ACS Biomaterials Science & Engineering - ACS Publications

Subscriber access provided by EPFL | Scientific Information and Libraries

Article

Transferrin conjugated biodegradable graphene for targeted radiofrequency ablation of hepatocellular carcinoma Divya Rani Bijukumar, Girish CM, Abhilash Sasidharan, Shantikumar V Nair, and Manzoor Koyakutty ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00184 • Publication Date (Web): 04 Nov 2015 Downloaded from http://pubs.acs.org on November 14, 2015

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Biomaterials Science & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Transferrin conjugated biodegradable graphene for targeted radiofrequency ablation of hepatocellular carcinoma Divya Bijukumar, Girish C M, Abhilash S, Shantikumar Nair, Manzoor Koyakutty* Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Amrita Vishwa Vidyapeethanm University, Cochin-682041 Corresponding Authors: Prof. Manzoor Koyakutty, PhD Corresponding Authors’ Institution: Amrita Centre for Nanosciences and Molecular Medicine, Amrita Institute of Medical Sciences and Research Centre, Kochi, Kerala, India. Tel: +91-484 2858750 Fax: +91-484 2802020 E-mail address: [email protected]

This work was supported by Department of Biotechnology (DBT), Government of India, under the project ‘In silico design, development, nanotoxicology and preclinical evaluation of theragnostic cancer nanomedicine Phase-II’ (BT/PR14920/NNT/28/503/2010).

Short title: Abstract word count: 158 Manuscript word count: 5,537 Total number of references: 40 Total number of figures in main article: 6

Disclosures/Conflicts of interest: None

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Radiofrequency ablation (RFA) is a clinically established therapy for hepatocellular carcinoma (HCC). However, due to poor radio-thermal conductivity of liver tissues, RFA is less efficient against relatively larger (> 5 cm) liver tumors. Recently, nanoparticle enabled RFA has emerged as a better strategy. Based on our recent understanding on biodegradability and novel electrothermal properties of graphene, herein, we report development of transferrin conjugated, biodegradable graphene (TfG) for RFA therapy. Cellular uptake studies using confocal microscopy and Raman imaging revealed significantly higher TfG uptake by HCC cells compared to bare graphene. TfG treated cancer cells upon 5 minutes exposure to 100 W, 13.5MHz RF showed > 85% cell death which was 4 times greater than bare graphene. Further evaluation in 3D (3 Dimensional) HCC culture system as well as in vivo rat models demonstrated uniform destruction of tumor cells throughout the 3D micro-environment. This study reveals the potential of molecularly targeted graphene for augmented RFA therapy of liver tumor.

Key words: Graphene, Hepatocellular carcinoma (HCC), RFA, 3D culture.


Page 2 of 31

Page 3 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction Hepatocellular carcinoma (HCC) is the sixth most common deadly malignancy in the world which is normally associated with liver injury, viral hepatitis infection, chronic alcoholism and cirrhosis1. Though surgical resection is considered successful, the candidates for surgery are limited because of the metastasis of tumor to other organs. Radio frequency ablation (RFA) is a standard treatment modality for liver tumors of nearly 3-5 cm, where surgical resection is not preferred 2. RFA uses high energy electrical current applied through an electrode, which creates ionic agitation in the tissue leading to thermal ablation3. The basic mechanism of RFA is hyperthermia mediated cellular destruction and protein denaturation leading to coagulation necrosis. Reports suggest that cells undergo apoptosis when heated up to 40-47°C, but further increase in temperatures up to 50 °C or more may cause immediate necrosis4. One of the major drawbacks of the current RFA is charring of the tissue adjacent to the RF probe due to the poor RF/thermal conduction of tissues. Because of this, RF mediated cancer therapy is not successful in patients having tumor lesions >5 cm. In addition, the current modality is invasive as it requires insertion of a probe into the tumor site. This may cause serious damage to the surrounding normal tissue and/or spread of tumor cells while withdrawing the probe. Non-invasive RFA is an emerging tool where radio frequency waves were irradiated to the tissue of interest (tumor). However for an effective hyperthermia, amplification of RF/thermal conduction within the exposed tissue region is required. 5

Recently, RF responsive nanoparticles were proposed to improve non-invasive RF therapy . Carbon nanotubes and gold nanorods/nanoshells were reported to be very effective in RF hyperthermia in tissues 6, 7, 8. Curley et al and his colleagues extensively studied the role of CNT and gold nanoparticle in evoking RF hyperthermia for cancer therapy applications 9-16. He reported the application of SWCNT in evoking lethal thermal injury to hepatic VX2 tumors in a rabbit model9. Anti-tumor effect of gold nanoparticles via RF mediated hyperthermia was extensively studied in gastrointestinal cancer, pancreatic cancer, breast cancer etc. using kanzius RF generator 10,13,14,16. Although CNT is a promising candidate for RFA, one of the major limitations is the toxicity associated with its fibrous nature. For the past few years, the 2-dimensional alternative allotrope of carbon, graphene, was dominating over CNT owing to its better biocompatibility together with novel electronic and thermal property. Recently Yang et al, reported the application of graphene for near infrared (NIR) mediated in vivo hyperthermic effect in mice and suggests the greater promise of graphene in cancer therapy17. SWCNTs have been reported to have NIR/pH responsive chemo-



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

photothermal therapy potential18. Another recent report demonstrated the ability of intravenous injection of PEGylated graphene particles for near infrared mediated laser ablation of tumor19. However, potential usage of graphene for RF mediated tumor killing is least explored. Recently our group has reported the potential of functionalized graphene for RF mediated hyperthermia against drug resistant CML cancer cell line K562R in 2D culture20. As RFA is a clinically approved therapeutic option for liver tumor and the current method is limited due to poor RF/thermal conductance of liver, we focused the present investigations on the potential of graphene mediated noninvasive RF hyperthermia in 3-dimentional (3D) culture of liver cancer. We demonstrated that bioconjugation of graphene with transferrin ligands (TfG) enabled targeted cellular uptake by HepG2 liver cancer cells and enhanced hyperthermia effects. Intracellular uptake of targeted graphene particles was noted throughout the 3D culture rendering enhanced anti-cancer effects under RF exposure. Regarding the clinical translation, in vivo biodegradability of graphene was a major concern. However our recent work using Raman imaging of tissue bound graphene in rat model suggests that macrophage engulfed graphene is degraded in vivo over a period of 3 months. In the present work we used this biodegradable graphene for RF hyperthermia. Experimental Section Functionalized and Transferrin conjugated graphene preparation We have used CNR Rao’s Arc discharge method for the synthesis of pristine graphene21. Briefly, Direct current arc discharge evaporation of graphite was carried out in a water-cooled stainless steel chamber filled with a mixture of hydrogen and helium in 200:500 Torr proportions without using any catalyst. The discharge current was 125 A, with an open circuit voltage of 60V, by maintaining cathode at a constant distance of 2mm from the anode. After the chamber had cooled to room temperature, pristine-G was collected from the chamber and used for functionalization20, 22. 25 mg of pristine graphene was mixed with concentrated H2SO4 and HNO3 in 3:1 ratio and stirred overnight 21. Then the solution was washed with distilled water by repeated centrifugation in order to remove acid environment and dispersed in water. The resultant solution was sonicated for 6 hours to form uniform dispersion of functionalized graphene (FG). Transferrin conjugation of graphene (TfG) was performed by means of EDC-sulfo NHS bio-conjugation chemistry. Briefly, 5mg/ml of FG was dispersed in conjugation buffer consist of 100mM 2(N-Morphilino) ethanesulfonic acid


