Carbon dot festooned and surface passivated graphene-reinforced chitosan construct for tumor-targeted delivery of TNF-α gene

Carbon dot festooned and surface passivated graphene-reinforced chitosan construct for tumor-targeted delivery of TNF-α gene

International Journal of Biological Macromolecules 127 (2019) 628–636 Contents lists available at ScienceDirect International Journal of Biological ...

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International Journal of Biological Macromolecules 127 (2019) 628–636

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Carbon dot festooned and surface passivated graphene-reinforced chitosan construct for tumor-targeted delivery of TNF-α gene Jumana Abdul Jaleel a, Shabeeba M. Ashraf b, Krishnan Rathinasamy b, K. Pramod a,⁎ a b

College of Pharmaceutical Sciences, Govt. Medical College, Kozhikode, Kerala, India School of Biotechnology, National Institute of Technology Calicut, Calicut, India

a r t i c l e

i n f o

Article history: Received 4 October 2018 Received in revised form 18 January 2019 Accepted 28 January 2019 Available online xxxx Keywords: Carbon dot Chitosan Graphene

a b s t r a c t Gene therapy is a promising alternative that ensures effective treatment and cure for cancer. Here, we report graphene-reinforced chitosan (CS) construct based non-viral vector for tumor-targeted gene therapy. The therapeutic gene, pDNA-TNF-α, was loaded on to chitosan-carboxylated graphene oxide (CS-CGO) construct via electrostatic interaction. The pDNA-TNF-α-CS-CGO thus obtained was further passivated with 4,7,10-trioxa-1,13tridecanediamine for protecting the vector from the mononuclear phagocyte system that contributes to the prolongation of circulation half-life. The surface passivated carrier (PEG-pDNA-TNF-α-CS-CGO) then festooned with the folic acid derived carbon dots (C-dots) for targeting folate receptors that are overexpressed in most of the cancer cells. The results of TEM images and zeta potential values ensured the occurrence of desired changes in each stage of C-dot-PEG-pDNA-TNF-α-CS-CGO formulation. After 14 days of incubation, the anti-angiogenesis effect was observed for final formulation in the chorioallantoic membrane. The results of in vitro gene expression study in cancer cell line show a comparatively higher transfection efficacy of the developed system (C-dot-PEGpDNA-TNF-α-CS-CGO) than pDNA-TNF-α. The efficiency of the developed gene delivery system was further confirmed using a developed and validated artificial tumor cell apparatus. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Cancer is one of the most dangerous diseases in the world. Conventional treatment like surgery, chemotherapy, radiation therapy etc. are effective, but are associated with severe side effects and chances of recurrence is more. Gene therapy involves the use of a therapeutic gene that is capable of expressing proteins which alters the disease condition. To ensure specificity and safety during gene therapy, it is desired to control its delivery to the target site [1]. Thus, in the present study, we aimed for a gene delivery system for tumor-targeted therapy. TNF-α gene that codes for the protein tumor necrosis factor (TNF α), a cytokine produced during immunological reactions by the immune cells mainly macrophages, have anti-tumor activity. Thus, TNF-α gene is a promising candidate for cancer gene therapy [2]. Biodegradable polymers are known for their low toxicity and high biocompatibility [3] and are preferred for gene delivery applications. Chitosan (CS) is a natural cationic polymer having no cytotoxic issues. The cationic nature of chitosan immensely contributes to their transfection efficiency [4]. They are superior to other widely reported cationic polymers such as polyethylenimine (PEI), owing to their biodegradable and biocompatible nature. The transfection efficiency of PEI is higher than that of ⁎ Corresponding author. E-mail address: [email protected] (K. Pramod).

https://doi.org/10.1016/j.ijbiomac.2019.01.174 0141-8130/© 2019 Elsevier B.V. All rights reserved.

