Development of a Graphene Oxide Nanocarrier for Dual-Drug Chemo

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Development of a Graphene Oxide Nanocarrier for Dual-Drug Chemo-phototherapy to Overcome Drug Resistance in Cancer Tuan Hiep Tran, Hanh Thuy Nguyen, Tung Thanh Pham, Ju Yeon Choi, Han-Gon Choi, Chul Soon Yong, and Jong Oh Kim ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b10426 • Publication Date (Web): 07 Dec 2015 Downloaded from http://pubs.acs.org on December 9, 2015

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ACS Applied Materials & Interfaces

Article Development of a Graphene Oxide Nanocarrier for Dual-Drug Chemophototherapy to Overcome Drug Resistance in Cancer

Tuan Hiep Trana, Hanh Thuy Nguyena, Tung Thanh Phama, Ju Yeon Choia, Han-Gon Choib***, Chul Soon Yonga**, Jong Oh Kima*

a

College of Pharmacy, Yeungnam University, 214-1, Dae-Dong, Gyeongsan 712-749, South

Korea. b

College of Pharmacy, Hanyang University, 55, Hanyangdaehak-ro, Sangnok-gu, Ansan 426-

791, South Korea

Authors to whom correspondence should be addressed:

*Corresponding author: Tel.: +82-53-810-2813; Fax: +82-53-810-4654 E-mail: [email protected] **Co-corresponding author: Tel.: +82-53-810-2812; Fax: +82-53-810-4654 E-mail: [email protected] ***Co-corresponding author: Tel.: +82-31-400-5802; Fax: +82-31-400-5958 E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT Despite the tremendous progress in chemotherapy, drug resistance remains a major challenge for anticancer treatment. The combinations of chemo-photothermal and chemo-chemo treatments have been reported to be potential solutions to overcome drug resistance. In this study, we developed a dual-in-dual synergistic therapy based on the use of dual anticancer drug-loaded graphene oxide (GO) stabilized with poloxamer 188 for generating heat and delivering drugs to kill cancer cells under near infrared (NIR) laser irradiation. The nanocomparable system is stable and uniform in size, generating sufficient heat to induce cell death. Dual drugs (doxorubicin and irinotecan)-loaded GO (GO-DI) in combination with laser irradiation caused higher cytotoxicity than that caused by the administration of a free single drug as well as a combination of drugs and blank GO in various cancer cells, especially in MDA-MB-231 cells, resistant breast cancer cells. Exposure to “hot” NIR and GO-DI activated the intrinsic apoptosis pathway, which was confirmed based on changes in the morphology of cell nuclei and overexpression of apoptosis-related proteins. Based on the results, the combined treatment showed a synergistic effect compared to the effect of chemotherapy or photothermal treatment alone, demonstrating higher therapeutic efficacy to overcome one of the most severe problem in anticancer therapy, that of intrinsic resistance to chemotherapeutics.

Keywords: graphene oxide, poloxamer 188, combination chemotherapy, NIR, drug resistance, irinotecan, doxorubicin


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INTRODUCTION Cancer is currently one of the most lethal diseases and a global health concern.1 Recently, several approaches such as radiotherapy, chemotherapy, photodynamic therapy (PDT), and photothermal therapy (PTT) have been applied for treatment of cancers.2 However, the clinical efficacy of anticancer drugs has been found to be limited in cases of treatment with a single therapeutic. Therefore, combination therapy is considered to be a potential strategy for anticancer therapy owing to its advantages of enhanced therapeutic efficacy, low possibility of drug resistance, and fewer side effects than those of monotherapy. Nanocarriers with two or more therapeutic actions targeted at different tumor sites in synergistic chemo-chemo, chemo-siRNA, chemo-thermal, and chemo-photodynamic therapies more efficiently induce apoptosis in cancer cells and inhibit the growth of cancer tissues.3 Among these, the combination of PDT and chemotherapy has a long application history in both preclinical research and clinical tumor treatment.4 Recently, the integration of PTT and chemotherapy and administration via one nanocarrier have attracted a lot of attention because it affords a synergistic effect and better therapeutic efficacy.5 Rational drug combinations hold great promise for countering drug resistance and help reduce tumor metastasis via various mechanisms of action involving inhibition of multiple tumor cell survival pathways.6 Recently, we demonstrated that the combination of doxorubicin (DOX) and irinotecan (IRI) produced a synergistic therapeutic effect by inducing the regulation of DNA topology/nucleic acid synthesis in the cell nucleus.7 Furthermore, graphene, a two-dimensional nanomaterial, has been widely investigated for biomedical application.8 It shows light absorption from UV to NIR regions, a characteristic that can be used in thermal ablation of malignant tissue at temperatures above 40ºC without any surgical resection in some conditions.9-10 In this study, we aimed to exploit the advantages of chemo-chemo as well as chemo-



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thermal approaches by developing a dual-in-dual carrier for a synergistic effect. For this purpose, two anticancer drugs, DOX and IRI, were loaded into the hydrophobic surface of the carrier, graphene oxide (GO), to form a well-dispersed solution. A pH-responsive release of the drugs from GO under acidic and physiological conditions was studied for safe in vivo circulation, productive drug release inside organelles, and synergistic effects with PTT. Cellular uptake, cytotoxicity studies and in vitro tumor ablation were used to determine the feasibility of using this nanocarrier in chemo-photothermal treatment of cancer.