Page 4 of 31

Page 5 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(MES) (Sigma Aldrich) and 500mM sodium chloride (pH 5) (Sigma Aldrich). In order to activate the FG, 20 mg of EDC (Sigma Aldrich), was added with mild shaking and followed by 80mg sulfo-NHS and incubated in dark for 30min at room temperature. To this, 1mg/ml transferrin (Sigma Aldrich) in PBS (pH 7.5) was added and incubated in dark for 4 hours in a shaker. Further the solution was stirred overnight at 4 ºC. After incubation, the unbound transferrin molecules were removed by repeated centrifugation using PBS. The pellet then re-suspended in 5ml of PBS and bath sonicated for 40min at 30% amplitude. Nanoparticle Characterization TEM images were obtained with a FEI Technai

TM

Transmission Electron Microscope (FEI, USA). TEM was

performed with an accelerating voltage of 120 kV and digital images were acquired using an FEI Imaging System software. Drops of nanoparticle suspensions were placed onto a coated copper grid for 1 h. Grids were blotted dry with filter paper and air dried before TEM observation.

AFM measurements were carried out in JEOLSPM 5200 instrument. 50 µg/ml graphene solutions were prepared in distilled water and 20 µl of the sample solution was drop casted on to freshly cleaned mica surface and air dried. The AFM topography images were acquired in tapping mode from a scan area of 2X2 µm using super sharp silicon probed with resonance frequency, 330 KHz and spring constant, 42 N/M. RF hyperthermia of Graphene, CNT and gold nanoparticles 1mg/ml solutions of Graphene, CNT and gold nanoparticles were prepared in MEM (minimum essential medium). 5 ml of 1mg/ml nanoparticle solutions were taken in a glass petridish. Initial temperature of the solutions was measured using an electronic temperature probe and then irradiated with radiofrequency waves of 13.5MHz, 100W for 1,2,3,4 and 5 minutes. After each time points the final temperature of the nanoparticle solutions was measured. All experiments were done in triplicate.

In vitro Cell Culture HepG2 cells were obtained from ATCC. The cells were maintained in MEM (Sigma Aldrich) containing 20% fetal bovine serum (Sigma Aldrich) and 1% penicillin and streptomycin (Invitrogen). The cells were cultured in 25 cm2 tissue culture flasks at 37 ºC in a 5% CO2 humidified environment. After 85% confluence, the cells were washed with sterile Phosphate Buffer Saline at pH 7.4 and treated with 0.25% trypsin/EDTA for 3 min and re-suspended



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

with fresh 20% MEM. Cell suspension was centrifuged at 3000rpm for 3min. Cell number was counted using hemocytometer (Sigma Aldrich) and desired cell density of each experiments were re-suspended in fresh medium.

Immunostaining for TfR Expression Cell surface expression of TfR on HepG2 cells was analyzed using flow cytometry. Human TfR mAb (R&D systems, USA) conjugated to APC (R&D systems, USA), was used for TfR immunostaining. 1×105 cells were counted, washed with 0.5% BSA in PBS at 25000rpm of 3 min. The cells were re-suspended with 25µL of 0.5% BSA in PBS, and incubated with 10µL of anti-TfR/CD71 antibody for 30−45 min at 2−8 °C. Later, the cells were washed and re-suspended in PBS pH 7.4 supplemented with 0.5% bovine serum albumin (BSA) for flow cytometric reading (FACS Aria II, USA). The analysis was performed using FACS Diva software. The fluorescence was detected using 633 nm excitation and emission was collected using 660/20 nm band-pass filter.

In vitro 3D alginate/collagen/HepG2 cell culture system 2.5% medium viscosity alginate (Sigma -Aldrich) solution was prepared in normal saline by overnight stirring. HepG2 cells were cultured as described above. After 85% confluency the cells were trypsinised, counted by hemocytometer and re-suspended in alginate solution such that the final concentration will be 1 X 105 cells/ml of 2% alginate (Sigma Aldrich) solution containing 10%v/v of 0.1% collagen (Sigma Aldrich). The alginate/collagen/cell suspension was then dropped in to 200mM CaCl2 solution prepared in basal medium with antibiotics and incubated for 30min at 37 °C. After incubation at 37°C, the cross-linked alginate beads encapsulated with HepG2 cells were washed with PBS for removing CaCl2 solution. After, 1ml of 20%MEM (Sigma Aldrich) was dispensed in the well plate which resulted in the medium to be cover above the beads. Media change was done every 24hrs. Cell viability of 3D encapsulated HepG2 cells The cell viability and proliferation of HepG2 cells encapsulated alginate beads were assessed using an alamarBlue assay. Briefly, cells were encapsulated in alginate beads at a density of 105 cells/bead and were placed in a 24 well plate and cultured for up to 14 days in MEM with 20% FBS under standard culture conditions. Cells with similar seeding density culture on cell culture 2D well plate were considered as control. At different time intervals, viz. days 1, 7 and 14 of incubation, the cells were incubated with 10% alamarBlue (Invitrogen, USA) in complete medium for 6 h. After incubation, the medium was pipetted into 96-well plates, and the optical density was recorded using a


Page 6 of 31

Page 7 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


microplate spectrophotometer at 570 nm, with 600 nm set as the reference wavelength. Results are presented as the mean ± standard deviation of three independent analysis performed in triplicates. Intracellular uptake studies To investigate intracellular uptake of both graphene systems in HepG2 cells, we employed a laser scanning confocal microscope (Leica Confocal microscope, DMI 6000 CS with Leica TCS SP5 II scanner, Leica Microsystems, USA) and confocal Raman spectral mapping (Alpha 300RA, Witec, Germany). HepG2 cells were grown on 13 mm cover slips, treated with both FG and TfG G, and analyzed. Z-plane stacks were acquired with the confocal Raman microscope to create 3D Raman spectral images. RF hyperthermia For 2D (2 Dimensional) cell culture, 105cells were seeded on 12 well plates. After 24 hours incubation, the cells were treated with both FG and TfG at a concentration of 20 and 50ug/ml. For RF hyperthermia studies in 3D cell culture condition, the cell encapsulated alginate beads were treated with FG and TfG for 6 hours. The cell encapsulated alginate beads without graphene treatment was considered as control. After 6 h of incubation with graphene, the beads were washed with PBS and added fresh medium. Then 2D and 3D cultured cells were further exposed to RF 100W for 1 min, 3min and 5 min. After RF exposure the medium was aspirated and incubated the cells for 24hours with fresh medium. The cells were further incubated with 10% alamarBlue in complete medium for 6 h. After incubation, the medium was pipetted into 96-well plates, and the optical density was recorded using a microplate spectrophotometer at 570 nm, with 600 nm set as the reference wavelength. Results are presented as the mean ± standard deviation of three independent analyses performed in triplicates. Apoptosis assay RF mediated apoptosis on HepG2 cells was assessed using flow cytometry as well as confocal microscopy. Apoptosis assay was performed on HepG2 cells using Annexin V-FITC Apoptosis Detection Kit (BD Pharmingen, USA) according to the manufacturer’s instructions. Propidium iodide (PI) was used as a nuclear stain for identifying late apoptotic and necrotic cells. Briefly, 105 cells cultured in a 24 well tissue culture plate were incubated in the presence of 20 and 50 µg/ml of functionalized and transferrin conjugated functionalized graphene for 3 hours under, standard culture conditions. Controls were cells cultured in the absence of graphene.