chitosan [4]. But, toxicity is associated with a higher transfection efficiency of polyethylenimines. Thus, chitosan was chosen in our study. Condensation of plasmid DNA with cationic polymers causes a size reduction, offers protection against degradation, and promote their cellular uptake [5]. Conjugation of polymer to graphene provides various advantages that favor stability and gene delivery efficiency [6–8]. Presence of graphene increases the molecular weight of the core of the gene delivery system and significantly improves the transfection efficiency [9,10]. Therefore, a graphene-based core was planned for the gene delivery system in this study. GO, being the oxidized form of graphene is rich in oxygen-containing functional groups on their surface. On carboxylation of GO, negatively charged carboxyl groups interact with positively charged molecules involving non-covalent bonding, while covalent bonding requires the activation [11]. PEG coat acts as protective stealth; reduces the interaction with serum proteins, thereby decreases the opsonization process and increases the circulation half-life of the nanoparticles. PEG coating also allows functionalization of the particle surface for selective delivery. PEG facilitates the attachment of various ligands such as antibodies, peptides, aptamers etc. for targeted therapy [12]. Here, we used 4,7,10trioxa-1,13-tridecanediamine (a diamine PEG) for providing an electrostatic coating over the pDNA condensed conjugate. The cationic nature of this surface passivating agent facilitates electrostatic interaction with the pDNA condensed conjugate [13].

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Folate receptors are overexpressed on the surface of most tumors and therefore folic acid is a commonly used and efficient molecule for tumor-targeted delivery. The use of carbon nanodots (C-dots) derived from folic acid would allow the gene delivery system to target the tumor site. Thus, in the present study, the folic acid derived C-dots are attached to the diamine PEG layer to facilitate tumor-targeted delivery [14]. The present study is an effort to develop a non-viral system comprising of various strategies discussed above and evaluate it for targeting TNF-α gene to cancer cells. In addition, we aimed to evaluate our developed gene delivery system using an artificial tumor cell apparatus. An animated presentation of the present work and the hypothesis is available as Appendix B.

2.4. Preparation of graphene-reinforced polymer construct (CS-CGO)

2. Materials and methods

2.6. Surface passivation with 4,7,10-trioxa-1,13-tridecanediamine (diamine PEG)

CGO and CS dispersions were stirred at 300 rpm on a magnetic stirrer for 30 min. The CGO dispersion was then added dropwise to the CS solution; stirred for another 30 min at 300 rpm. The CS-CGO conjugate was prepared with three different CS:CGO ratios, 1:1, 5:1 and 10:1. 2.5. Condensation of plasmid DNA with CS-CGO conjugate To 1 mL of CS-CGO conjugate, 10 μL pDNA-TNF-α (2.5 μg/mL in TrisEDTA buffer, pH 8) was added and vortex mixed for 30 s. The product was then incubated at room temperature for 30 min [16]. The experiment was carried out with the CS-CGO conjugates prepared with all the three different CS:CGO ratios.

2.1. Materials Graphene nanoplatelets (GNPs) were gifted from Cheap Tubes Inc., Brattleboro, USA. Folic acid was obtained from Nice chemicals (P) Ltd., Kochi, India. Acrylamide and N,N′-methylenebisacrylamide were procured from Central Drug House (P) Ltd., New Delhi, India. Chitosan (deacetylation degree, 90%), and diamine PEG were obtained from Sigma-Aldrich Co., MO, USA. Dimethyl sulfoxide (DMSO) was purchased from Merk Specialities Pvt. Ltd., India. Ammonium persulfate was purchased from Spectrum Reagents and Chemicals Pvt. Ltd. India. N,N′methylenebisacrylamide, acrylamide, sodium dodecyl sulfate, were purchased from Central Drug House (P) Ltd., New Delhi. Dialysis membrane benzoylated, MWCO 2000 Da, was purchased from Sigma-Aldrich Co., MO, USA. Dialysis tube (MWCO 12000) was purchased from HiMedia Laboratories Pvt. Ltd., Mumbai, India. L-Cysteine was obtained from Sisco Research Laboratories Pvt. Ltd., India. Unless otherwise stated, all chemicals were of analytical grade obtained from commercial suppliers and used without further purification.

