapid dynamic exchange amongst free and bound DNA 70 or siRNA with respective polyplexes is reported. Even, competitive binding of any plasma membrane components, intracellular RNA, lipids, and proteins to nanocargo over siRNA or change in pH may be an effective aid to assist the release of siRNA. Consequently, a combined research effort is desired to ascertain the mechanism for release of siRNA from the cargo that demands further detailed investigations.

4. CONCLUSIONS In current work, we systematically explored the capability of improved GO to covalently linked PEG and PAMAM via alternative synthetic methodology to form GPD nanoparticles. The formation of self-assembled GPD nanoarchitecture is supported by SEM and TEM images. A remarkable stability of GPD at physiological pH without extensive purification of GPD carrier is the key for the exploration at clinical level. The efficacy of GPD as a nonviral 39 ACS Paragon Plus Environment


vector for EPAC1 siRNA delivery was determined in breast cancer and HUVEC cells. GPD revealed a lower cytotoxicity with the ability to condense siRNA into nanosized complexes. The designed nanocarriers showed a very high transfection efficiency of GPD/siRNA complexes and effective siRNA release with appreciable gene silencing efficiency. Moreover, effect of FBS concentration on transfection was also analyzed and 10% FBS showed the highest transfection efficiency. A pH-responsive siRNA release behaviour is observed, with a sustained siRNA release rates under acidic pH conditions. Our investigations clearly suggests that the GPD exhibited reduced cytotoxicity, efficient cellular uptake and effectiveness towards metastasis and invasion properties than PAMAM and Lipofectamine 2000 based siRNA complexes. Thus, present work highlights the ability of modified GO as a powerful vector for the efficient delivery of siRNA with improved therapeutic potential. ASSOCIATED CONTENT Supporting Information Treatment of GO with PAMAM and EDC; Deconvolution of FTIR spectrum of GO; XPS traces of functionalized GOs: (a) wide survey spectra, (b) N(1s) spectrum in GP; Elemental raman and zeta potential analysis of GO functionalized with PEG and PAMAM; Composition of GP and GPD obtained from different analytical techniques; Flow cytometry analysis on the transfection efficiency of different volumes of GPD complex with EPAC1 siRNA in MDA-MB-231 cells; Cell viability and transfection efficiency of Lipofectamine 2000 at different concentrations with EPAC1 siRNA in MDA-MB-231 cells; Effect of FBS percentage on transfection efficiency, zeta potential, and hydrodynamic radii of GPD/siRNA in MDA-MB-231 cells (PDF). AUTHOR INFORMATION Corresponding Author


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*Email: [email protected]; [email protected]; [email protected] Author Contributions The manuscript was written through contributions of NY, N.K., S.S. and BL. All authors have given approval to the final version of the manuscript. The authors would like to acknowledge the financial support from Shiv Nadar Foundation. S. Sh. work was funded through the Opportunities for Undergraduate Research (OUR) scheme. We are thankful to Malvern and Sprint testing solutions for providing assistance in characterization facility. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by Shiv Nadar Foundation. REFERENCES 1) http://www.who.int/cancer/detection/breastcancer/en/index1.html accessed on 2018, Feb 28. 2) Sehrawat, S.; Ernandez, T.; Cullere, X.; Takahashi, M.; Ono, Y.; Komarova, Y.; Mayadas, T.N. AKAP9 regulation of microtubule dynamics promotes Epac1-induced endothelial barrier properties. Blood 2011, 2, 708-718. 3) Kumar, N.; Gupta, S.; Dabral. S.; Singh. S.; Sehrawat. S. Role of exchange protein directly activated by cAMP (EPAC1) in breast cancer cell migration and apoptosis. Mol. Cell Biochem. 2017, 430,115-125. 4) Kumar, N.; Prasad, P.; Jash. E; Saini, M., Husain, A., Goldman, A. and Sehrawat, S. Insights into exchange factor directly activated by cAMP (EPAC) as potential target for cancer treatment. Mol. Cell Biochem. 2018, 1-6. 5) Keles, E.; Song, Y.; Du, D.; Dong, W.J.; Lin, Y. Recent progress in nanomaterials for gene delivery applications. Biomater. Sci. 2016, 9, 1291-1309. 6) Asai, T.; Matsushita, S.; Kenjo, E.; Tsuzuku, T.; Yonenaga, N.; Koide, H.; Hatanaka, K.; Dewa, T.; Nango, M.; Maeda, N.; Kikuchi, H. Dicetyl phosphatetetraethylenepentamine-based liposomes for systemic siRNA delivery. Bioconjugate Chem. 2011, 22, 429-435.



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Graphical Abstract


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Stable dispersions of covalently tethered polymer improved graphene

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