After incubation the cells

were washed with PBS in order to remove the free graphene particles from solution and fresh media was added and



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

further exposed to RF 100W for 1 min, 3min and 5 min. After RF exposure immediately aspirated the medium and treated with fresh medium and cultured for 24 hours. Later the cells were harvested, washed in 1X binding buffer containing 0.01 M HEPES (pH 7.4), 0.14 M NaCl, 2.5 mM CaCl2 provided along with the kit and stained with Annexin V-FITC and PI according to the manufacturer’s instructions. The cells were further analyzed using flow cytometry. Staining the samples with Annexin V and PI individually performed the compensation for double fluorescence. For confocal microscopic imaging, the cells were harvested, washed in 1X binding buffer, and stained with Annexin V-FITC and PI. After the incubation period, the cells were washed again to remove the unconjugated dye, and observed under a microscope. Annexin V-FITC fluorescence was detected using 488nm laser excitation, and PI fluorescence was detected using 543nm laser excitation. CalceinAM/EtBr Live Dead Assay To investigate cell viability in HepG2 cells cultured in alginate 3D in vitro culture after RF treatment, CalcienAM/EtBr Live/dead assay were done. HepG2 cells cultured in 3D alginate beads were incubated with graphene; both FG and TfG. After incubation, the beads were collected and washed with PBS. Further, 0.5µg/ml Calciein and 2µg/ml EtBr in PBS were added and incubated for 1h in dark at room temperature. Then the beads were washed with PBS and confocal images were taken with Excitation and Emission 494/517 and 528/617 for calcein and EtBr respectively. A z- series images was collected with Leica Confocal microscope, DMI 6000 CS with Leica TCS SP5 II scanner at a depth of 800 µm with a step size of 17.9 µm using Leica 10X objective. Differential Interference Contrast (DIC) Microscopic images were also taken at similar focal planes and merged using LAS AF software to obtain live dead cells in a single bead with perfect contrast. In vivo evaluation of RF thermal response Site specific RF thermal response at graphene injected regions within the liver was evaluated using Wistar Rat models. All the animal experiments were carried out after Ethical Committee approval from the Animal Ethical Committee at Amrita Institute of Medical sciences and Research Centre, Kochi. The animals were anaesthetized with 0.5 ml of Xylazine (20mg/ml, 0.2ml) and Ketamine (50mg/ml, 0.3ml) and shaving the fur over dorsal aspect of the animal (to allow better contact with grounding pads). Under sterile conditions, a central incision was made over abdomen and flaps lifted to expose the liver. 50 µg/ml of transferrin conjugated functionalized graphene was injected directly to the liver at a depth of 1cm. 30 minutes after graphene injection then the animal was exposed to a


Page 8 of 31

Page 9 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


uniform RF field. Through the central incision 17 gauge sterile RF electrode was introduced into the liver and RF (100 W, 3 minute) energy was administered. This was followed by intracardiac blood draw and euthanasia of the animal (with overdose of anesthesia) to harvest its liver. Tetrazolium chloride (TTC) staining was performed to assess accurate size of ablated tissue. TTC was procured from Sigma Aldrich India. Vertical section of the liver samples were taken and incubated in freshly prepared 1% (w/v) solution of TTC in normal saline, for 20 minutes. Following this the tissue samples were fixed in 10% formalin for 20 minutes. Results Characterization of graphene Graphene was synthesized by thermal exfoliation of highly pure graphite oxide18. Figure 1A (i) show the TEM image of carboxyl functionalized graphene. Corresponding AFM image and height profile (Figure 1A (ii, iii)) shows actual thickness of nearly 0.8-1nm indicating the bi-layer structure. This functionalized graphene was conjugated with transferrin ligands using EDC/NHS overnight reaction in dark as discussed in our earlier report19. The morphology of as prepared transferrin conjugated graphene (TfG) is shown in the TEM image (Figure 1B (i & ii)). Due to Tf conjugation, graphene sheets appears to bed folded into 3D particle morphology and a thin layer of transferrin protein can be seen around the TfG nanoparticles (arrow mark).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 31

Figure 1. (A) (i) TEM, (ii) AFM and (iii) AFM height profiling of functionalized graphene (B) TEM image of Tf conjugated graphene, Tf-G and (ii) HR-TEM showing a thin layer transferrin (arrows) around graphene NP (C) Comparative evaluation of RF induced temperature increase by gold NPs, Single walled CNT and graphene Tf-G, all at 20ug/ml.

The particle size of the TfG nanoparticle (124nm) and the conjugation efficiency of the transferrin to functionalized graphene recorded spectrophotometrically as 61.76% were reported in our previous work

19

. Further, the heating

effect of graphene was evaluated and compared with gold nanoparticle and CNTs under a constant RF field of 13.56 MHz at a power 100 W in MEM. Graphene produced a temperature increase of ~ 10°C and 7 °C as compared to gold and CNT nanoparticles respectively after 5 minutes of RF exposure. From the room temperature of 24 °C graphene nanoparticle reaches a temperature of 50°C within 5 minutes, whereas, gold nanoparticles or CNTs produced a temperature rise up to 40-42 °C (Figure 1c) only. Transferrin expression and cytotoxicity studies Cell surface receptor expression of transferrin was evaluated employing flow cytometry using antibody specific to transferrin receptor. Flow cytogram data revealed that 90% of hepatocarcinoma cells (HepG2) cells used in the current study expressed transferrin receptors (Figure 2A). Further, we evaluated the cytotoxic effects of FG and TfG using HepG2 cells (20 µg/ml and 50 µg/ml). Compared to untreated control cells, as seen in figure 2B, both the samples remain non-toxic for a period of 24 hours.