2.2. Preparation of graphene oxide (GO) from graphene nanoplatelets (GNP) by modified Hummer's method GNP (100 mg) was dispersed in 20 mL of concentrated sulfuric acid and stirred for 2 h. Then 600 mg of potassium permanganate was added under ice bath (0–5 °C); maintained for half an hour to avoid a vigorous reaction. The mixture was then subjected to overnight stirring. Distilled water (30 mL) was then slowly added to the slightly viscous dark green solution obtained after overnight stirring. The temperature was then raised to 70 °C and maintained for 1 h. A dark brown solution was obtained which turned to yellow on the addition of 10 mL of 30% hydrogen peroxide (H2O2). The obtained solution was centrifuged at 11,000 rpm and washed 4 times with 0.5 M HCl, followed by distilled water to make it neutral in pH. The resultant dispersion was then subjected to freeze-drying to get the dried graphene oxide powder [8,15]. Fig. S1 provides a diagrammatic presentation of the procedure involved in GO synthesis.

A 10% solution of diamine PEG in acetate buffer pH 5.5 was used for coating the pDNA-TNF-α loaded construct (pDNA-TNF-α-CS-CGO). Diamine PEG (100 μL) solution was added to 1 mL of pDNA-CS-CGO conjugate, using a micropipette and vortexed for 30 s. The sample was then allowed to stand at room temperature for 30 min to ensure effective coating. 2.7. Preparation and characterization of C-dots C-dots were prepared by heating folic acid solution (100 mg in 5 mL water) with 400 μL, 20 M sodium hydroxide solution at ~80–90 °C for 1.5 h. The resultant solution was purified by dialysis against water by using benzoylated dialysis membrane of MWCO 2000 Da [14]. 2.8. Festooning C-dots to the surface passivated system 100 μL of C-dot preparation obtained after dialysis was added to 1.1 mL of the surface passivated system and vortexed for 30 s. The obtained formulation was then kept at room temperature for 30 min. 2.9. Anti-angiogenesis study in chorioallantoic membrane (CAM) Three days incubated “Gramasree” eggs were collected from the Regional Poultry Farm, Chathamanglam, Calicut, India. On the fourth day of embryo development, 2–3 mL of albumin was withdrawn from the end opposite to air sac and sealed aseptically with sterilized cotton and transparent tape. On the 8th day, a small window was made in the air sac portion and then a sterilized circular tissue paper disc soaked in Minimum Essential Medium (MEM) containing HeLa cell suspension was placed over the CAM and then sealed aseptically with transparent tape. On the 11th day of incubation, 20 μL formulation (with and without pDNA-TNF-α and pDNA-TNF-α alone as blank) was added to the tissue paper disc, again sealed and incubated. A control is also maintained without the formulation. The eggs are then visualized on the 14th day for anti-angiogenesis activity [17]. 2.10. In vitro protein expression study

2.3. Preparation of carboxylated graphene oxide (CGO) from GO Graphene oxide (50 mg) was dispersed in 50 mL of distilled water and subjected to bath sonication for 1 h. A brown colored dispersion was obtained which was made up to 250 mL with 2 M chloroacetic acid in 4 M NaOH and sonicated for another 75 min [11]. The carboxylated sample was then washed with distilled water (12,000 rpm) to get a neutral dispersion and finally sonicated (bath sonication) for 30 min. The resultant dark colored dispersion was freeze-dried to get the product.

Human cervical cancer cell line (HeLa) was obtained from the National Centre for Cell Science, Pune, India. HeLa cells were generally grown in 25 cm2 tissue culture flasks in a humidified atmosphere containing 5% CO2 and 95% air at 37 °C in a CO2 incubator (New Brunswick). HeLa cells were grown and maintained in minimal essential medium (MEM) supplemented with 10% (v/v) FBS, sodium bicarbonate and an antibiotic solution containing 100 units of penicillin, 100 μg of streptomycin, and 0.25 μg of amphotericin B per mL. HeLa cells were seeded at a concentration of 0.5 × 105 cells/mL in 25 cm2 tissue culture flasks. Twenty four hours after seeding, cells

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were treated with 0.5 mL of the final formulation and incubated further. After 62 h of incubation, the cells were harvested using a cell scraper and spun down at 1000 ×g for 5 min at 4 °C and washed twice with phosphate buffered saline (PBS). The supernatant was decanted and 200 μL of PBS was added to it and mixed thoroughly. The cells were then subjected to probe sonication under ice bath for 50 s to cause cell lysis and to release the intracellular protein. The cell lysate was collected by centrifugation and the TNF-α protein was quantified using the chemiluminescence method. A similar procedure was repeated for the control cells which were treated with pDNA-TNF-α instead of the final formulation. 2.11. Evaluation of C-dot-PEG-pDNA-TNF-α-CS-CGO using developed and validated artificial tumor cell apparatus – a proof of concept study Here, the molecularly folic acid imprinted polymer was prepared to simulate artificial folate receptor. Acrylamide (9.5 g), N,N′methylenebisacrylamide (MBA) (0.5 g) and ammonium persulfate (0.102 g) were taken in a 250 mL round bottom flask. To them, 100 mL of sodium dihydrogen phosphate buffer (p H 6.8, 50 mM) was added. Folic acid (0.3 g) dissolved in 10 mL of DMSO was added to the above mixture and stirred under a nitrogen atmosphere for 45 min. 10 mL of water containing 40 mg of dissolved sodium bisulfite (NaHSO 3 ) was added and stirring was continued under the same condition to obtain the molecularly folic acid imprinted polymer [18]. The imprinted folic acid was removed by immersing in sodium phosphate buffer p H 8, 50 mM until a clear gel was obtained. The obtained molecularly folic acid imprinted polymer (MFAIP) was used in the gel state without further drying. The gel was cut into small pieces of possible uniform size and stored. The developed and validated artificial tumor cell apparatus (see Supplementary data Sec. 1.14 and 2.1) was used for the evaluation of C-dotpDNA-TNF-α-CS-CGO. A suspension of C-dot-pDNA-TNF-α-CS-CGO (0.5 mL equivalent to 10.416 ng of pDNA-TNF-α) was filled into a 10 mL disposable syringe containing 1.0 g of molecularly folic acid imprinted polymer, mixed well and incubated for 1 h. After incubation with the molecularly imprinted polymer, the sample was syringed onto a benzoylated membrane (1.1304 cm2) placed on dialysis membrane (MWCO 12,000 Da), tied at one end of diffusion tube. The benzoylated membrane simulating plasma membrane compartmet along with the added sample was incubated for 30 min. After incubation, 100 μL artificial cell cytosol fluid (pH 7.2 phosphate buffer with cysteine) was added and kept in contact with the nucleus compartment (containing 1 mL of pH 7.2 phosphate buffer without cysteine). The nucleus compartment was maintained at 37 °C and stirred at 500 rpm. The sample was withdrawn from the nucleus compartment at a time interval of 2 h and was subjected to gel electrophoresis (with 1.4% agarose gel at 150 V for 1 h). The samples at different stages (samples just before and after incubation with molecularly folic acid imprinted polymer) of the study were also subjected to gel electrophoresis for comparison. The entire experiment was repeated with an equivalent amount of pDNA-TNF-α alone. An animated presentation on the working of artificial tumor cell apparatus is available as Appendix C. 3. Results and discussion 3.1. Preparation of graphene oxide (GO) from GNP The FTIR spectrum of GNP shows O\\H stretching vibration (3000–3700 cm−1) and C_C stretching vibration (1647 cm−1) (Fig. S2) [19,20]. The TEM image of GNP (Fig. 1a) confirmed the presence of graphene layers in platelet form [21]. The FTIR spectrum (Fig. S3) of graphene oxide (GO) dispersion (Fig. S4) shows OH stretching vibration (broad peak at 3405 cm−1), C_C stretching vibration of carboxyl and carbonyl groups (1636 cm−1), and C\\O and C\\OH stretching vibrations (1375 cm−1 and 1159 cm−1) [8,15,22,23]. The UV–Vis spectrum of