Figure 2. (A) Flow cytogram showing percentage expression of transferrin receptors by liver cancer cells (HepG2). (B) Biocompatibility of functionalized(FG) and Transferrin conjugated graphene (TfG) at 20 µg/ml and 50 µg/ml with HepG2 cells treated for 24 hours (C) (i & ii) DIC images representing varied cellular


Page 11 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


uptake of FG and TfG by HepG2 cells, (iii & iv) corresponding Raman image confirming the presence of graphene (pink) inside the cells (blue). Targeted cellular uptake study Cellular uptake study of graphene was carried out by treating HepG2 cells with both functionalized graphene (FG) and transferrin conjugated graphene (TfG) for a period of 3 hours. Both FG and TfG at a concentration of 50µg/ml were treated with HepG2 cells in normal cell culture condition. Confocal microscopic images show that graphene uptake was significantly higher when the particles were conjugated with transferrin (Figure 2C (i & ii)). We further confirmed the result using 2D confocal Raman spectral imaging. The characteristic G-band vibration of graphene at 1580 cm-1 and 2640 cm-1 were detected and imaged which is seen as pink colored (false color) dots against the blue background (auto fluorescence) of the cell (Figure 2C (iii & iv)). RF hyperthermia in 2D culture The effect of RF mediated hyperthermia to cells cultured in 2D culture condition was studied. Initially we performed an experiment to optimize the power and time duration of RF exposure, which does not evoke any cell death in normal culture conditions without graphene treatment. Cells were exposed to 13.5 MHz radiofrequency waves of power 50 and 100 W for 1, 3 and 5 minutes. The results revealed that RF power up to 100 W for a period of 5 minutes did not evoke any toxic effect to the untreated cells (Figure3A). In the next step the cytotoxicity of graphene treated cells was evaluated using RF power 100 W, for different durations such as 1, 3 and 5 minutes (Figure 3B). When exposed to radiofrequency waves, at 100 W for 1 minute, FG or TfG treated cells did not show any toxicity as 98% of cells remain viable. However, as RF exposure time increased to 3 minutes, toxicity of TfG treated cells increased to 20% and 25% for 20µg/ml and 50µg/ml respectively. In contrast, FG nanoparticles exerted only 10% toxicity. Upon further increase in exposure time to 5 minutes, TfG treated cells registered 82% toxicity at 50µg/ml whereas FG treated cells showed only marginal toxicity of 25% for the same concentration Quantitative determination of viable versus apoptosis cell by Annexin V/propidium iodide staining was done using flow cytometry. Apoptosis study was carried out after 3 and 5 minutes RF exposure to both FG and TfG treated cells at a concentration of 50 µg/ml. From the flow cytogram it was found that ~ 49% cells were in early/late stage of apoptosis, while 3% underwent necrosis in FG treated cells after RF exposure at 100 W for 5 minutes (Figure



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 31

3D). Although we could not see a significant difference in the percentage of apoptosis in between 3 or 5 minutes of exposure necrotic cell percentage was observed to be higher after 5 minutes of RF exposure. Interestingly, when graphene was targeted using transferrin, total of 74% cells underwent early /late stage of apoptosis while 2.6% remain necrotic even by 3 minutes of RF exposure. By 5 minutes the rate of apoptosis increased to 92% with a marginal increase in the necrotic cell death. Over all the percentage of viable cells were reduced to 3% in case of TfG compared to 48% in FG after 5 minutes of RF exposure. The results were consistent with our confocal images (Figure 3C) wherein, the cells were found to have lost their morphology and showed cytoplasmic and nuclear condensation after 5 minute exposure in TfG treated cells. Complete cell membrane destruction were observed in the case of necrotic cells (enlarged section of Figure 3C).

Figure 3. (A) Percentage cell viability of HepG2 cells by alamar blue assay after RF exposure of 50 W-1 minute, 100 W-1 minute, 100 W-3 minutes, 100 W-5 minutes. (B) Percentage cell viability of FG and TfG treated HepG2 cells after RF exposure at a power of 100 W for 1, 3 and 5 minutes using alamar blue assay. (C) Confocal microscopic images of Annexin V/propidium iodide staining of apoptosis assay. The images reveal apoptosis in HepG2 cells upon RF exposure of 100 W- (i) 1min, (ii) 3 minutes and (iii)5 minutes after treating cells with 50 µg/ml concentration of FG (Lane 2) and TfG (Lane 3), in comparison to untreated cells (Lane 1). (D)Flow cytogram showing the apoptosis in HepG2 cells upon RF exposure for (i) 100 W-3 and (ii) 5 minutes at 50µg/ml FG (Lane 2) and TfG (Lane 3) compared to untreated cells (Lane 1).


Page 13 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


RF hyperthermia in 3D alginate cell culture system We further investigated the extent of cell destruction by graphene mediated RF hyperthermia in 3D alginate/collagen liver tumor culture system.

Figure 4A shows the stereomicroscopic images of acellular and cellular

alginate/collagen 3D microbeads. Figure 4B shows the cellular proliferation upto 7 days of 3D alginate and 3D alginate/collagen system by keeping 2D culture as control. It can be seen that compared to 2D culture the 3D alginate microbead did not show significant increase in cellular proliferation. However, when collagen was incorporated in to the alginate matrix excellent cellular proliferation was seen after 5th and 7th days of culture. This result was confirmed using live/dead assay in confocal imaging. After 48 hours of cell encapsulation, the alginate micro beads were found completely filled with viable cells (green florescence) (Figure 4C). Further we have developed cell encapsulated macro beads of 1cm2 diameter and the same also showed viable cells throughout the bead as evident from the alamarBlue assay in Figure 4D(I & ii). Viable cells containing beads showed pink coloration due to the cell mediated reduction of alamarBlue reagent, resazurin to resorufin. In order to study the phenotypic expression of transferrin by HepG2 cell in macro culture, we have stained the cells by FITC transferrin antibody and imaged using Kodak Multispectral in vivo imaging system (FX pro, USA). The samples were kept in a petriplate inside the imaging chamber and exposed to fluorescence band pass filters e excitation filter: 490 nm ± 15 nm, emission filter: 530 nm ± 15 nm (Figure.4D). A high fluorescent intensity of transferrin antibody was observed throughout the cell encapsulated bead (Figure 4D (iv)), which confirm the presence of HepG2 cells throughout the bead architecture.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 31

Figure 4. (A) Stereomicroscopic images of alginate micro beads acellular and after encapsulation of HepG2 Cells, (B) Cell viability of HepG2 cells in 2D and 3D alginate-collagen culture systems after 1, 5 and 7 days of incubation. (C) confocal microscopic images (i) Differential interference contrast (DIC) image (ii) calceinTM/EtBr staining for live-dead assay of HepG2 cell encapsulated micro bead (iii) merged image of DIC and Live-dead assay of cell encapsulated micro bead (scale bar = 100µm, magnification 10x ),(D) acellular and cellular macro bead (i) macro bead without cell showing the color of alamar blue (blue color) (ii)cellular macro bead showing the color of resorufin (pink) after incubation with alamar blue reagent (iii) transferrin antibody incubated acellular alginate (iv) Fluorescence emitted by the HepG2 cell encapsulated alginate macro bead.