Fig. 1. TEM photomicrographs (a) GNP (b) GO.

graphene oxide dispersion in water shows a characteristic peak at 230 nm and a shoulder peak around 300 nm (Fig. S5) [8,11,15,22,23]. TEM image (Fig. 1b) shows that GO was single layered and more transparent than GNP [24]. The reduction in the absorbance during methylene blue assay of the sample confirmed the effective oxidation of GNP to GO (Table S1) (Fig. S6). 3.2. Preparation of carboxylated graphene oxide from GO Fig. 2a shows a schematic presentation of the formation of carboxylated graphene oxide (CGO) from GNP. The FTIR spectrum of CGO (Fig. S7) shows OH-stretching vibration (3500–3400 cm−1) and the deformation of CH2 group (1400–1300 cm−1) [11,25]. The UV spectrum of CGO (Fig. S8) shows a characteristic peak at 235 nm, corresponding to the π-π* transition in the C_C bond. TEM image (Fig. 2b) further confirmed the formation of thin layered CGO [25]. The reduction in the absorbance of methylene blue is comparatively greater than GO and confirmed the presence of additional carboxyl groups in CGO (Table S2). 3.3. Preparation and characterization of graphene-reinforced polymer construct (CS-CGO) The FTIR spectrum of chitosan (Fig. S9) was in agreement with reported data [26,27]. Fig. 2c shows a schematic presentation of the formation of CS-CGO conjugate. Zeta potential of the CS-CGO conjugate

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Fig. 2. (a) Schematic representation of formation of CGO from GNP (b) TEM image of CGO (c) Schematic representation of formation of CS-CGO conjugate (d) Representative TEM image of CS-CGO conjugate.

was found to be 13.0 mV (Fig. S10). The average particle size of CS-CGO conjugate was measured and found to be 259.0 nm with a polydispersity index of 0.357 (Fig. S11). From the TEM image (Fig. 2d), we can see that there exists a thin layered coating on the surface of CGO thus confirming CS coating of CGO [28].

3.4. Condensation of plasmid DNA (pDNA-TNF-α) with CS-GO conjugate The UV–Vis spectrum (Fig. 3a) of pDNA-TNF-α shows a characteristic peak at 257 nm [29,30]. Fig. 3b provides a diagrammatic presentation of the condensation of pDNA-TNF-α with CS-CGO conjugate. The results of agarose gel electrophoresis confirmed the effective condensation of pDNA-TNF-α at CS: CGO concentration ratios of 5:1 and 10:1. The intensive bands of the DNA remained within the well without any migration at these two ratios, while the band of CS-CGO at 1:1 ratio showed migration, which indicates the presence of free DNA. Thus CS-CGO at 1:1 ratio is not efficient for complete condensation of the added pDNA-TNF-α (Fig. 3c). The details of other lanes are discussed in coming sections. The zeta potential of the pDNA-TNF-α-CS-CGO conjugate was −1.27 (Fig. S12). The negative value confirms the condensation of pDNA-TNFα with CS-CGO conjugate. The positive zeta potential value of CS-CGO conjugate changed to the negative sign, after condensation with negatively charged pDNA-TNF-α.