The 3D tumor culture was treated with FG or TfG (50ug/ml) nanoparticles for a period of 3 hours under continuous agitation for proper flow of medium throughout the beads. When exposed to radiofrequency waves of 100W for 1 minute, we couldn’t observe any significant toxicity in 3D tumor beads after FG and TfG treatment. However, as the duration of exposure increased to 3 minutes, FG and TfG evoked 45 and 60% of cytotoxicity, respectively. Further increase of exposure to 5 minutes caused no significant change in the level of toxicity in FG treated 3D culture, whereas cytotoxicity increased to 85% in TfG treated culture (Figure 5A). This is in correlation with the RF induced temperature increase shown by TfG (Figure 1C). Figure 1 C showed that graphene produced an increase in temperature of 50 °C after 5 min RF exposure. In cell culture experiments, the initial temperature before RF exposure is 37 °C and after 3 minutes and 5 minutes RF exposure, the temperature will increase above 50 °C which is sufficient enough to kill the cells. In order to visualize these results, we have performed a confocal imaging of calceinTM live/dead assay from the middle section of the 3D spheroid after 24 hours of RF exposure (Figure.5B). From the images, it was clear that even after 3 minutes of RF exposure, cells found dead in TfG treated spheroid and by 5 minutes most of the cells were dead as evident from the enlarged image. More importantly, this cell death happened all throughout the 3D culture as seen from the Z-stacked image of the spheroid shown in Figure 5C. Image taken from the top to bottom layer of the spheroid showed uniform cell death, indicating that TfG affected almost all cells in the 3D culture.


Page 15 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. (A) Percentage viability of FG and TfG treated HepG2 cells in 3D culture system after RF exposure by alamar blue assay. (B) Confocal microscopic images of CalceinTM/EtBr staining for Live/Dead assay. The images clearly revealing the dead and necrotic cells after RF exposure of 100W- 3 and 5 minutes after treating cells with 50 µg/ml of FG and TfG, in comparison to untreated cells. (C) Confocal images of CalceinTM/EtBr staining on cell encapsulated 3D culture system after 100W-3 minutes of RF exposure (Scale bar= 150µm, 10x magnification) In vivo evaluation of RF thermal response In order to confirm the in vitro finding we further tested the efficacy of Transferrin conjugated graphene for its RF mediated hyperthermia in liver using Wistar Rat model. We injected the graphene sample (10µg/kg body weight) in a localized region of one of the lobes and applied 100W RF for 630sec over the whole liver region (Fig. 6a, b). Interestingly, after the RF irradiation, the graphene injected region got ablated readily as indicated by the whitish coloration as seen in Fig. 6c. Live/dead assay of the vertical section of this region of liver using TTC staining, an indicator of respiration, clearly shows the necrosis in the graphene injected area (Fig 6d). Approximately 95% of the graphene containing region were dead due to enhanced hyperthermia effect whereas other regions remains live with no pathological change. Clearly, this indicates the specific effects due to the presence of graphene in the local region.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 31

Figure 6. Photographs of (a) TfG injected liver of Wistar Rat. (b)Whole liver before the radiofrequency ablation (c) Portion of liver showing immediate color change due to hyperthermic ablation in graphene injected site upon RF exposure (d) Vertical section of ablated area stained with TTC, showing clear necrotic region.

Discussion Nanoparticle mediated targeted therapy by utilizing their intrinsic properties such as induction of localized heating effect is considered as the current advancement in the field of radiofrequency ablation

23

. The role of carbon

nanotubes and gold nanoparticles for RFA was successfully investigated by various researchers

6,9-16

. Under

capacitively coupled radiofrequency fields, Curley et al reported an intense heat release by CNTs compared to gold nanoparticle. Interestingly compared to gold nanoparticles ionic gold was found better active under RF field. In case of CNT a major limitation was the toxicity concern. Recently Markovic et al reported the NIR mediated photothermal anti-cancer activity of the 2D allotrope of carbon, graphene24. It was found that compared to CNTs, graphene exhibited a higher NIR induced heat generation. Based on these previous studies, our focus was to investigate the RF thermal response of graphene for its utility in cancer therapy. A major concern in the use of


Page 17 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


graphene is its biocompatibility in vivo. Previously we reported the differential interaction of pristine and functionalized graphene with various cell types. Functionalized graphene was found better internalized by the cells with minimum toxicity24. More importantly, we have also demonstrated the in vivo biodegradability of graphene using Raman Spectral imaging in rat model. We found that graphene engulfed by alveolar macrophages, kupffer cells and spleen bound macrophages were degraded over a period of 1-3 months25. These results indicate potential utility of graphene for in vivo biomedical applications24, 25. In the present experiment we used this biodegradable graphene for RF hyperthermia. Initially the RF response of graphene was compared with that of same concentrations of CNT and gold nanoparticles. By 5 minutes of exposure, the graphene clearly showed enhanced hyperthermic effects with ∆T of 710°C compared to CNT/gold nanoparticle. Within 5 minutes of RF exposure the graphene solution registered a temperature increase upto 50°C, which is well above the required temperature for achieving cytotoxicity (4245°C)26,27,28. This suggests that graphene is a promising candidate for RF hyperthermia application. A major challenge of nanomaterial mediated anti-cancer therapy is associated with targeted delivery of particles, specifically to tumor cells. In the present work we studied the effect of transferrin conjugation on graphene for its specific delivery to liver tumor cells which over express the transferrin receptor. Transferrin (Tf), specifically TfR1 expression is restricted mainly to liver cells. Our studies revealed more than 90% transferrin in HepG2 cells. Since transferrin serves the entry of iron into the cells for normal cellular metabolism, the expression levels are several folds higher in the case of malignant cells29. Hence transferrin mediated tumor targeting is an attractive means for cancer therapy and currently three transferrin targeted nanomedicines are under clinical trials30,31,32. Our studies on bare and transferrin conjugated graphene by confocal microscopy and Raman imaging showed excellent cellular uptake by the latter. We further evaluated the RF hyperthermia mediated cell death by FG and Tf targeted graphene. The results indicate that compared to FG, TfG treated cells showed enhanced cytotoxicity after 3 and 5 minutes of RF exposure. As the time of RF exposure increases, the percentage of cell death also increased. This increase in toxicity in TfG can be mainly attributed to the enhanced cellular uptake of graphene due to transferrin conjugation. Since the concentration of graphene nanoparticle taken up by cells was significantly higher in TfG, the heat energy contributed by those particles also must be higher and thereby increasing the cell death.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 31

It was reported that the mechanism of cell death by RF hyperthermia is primarily due to necrosis33. Classically, hyperthermia mediated tumor destruction occurs due to irreversible denaturation of proteins, and lipids, rendering necrosis33. However our findings suggest that TfG mediated RF hyperthermia caused mainly apoptosis than necrosis. Flow cytometry data suggest that, with increase in exposure time under constant RF power of 100W, the apoptotic cell fraction in TfG treated cells increased from 74 to 92% whereas the necrotic fraction remained largely insignificant. This suggests an altered mode of cell death in graphene mediated RF hyperthermia. Leber et al, studied the impact of temperature on cell death using HCC cells and reported that heat treatment of 65 °C for 15 minutes and 75 °C for 5 minutes can cause late apoptosis in 2D culture34. In our case, enhanced cell death happened in Tf-G treated cells with 5min RF exposure at 100W, which results heating up to 500C. We believe that, with enhanced intracellular localization, graphene caused more intracellular heating and coagulation damages on intracellular proteins or organelles than the whole cell destruction. It was reported that increase in temperature at intracellular regions may lead to activation of pro-caspase 2 as well as Bax and Bak pathways26. Further, mitochondrial membrane damage mediated release of cytochrome c also may contribute to the triggering of apoptosis. Clearly, graphene mediated RFA caused programmed cell death than physical destruction of cells and compared to the results of Leber et al., the apoptosis happened at an early stage34. Further, as an anti-cancer therapeutics, apoptosis is better preferred over necrosis which may cause acute inflammatory response and edema. We observed a higher percentage of cell death in TfG treated cells than FG. Moreover, as the concentration of TfG increased, a threefold higher cell death was obtained. This is attributed to the higher percentage of TfG uptake by the cells at 50µg/ml and corresponding enhanced heat generation by 5 minutes of RF exposure. We have verified our results on 2D culture with 3D spheroids of HCC. Since human tissue is three dimensional (3D) in nature, normal monolayer culture fails to mimic the in vivo conditions precisely. Currently, a number of 3D in vitro cell culture models are under development for various drug toxicity analyses. Presently biomaterial based 3D culture systems are established to confine cells three dimensionally into a scaffold35,36 or encapsulating cells in hydrogels37,38,39. Consistent with earlier reports40, the cellular proliferation was restricted in the alginate matrix (Figure 4B). In comparison to 2D cell culture system, 3D alginate/collagen encapsulated HepG2 cells showed better cell viability. The presence of collagen makes the surface more cell-friendly, which helps the HepG2 cells to adhere and forms a uniform cell growth in 3D. Consistent with the results of 2D culture, our results in 3D cell culture system also showed enhanced hyperthermia mediated apoptosis in TfG treated HepG2 cancer cells. Interestingly we


Page 19 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


could observe a uniform cell death all throughout the 3D spheroid. This suggest, intracellular uptake of nanoparticles within the 3D culture system and effective hyperthermia within the 3D microenvironment. Further in vivo evaluation of graphene mediated RF hyperthermia also revealed uniform ablation in rat liver model using TTC staining (Fig. 6). 95% of the ablated tissue area was found to be dead after live dead staining (Fig 6d). Thus, the augmented RF thermal response of Transferrin conjugated graphene was successfully demonstrated in vivo. Conclusions In conclusion, our study focused on the application of graphene for anti-cancer therapy via RF hyperthermia mediated tumor destruction with primary focus on liver tumor. The effect of both functionalized (FG) as well as transferrin conjugated graphene (TfG) was evaluated in 2D and 3D cell culture environments. This study is the first report on RF hyperthermia of graphene and its cell death mechanism. Our observation showed that graphene mediated cell death is mainly through programmed cell death over necrosis. Moreover, results of the in vivo studies clearly suggest that graphene can be used for targeted RF mediated tumor destruction.

Acknowledgements The authors would like to gratefully acknowledge the financial assistance from Department of Science and Technology, Government of India, for supporting this work under the Nano centre Grant no. SR/S5/NM-51/2005, National Nanotechnology Initiative. The authors are also grateful for the initial support of this work from Department of Biotechnology, Government of for financial support. We are extremely grateful to Amrita Vishwa Vidyapeetham for providing all the infrastructural support for basic research in nanobiomedical sciences. Authors acknowledge Mr. Sajin P Ravi, Mr. Sarath S and Ms. Sreerekha P R for their technical assistance.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 31

References 1.

Parkin, D.M.; Bray, F.; Ferlay, J.; Pisani, P. Global cancer statistics, 2002. CA- A Cancer Journal for Clinicians 2005, 55(2), 74–108, DOI: 10.3322/canjclin.55.2.74.

2.

Bleicher, R. J.; Alleqra, D. P.; Nora, D.T.; Wood, T.F.; Foshaq, L.J.; Bilchik, A.J. Radiofrequency ablation in 447 complex unresectable liver tumors: lessons learned. Annual Surgical oncology 2003, 10(1), 52-8, DOI: DOI: 10.1245/ASO.2003.03.018.

3.

Goldberg, S.N.; Salddlnger, P.F.; Gazelle, G.S.; Huertas, J.C.; Stuart, K.E.; Jacobs, T.; Kruskal, J.B. Percutaneous tumor ablation: Increased necrosis with combined radio-frequency ablation and intratumoral doxorubicin injection in a rat breast tumor model. Radiology 2001, 220, 420-427, DOI: http://dx.doi.org/10.1148/radiology.220.2.r01au44420.

4.

Kampinga, H.H.; Dynlacht, J.R; Dikomey, E. Review Mechanism of radiosensitization by hyperthermia (> or = 43 degrees C) as derived from studies with DNA repair defective mutant cell lines. International Journal of Hyperthermia 2004, 20(2),131-9, DOI: 10.1080/02656730310001627713.

5.

Tamarov, K.P.; Osminkina, L.A.; Zinovyev, S.V.; Maximova K.A.; Kargina, J.V.; Gongalsky, M.B.; Ryabchikov, Y.; Al-Kattan, A.; Sviridov, A.P.; Sentis, M.; Ivanov, A.V.; Nikiforov, V.N.; Kabashin, A.V.; Timoshenko, V.Y. Radio frequency radiation-induced hyperthermia using Si nanoparticle-based sensitizers for mild cancer therapy. Nature scientific reports, 2014, DOI: 10.1038/srep07034.

6.

Kam, N.W.S.; O’Connell, M.; Wisdom, J.A.; Dai, H. Carbon nanotubes as multifunctional biological transporters and near-infrared agents for selective cancer cell destruction. PNAS 2005, 102(33), 1160011605, DOI: 10.1073pnas.0502680102

7.

Burke, A.; Ding, X.; Singh, R.; Kraft, R. A.; Levi-Polyachenko, N.; Rylander, M. N.; Szot, C.; Buchanan, C.; Whitney, J.; Fisher, J.; Hatcher, H. C.; D’Agostino, R.; Kock, N.D.; Ajayan, P.M.; Carroll, D.L.; Akman, S.; Torti, F.M.; Torti, S.V. Long-term survival following a single treatment of kidney tumors with multiwalled carbon nanotubes and near-infrared radiation. PNAS 2009; 106(31), 12897-12902, DOI: 10.1073pnas.0905195106.

8.

Dickerson, E. B.; Dreaden, E. C.; Huang, X.; Ivan, H.; Chu, H.; Pushpanketh, S.; McDonald, J. F.; ElSayed. Gold nanorod assisted near-infrared plasmonic photothermal therapy (PPTT) of squamous cell carcinoma in mice. Cancer Letters 2008, 269, 57–66, DOI: 10.1016/j.canlet.2008.04.026.