3.5. Surface passivation with 4,7,10-trioxa-1,13-tridecanediamine (diamine PEG) Fig. 3d shows a diagrammatic representation of the surface passivation of pDNA- TNF-α-CS-CGO with diamine PEG. The obtained zeta potential value for PEG-pDNA-TNF-α-CS-CGO was −34.2 (Fig. S13). Thus, after surface passivation, the zeta potential moved to a more negative value; ensuring the efficient coating of pDNA-TNF-α-CS-CGO with diamine PEG. The Fig. 3e shows the TEM image of PEG-coated pDNATNF-α-CS-CGO. After coating, an increase in the overall size of the conjugate was observed. Fig. 3c shows the results of gel electrophoresis of the pDNA-TNF-αCS-CGO after surface passivation with diamine PEG. The lanes 5, 6, and 7 represent diamine PEG-coated systems with different CS-CGO concentration ratio; 1:1, 5:1 and 10:1 respectively. For 1:1 ratio, there observed the migration of the band; indicating that the PEG-pDNA-TNF-α-CSCGO failed to condense the pDNA-TNF-α effectively. At the same time, for the ratio of 5:1 and 10:1, intense bands were observed within the well; confirms the effective condensation of pDNA-TNF-α. The comparatively higher band migration in lane 8 (PEG-pDNA-TNF-α mixture) shows that diamine PEG is not capable of condensing the pDNA-TNFα, on the other hand, it just acts as a surface passivating agent. This was a very important observation in the present study. Any unintended interaction between diamine PEG with pDNA-TNF-α would affect the desired performance of our developed system.

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Fig. 3. (a) UV–Vis spectrum of pDNA-TNF-α (b) Schematic representation of condensation of pDNA-TNF-α with CS-CGO conjugate (c) The bands obtained after electrophoresis [Lane (1) pDNA-TNF-α, (2)–(4) pDNA-TNF-α-CS-CGO conjugate at different ratios: 1:1, 5:1, 10:1, (5) (6) and (7) represent diamine PEG-coated systems (PEG-pDNA-TNF-α-CS-CGO) with different CS-CGO concentration ratio; 1:1, 5:1 and 10:1 respectively, (8) PEG-pDNA-TNF-α mixture] (d) Schematic representation of surface passivation of pDNA-TNF-α-CS-CGO with diamine PEG (e) TEM image of PEG-pDNA-TNF-α-CS-CGO.

3.6. Preparation and characterization of C-dots The FTIR spectrum of folic acid has an intense narrow peak at 1695 cm−1 is due to C\\O stretching vibration (Fig. S14) [31–34]. C-dots were prepared from folic acid solution and purified by dialysis. After the formation of C-dots, the solution turned to dark brown from bright orange (Fig. 4a and b). Fig. 4c–e shows the images of Cdots under short, long UV rays and under daylight. There was a pronounced increase in the fluorescence emission intensity at this region of 440–460 nm confirms the formation of C-dots (Fig. 4f). The UV–Vis absorption spectrum (Fig. 4g) shows an intense peak at 280 nm arising from π-π* transition of aromatic sp2 carbon within the

core and a shoulder peak around 360 nm corresponds to folic acid residue on the C-dot surface [14]. Fig. 4h shows the TEM image of C-dots. The presence of dark circular particles confirms the formation of C-dots. The FTIR spectrum of C-dot sample (Fig. S15) confirmed the presence of different functional groups on the surface of C-dot [14]. A broad peak between 3500 and 3000 cm−1 resulted from the OH and NH stretching vibrations. A sharp peak at 1607 cm−1 indicated C_C, C_O, and C_N vibrations. The quantum yield of C-dots usually varies depending upon the carbon source used and the method of synthesis involved [35]. On the other hand, surface passivation and doping enhance their quantum yield [25,36]. The quantum yield of the prepared C-dots was calculated

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Fig. 4. The color of folic acid solution (a) before and (b) after C-dot formation; Images of C-dot dispersion under (c) short UV, (d) long UV, and (e) daylight; (f) fluorescence emission spectrum of C-dots (g) UV–Visible spectrum of C-dot (h) TEM image of C-dots.