Page 21 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


9.

Gannon, C.J.; Cherukur, P.; Yakobson, B.I.; Cognet, L.; Kanzius, J.S.; Kittrell, C.; Weisman, R.B.; Pasquali, M.; Schmidt, H.K.; Smalley, R.E.; Curley, S.A. Carbon nanotube- enhanced thermal destruction of

cancer

cells

in

a

noninvasive

radiofrequency

field.

Cancer.

2007,

110,

2654–65,

DOI:10.1002/cncr.23155 10. Gannon, C.J.; Patra, C. R.; Bhattacharya, R.; Mukherjee, P.; Curley, S. A. Intracellular gold nanoparticles enhance non-invasive radiofrequency thermal destruction of human gastrointestinal cancer cells. Journal of Nanobiotechnology 2008, 6, 2-8, DOI: 10.1186/1477-3155-6-2 11. Glazer, E.S.; Curley, S.A. Non-invasive radiofrequency ablation of malignancies mediated by quantumdots, gold nanoparticles and carbon nanotubes. Therapeutic Delivery 2011, 2(10), 1325-30, DOI: 10.4155/tde.11.102 12. Raoof, M.; Curley, S. A. Non-Invasive Radiofrequency-Induced Targeted Hyperthermia for the Treatment of Hepatocellular Carcinoma. International Journal of Hepatology 2011, 1-6. DOI:10.4061/2011/676957 13. Glazer, E. S.; Massey, K. L.; Zhu, C.; Curley, S. A. Pancreatic carcinoma cells are susceptible to noninvasive radiofrequency fields after treatment with targeted gold nanoparticles. Surgery 2010, 148(2), 319324. DOI: 10.1016/j.surg.2010.04.025. 14. Glazer, E. S.; Curley, S. A. Radiofrequency field-induced thermal cytotoxicity in cancer cells treated with fluorescent nanoparticles. Cancer 2010, 116(13), 3285-3293, DOI: 10.1002/cncr.25135. 15. Cherukuri, P.; Glazer, E. S.; Curley, S. A. Targeted Hyperthermia Using Metal Nanoparticles. Advanced Drug Delivery Review 2010, 62(3), 339-345, DOI: 10.1016/j.addr.2009.11.006. 16. Cherukuri,

P.; Curley, S. A. Use of nanoparticles for targeted, non-invasive thermal destruction of

malignant cells. Methods in Molecular Biology 2010, 624, 359-373, DOI: 10.1007/978-1-60761-609-2_24. 17. Yang, W.; Ratinac, K.R.; Ringer, S. P.; Thordarson, P.; Gooding, J. J.; Braet, F. Carbon nanomaterials in biosensors: should you use nanotubes or graphene? Angewandte Chemie International Edition 2010, 49, 2114–2138, DOI: 10.1002/anie.200903463 18. Wang, L.; Shi, J.; Jia, X.; Liu, R.; Wang, H.; Wang, Z.; Li, L.; Zhang, J.; Zhang, C.; Zhang, Z. NIR-/pHResponsive drug delivery of functionalized single-walled carbon nanotubes for potential application in cancer chemo-photothermal therapy. Pharmaceutical Research 2013;30(11),2757-71.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 31

19. Yang, K.; Zhang, S.; Zhang, G. X.; Sun, X. M.; Lee, S. T.; Liu, Z. Graphene in mice: ultrahigh in vivo tumor uptake and efficient photothermal therapy. Nano Letters 2010, 10, 3318–3323, DOI: 10.1021/nl100996u 20. Sasidharan, A.; Sivaram, A.J.; Ratnakumari, A.P.; Chandran, P.; Malarvizhi, G.L.; Nair, S.; Koyakutty, M. Radiofrequency ablation of drug resistant cancer cells using molecularly targed carboxyl-functionlized biodegradable graphene. Advanced Helathcare Materials 2015;4,679–684 21. Subrahmanyam, K.S.; Panchakarla, L. S.; Govindaraj, A.; Rao, C.N. Simple method of preparing graphene flakes by an arc-discharge method. Journal of Physical Chemistry C. 2009; 113, 4257-4259. 22. Sasidharan, A.; Panchakarla, L. S.; Sadanandan, A. R.; Asokan, A.; Chandran, P.; Girish, C.M.; Menon, D.,Nair, S.; Rao, C.N Koyakutty, M. Hemocompatibility and Macrophage Response of Pristine and Functionalized Graphene. Small 2012; 8(8), 1251-63. 23. Sviridov, A. P.; Andreev, V. G.; Ivanova, E. M.; Osminkina, L. A.; Tamarov, K. P.; Timoshenko, V. Porous silicon nanoparticles as sensitizers for ultrasonic hyperthermia. Applied Physics Letters 2013; 103, 193110-193114. 24. Markovic, Z.M.; Harhaji-trajkovic, L.M.; Todorovic-Markovic, B.M.; Depic, D.P.; Arsikin, K.M.; Jovanovic, sp.; Pantovic, A.C.; Dramicanin, M.D.; Trajkovic, V.S. In vitro comparison of the photothermal anticancer activity of graphene nanoparticles and carbon nanotubes. Biomaterials 2011, 32(4), 1121-1129, DOI: 10.1016/j.biomaterials.2010.10.030 25. Sasidharan, A.; Panchakarla, L.S.; Chandran, P.; Menon, D.; Nair, S.; Rao, C.N.; Koyakutty, M. Differential nano-bio interactions and toxicity effects of pristine versus functionalized graphene. Nanoscale 2011, 3(6), 2461-4, DOI: 10.1039/C1NR10172B 26. Girish, C. M.; Sasidharan, A.; Gowd, G. S.; Nair, S.; Koyakutty, M. Confocal Raman Imaging Study Showing Macrophage Mediated Biodegradation of Graphene In Vivo. Advanced Healthcare Materials 2013, 2(11),1489-500, DOI: 10.1002/adhm.201200489. 27. Milleron, R.S.; Bratton, S.B. ‘Heated’ debates in apoptosis. Cellular and Molecular Life Sciences 2007, 64, 2329–33, DOI: 10.1007/s00018-007-7135-6. 28. Wust, P.; Hildebrandt, B.; Sreenivasa, G.; Rau, B.; Gellermann, J.; Riess, H.; Felix, R.; Schlag, P.M. Hyperthermia in combined treatment of cancer. The lancet oncology 2002, 3, 487–97, DOI: 10.1016/S1470-2045(02)00818-5. 29. Hildebrandt, B.; Wust, P.; Ahlers, O.; Dieing, A.; Sreenivasa, G.; Kerner, T.; Felix, R.; Riess, H. The cellular and molecular basis of hyperthermia. Critical reviews in oncology/hematology 2002, 43, 33–56, DOI: 10.1016/S1040-8428(01)00179-2.