with reference to quinine sulfate (Eq. (S1)) and was found to be 3.5598%. The absorbance vs. integrated fluorescence intensity plots of quinine sulfate (Fig. S16) and C-dots (Fig. S17) are provided in the Supplementary data. 3.7. Festooning C-dots to the surface passivated system Fig. 5a shows the schematic presentation of the formation of C-dotPEG-pDNA-TNF-α-CS-CGO. After festooning PEG-pDNA-TNF-α-CS-CGO with C-dot, the zeta potential moved to a more negative value (−34.5) (Fig. S18), thus confirming their attachment on PEG-pDNA-TNF-α-CSCGO. The average particle size of the C-dot-PEG-pDNA-CS-CGO was 403 nm with a polydispersity index of 0.431 (Fig. S19). Fig. 5b shows the TEM image of the final system (C-dot-PEG-pDNA-TNF-α-CS-CGO). After the addition of C-dot, it gets attached to the surface of the PEGpDNA- TNF-α-CS-CGO, forming a decorated surface. Fig. 5c shows the image of the gel electrophoresis of C-dot-PEG-pDNA-CS-CGO. The results of agarose gel electrophoresis confirm the effective condensation of pDNA-TNF-α in the final system; C-dot-PEG-pDNA-TNF-α-CS-CGO. 3.8. Anti-angiogenesis study in the chorioallantoic membrane Angiogenesis is the process by which new blood vessels are formed from already existing one. It plays a great role in cancer development [37]. Among the several studies available for angiogenesis, chorioallantoic membrane (CAM) is a commonly used platform that provides a simple, economical model for angiogenesis study. For studying anticancer

activity, angiogenesis can be induced on CAM using tumor cells, owing to their immature immune system [38]. The anti-angiogenic effect of anti-cancer agents can be evaluated by observing the morphological changes in the CAM [39]. Thus, the anticancer activity can be evaluated by the anti-angiogenesis effect on CAM [40]. Interestingly, graphene and GFN are well known for its anticancer activity [41,42]. Our developed formulation contains CGO, which could further enhance the anticancer activity of the TNF-α protein. Here we used, HeLa cell line cultured in MEM for inducing angiogenesis on CAM. After 14 days of incubation, anti-angiogenesis effect was observed for final formulation with pDNA-TNF-α, but no characteristic anti-angiogenesis effect for the membrane treated with control (pDNATNF-α). Fig. 6 shows the images of CAM treated with control, blank and final formulation. 3.9. In vitro protein expression study The expression of the protein TNF-α 62 h of post-treatment with the formulation was quantified by chemiluminescence assay method. The TNF-α expressed by cancer cells seeded with the formulation (C-dotPEG-pDNA-TNF-α-CS-CGO) was higher than the cells seeded with control (pDNA-TNF-α alone). The concentrations of TNF-α in the formulationtreated sample and control were 10.6 and 10.1 pg/mL respectively. This indicates a slightly higher gene transfection efficiency of the developed vector than the pDNA-TNF-α alone. But further detailed pre-clinical and clinical studies are warranted to establish the utility of the developed system.

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Fig. 5. (a) Schematic representation of formation of C-dot-PEG-pDNA-TNF-α-CS-CGO (b) Representative TEM image of C-dot-PEG-pDNA-TNF-α-CS-CGO (c) The bands obtained after electrophoresis. Lane (1) pDNA-TNF-α (2) C-dot-PEG-pDNA-TNF-α-CS-CGO.

3.10. Evaluation of C-dot-PEG-pDNA-TNF-α-CS-CGO using developed and validated artificial tumor cell apparatus – a proof of concept study By this study, we have to ensure that the developed artificial tumor cell apparatus is suitable for evaluation of C-dot-PEG-pDNA-TNF-α-CSCGO. We have to show that our developed C-dot-PEG-pDNA-TNF-αCS-CGO is having higher transfection capability than the naked plasmid DNA (pDNA-TNF-α). For confirming the presence of pDNA-TNF-α- in the artificial nucleus compartment, we employed agarose gel electrophoresis, since it can detect the presence of pDNA-TNF-α more precisely and accurately even in the presence of other molecules and is sensitive enough to detect the presence of DNA. On the other hand, the presence of cysteine in the cytosol compartment may influence the results from UV–Vis spectroscopy. Fig. 7 shows the results of agarose gel electrophoresis obtained after 2 h diffusion. The presence of free pDNA-TNF-α bands from the samples of C-dot-PEG-pDNA-TNF-α-CS-CGO, confirms the suitability of the developed artificial tumor cell apparatus and the absence of free pDNA-TNF-α bands from the samples of pDNA-TNF-α alone, persuade the capability of C-dot-PEG-pDNA-TNF-α-CS-CGO in gene delivery. 4. Conclusions Cancer gene therapy is an ongoing area of research with great future potential. Several vectors have been developed for cancer targeted gene

therapy, among which viral vectors have a supreme position. Even though viral vectors are efficient in gene delivery, they are practically limited owing to severe immunological reactions. Non-viral vectors are devoid of this limitation but their transfection efficiency is usually poor. In order to enhance the gene transfection efficiency, and to achieve a targeted therapy, we developed a non-viral gene delivery carrier that consists of functionalized graphene reinforced polymer constructs (CS-CGO) that condenses the pDNA-TNF-α. The formation of polymer construct was confirmed by transmission electron microscopy and the effective condensation of pDNA-TNF-α by the construct by agarose gel electrophoresis. Surface passivation of the developed pDNATNF-α condensed system (pDNA-TNF-α-CS-CGO) with diamine PEG protects the system from the reticuloendothelial system and prolongs the circulation half-life. The attachment of C-dots helps in targeting the cancer cells via folate receptors which are overexpressed in most of the tumor cells. The developed system forms a biocompatible gene delivery vector with targeting benefits. On the other hand, the presence of C-dots in the system allows the in vivo bioimaging owing to their excellent fluorescent properties thereby opening the way to cancer theranostics. The results of in vitro gene expression study in cancer cell line ensure comparatively higher transfection efficacy of the developed system (C-dot-PEG-pDNA-TNF-α-CS-CGO) than pDNA-TNF-α. We also developed an artificial tumor cell apparatus involving MFAIP that acted as folate receptors allowing the evaluation of C-dot-PEGpDNA-TNF-α-CS-CGO for receptor-mediated gene delivery. Further studies are necessary to enhance transfection capability. Moreover,

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Fig. 6. Images of anti-angiogenesis study using CAM after 14 days (a) blank, (b) C-dot-PEG-pDNA-TNF-α-CS-CGO (formulation), (c) pDNA-TNF-α (control), and (d) to (f) represents corresponding inverted images.

in vivo studies are necessary to evaluate the system effectively prior to their establishment in clinical settings. Supplementary data (Appendix A) to this article can be found online at https://doi.org/10.1016/j.ijbiomac.2019.01.174.

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Acknowledgments The authors gratefully acknowledge Dr. Dhanasooraj, Ms. Shammy S, and Mr. Sanal, at Multidisciplinary Research Unit, Govt. Medical College, Kozhikode, India for their help in gel electrophoresis studies. The authors thank Dr. Rajendra P. Pant, Principal Scientist, Division of Plant Pathology, Indian Agricultural Research Institute, for his help and cooperation in TEM studies. Funding The authors declare that there are no sources of financial funding and support. Conflict of interest JAJ has made a patent application (Indian Patent Application No. 201741033433, dated 21st September 2017) related to a part of this work. References

Fig. 7. The bands obtained after electrophoresis [Lane (1) C-dot-PEG-pDNA-TNF-α-CSCGO (2) pDNA-TNF-α (3) samples of pDNA-TNF-α and (4) C-dot-PEG-pDNA-TNF-α-CSCGO after 1 h incubation with MFIP (5)–(7) samples of C-dot-PEG-pDNA-TNF-α-CSCGO after 2 h diffusion and (8) sample of pDNA-TNF-α after 2 h diffusion].

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