Page 23 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


30. Calzolari, A.; Oliviero, I.; Deaglio, S.; Mariani, G.; Biffoni, M.; Sposi, N. M.; Malavasi, F.; Peschle, C.; Testa, U. Transferrin receptor 2 is frequently expressed in human cancer cell lines. Blood Cells, Molecules, and Diseases 2007, 39, 82–91, DOI: 10.1016/j.bcmd.2007.02.003. 31. Safety

Study

of

CALAA-01

to

Treat

Solid

Tumor

Cancers.

http://clinicaltrials.gov/ct2/show/NCT00689065?term=NCT00689065&rank=1 32. Study of MBP-426 in Patients With Second Line Gastric, Gastroesophageal, or Esophageal Adenocarcinoma.http://clinicaltrials.gov/ct2/show/NCT00964080?term=NCT00964080&rank=1 33. Safety

Study

of

Infusion

of

SGT-53

to

Treat

Solid

Tumors.http://clinicaltrials.gov/ct2/show/NCT00470613?term=NCT00470613&rank=1 34. Leber, B.; Mayrhauser, U.; Leopold, B.; Koestenbauer, S.; Tscheliessnigg, K.; Stadlbauer, V.; Stiegler, P. Impact of temperature on cell death in a cell-culture model of hepatocellular carcinoma. Anticancer Research 2012, 32, 915-922. 35. Curley, S.A. Radiofrequency ablation of malignant liver tumors. The Oncologist 2003, 10, 338–47, DOI: 10.1634/theoncologist.6-1-14. 36. Fischbach, C.; Chen, R.; Matsumoto, T.; Schmelzle, T.; Brugge, J.S.; Polverini, P.J.; Mooney, D.J. Engineering tumors with 3D scaffolds. Nature Methods 2007, 4(10), 855–60, DOI: 10.1038/nmeth1085 37. Dhiman, H.K.; Ray, A.R.; Panda, A.K. Three-dimensional chitosan scaffold-based MCF-7 cell culture for the determination of the cytotoxicity of tamoxifen. Biomaterials 2005, 26(9), 979–86, DOI: 10.1016/j.biomaterials.2004.04.012. 38. Weaver, V.M.; Lelievre, S.; Lakins, J.N.; Chrenek, M.A.; Jones, J.C.R.; Giancotti, F., Werb, Z.; Bissell, M.J. Giancotti, F. β4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium. Cancer Cell 2002, 2(3), 205–16, DOI: 10.1016/S1535-6108(02)00125-3. 39. Zhang, X.; Wang, W.; Yu, W.; Xie, Y.; Zhang, X.; Zhang, Y.; Ma, X. Development of an in vitro multicellular tumor spheroid model using microencapsulation and its application in anticancer drug screening and testing. Biotechnology Progress 2005, 21(4), 1289–96, DOI: 10.1021/bp050003l. 40. Ong, S.M.; He, L.; Thuy Linh, N.T.; Tee, Y.H.; Arooz, T.; Tang, G.; Tang,C.H.; Yu, H. Transient intercellular

polymeric

linker.

Biomaterials

2007,

28(25),

3656–67,

DOI:

10.1016/j.biomaterials.2007.04.034.

Figure legends Figure 1. (A) (i) TEM, (ii) AFM and (iii) AFM height profiling of functionalized graphene (B) TEM image of Tf conjugated graphene, Tf-G and (ii) HR-TEM showing a thin layer transferrin (arrows) around graphene NP (C) Comparative evaluation of RF induced temperature increase by gold NPs, Single walled CNT and graphene Tf-G, all at 20ug/ml.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 31

Figure 2. (A) Flow cytogram showing percentage expression of transferrin receptors by liver cancer cells (HepG2). (B) Biocompatibility of functionalized(FG) and Transferrin conjugated graphene (TfG) at 20 µg/ml and 50 µg/ml with HepG2 cells treated for 24 hours (C) (i & ii) DIC images representing varied cellular uptake of FG and TfG by HepG2 cells, (iii & iv) corresponding Raman image confirming the presence of graphene (pink) inside the cells (blue).

Figure 3. (A) Percentage cell viability of HepG2 cells by alamar blue assay after RF exposure of 50 W-1 minute, 100 W-1 minute, 100 W-3 minutes, 100 W-5 minutes. (B) Percentage cell viability of FG and TfG treated HepG2 cells after RF exposure at a power of 100 W for 1, 3 and 5 minutes using alamar blue assay. (C) Confocal microscopic images of Annexin V/propidium iodide staining of apoptosis assay. The images reveal apoptosis in HepG2 cells upon RF exposure of 100 W- (i) 1min, (ii) 3 minutes and (iii)5 minutes after treating cells with 50 µg/ml concentration of FG (Lane 2) and TfG (Lane 3), in comparison to untreated cells (Lane 1). (D)Flow cytogram showing the apoptosis in HepG2 cells upon RF exposure for (i) 100 W-3 and (ii) 5 minutes at 50µg/ml FG (Lane 2) and TfG (Lane 3) compared to untreated cells (Lane 1).

Figure 4. (A) Stereomicroscopic images of alginate micro beads acellular and after encapsulation of HepG2 Cells, (B) Cell viability of HepG2 cells in 2D and 3D alginate-collagen culture systems after 1, 5 and 7 days of incubation. (C) confocal microscopic images (i) Differential interference contrast (DIC) image (ii) calceinTM/EtBr staining for live-dead assay of HepG2 cell encapsulated micro bead (iii) merged image of DIC and Live-dead assay of cell encapsulated micro bead (scale bar = 100µm, magnification 10x ),(D) acellular and cellular macro bead (i) macro bead without cell showing the color of alamar blue (blue color) (ii)cellular macro bead showing the color of resorufin (pink) after incubation with alamar blue reagent (iii) transferrin antibody incubated acellular alginate (iv) Fluorescence emitted by the HepG2 cell encapsulated alginate macro bead.

Figure 5. (A) Percentage viability of FG and TfG treated HepG2 cells in 3D culture system after RF exposure by alamar blue assay. (B) Confocal microscopic images of CalceinTM/EtBr staining for Live/Dead assay. The images clearly revealing the dead and necrotic cells after RF exposure of 100W- 3 and 5 minutes after treating cells with 50 µg/ml of FG and TfG, in comparison to untreated cells. (C) Confocal images of CalceinTM/EtBr staining on cell encapsulated 3D culture system after 100W-3 minutes of RF exposure (Scale bar= 150µm, 10x magnification)

Figure 6. Photographs of (a) TfG injected liver of Wistar Rat. (b)Whole liver before the radiofrequency ablation (c) Portion of liver showing immediate color change due to hyperthermic ablation in graphene


Page 25 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


injected site upon RF exposure (d) Vertical section of ablated area stained with TTC, showing clear necrotic region.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1 254x190mm (300 x 300 DPI)


Page 26 of 31

Page 27 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2 254x190mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3 254x190mm (300 x 300 DPI)


Page 28 of 31

Page 29 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4 254x190mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5 254x190mm (300 x 300 DPI)


Page 30 of 31

Page 31 of 31

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6 254x190mm (300 x 300 DPI)


ACS Biomaterials Science & Engineering - ACS Publications

Recommend Documents