MATERIALS AND METHODS Materials Doxorubicin hydrochloride (DOX) was a gift from the Dong-A Pharmaceutical Company (Yongin, South Korea). Irinotecan hydrochloride (IRI) was a kind gift from Hanmi Pharmaceuticals, Co. Ltd. (Hwaseong, South Korea). Graphite flakes were purchased from Alfa Aesar (Ward Hill, MA, USA). All other chemicals were of reagent grade and were used without further purification. SCC-7, MCF-7, and MDA-MB-231 cell lines were originally obtained from the Korean Cell Bank (Seoul, South Korea). Cell lines were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and incubated at 37°C in a 5% CO2 humid incubator.

Preparation of dual drug-loaded graphene oxide (GO-DI) GO was first prepared according to the modified Hummers’ method by using natural graphite flakes.11 To obtain nanosized GO, GO was cracked using an ultrasonic probe at 570 W for 2 h. To prepare a poloxamer 188-functionalized graphene nanohybrid, 200 mg of poloxamer 188


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was added to 10 mL of GO aqueous suspension (1.0 mg/mL) and the mixture was stirred for 30 min at room temperature. The loading of DOX/IRI onto GO (GO-DI) was conducted by simply mixing GO in poloxamer solution with DOX/IRI in a molar ratio of 1:1, and then stirred overnight at room temperature in the dark.

Dynamic light scattering (DLS) The GO-DI nanoparticles were characterized by measuring their mean size, polydispersity index (PDI), and zeta potential (ZP) using a dynamic light scattering (DLS) system, Zetasizer Nano–Z (Malvern Instruments, Worcestershire, UK). Samples were diluted with distilled water prior to analysis.

Morphology characterization GO dispersions were characterized by transmission electron microscopy (Hitachi H7600, Tokyo, Japan). A small drop of dilute GO solution was poured on a carbon-coated copper grid, allowed to dry in air under exposure to a UV lamp. Atomic force microscopy images were collected in the tapping mode by using Nanoscope IIIa (Digital Instruments Co., USA). The optical images of GO-DI in different media (distilled water, PBS or DMEM) was captured using a digital camera.12

Drug loading capacity The drug entrapment efficiency (EE) and drug loading capacity (LC) of GO-DI were determined indirectly by measuring the amount of free drug in the dispersion medium by using Amicon centrifugal tube (Millipore, USA). The EE and LE were calculated using the following equations13. EE (%) = (Winitial drug− Wunbound drug)/Winitial drug × 100



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LC (%) = (Winitial drug− Wunbound drug)/WGO-DI × 100 where, EE is the entrapment efficiency, LC is the drug loading capacity, and W is the weight (in mg). The concentrations of DOX and IRI in the filtrates and in the nanoparticles were determined by high-performance liquid chromatography (HPLC). The mobile phases for DOX and IRI were methanol:water:acetic acid (50:49:1), and (water:acetonitrile:methanol, 50:25:25, pH 3.5), respectively and were run at 1 mL/min with absorbance detected at 254 nm.7

In vitro drug release The drug release profiles were characterized in medium under different pH conditions (7.4, and 5.0) by using the dialysis method. Briefly, 1 mL of GO-DI was transferred into a dialysis bag (MWCO, 3.5 KDa). The dialysis bag was placed in 35 mL of release medium at 37°C inside a shaking water bath (HST – 205 SW, Hanbaek ST Co., Seoul, Korea) with continuous shaking at 100 rpm. At a predetermined time, 0.5 mL of the incubated solution was taken out and replaced with an equal volume of fresh media. The amounts of DOX and IRI released were analyzed using the HPLC method described above. The release experiments were performed in triplicate.14

In vitro cellular uptake and localization of GO-DI The internalization of the GO-DI into the cancer cells was characterized via flow cytometric measurements. Cancer cells grown in 12-well plates at a density of 1 × 105 for 24 h were incubated with GO-DI at a concentration of 10 µg/mL for an incubation time of 0.5, 1, and 1.5 h. Then, the cells were trypsinized, washed with phosphate-buffered saline (PBS), and analyzed for intracellular fluorescence by using FACS Verse (BD Biosciences, San Jose, CA, USA).15 Untreated cells were used as an internal control. At least 10,000 events were


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acquired and analysed per sample. Further, each cover slip kept in the 12-well plate was loaded with cancer cells at a density of 5000 cells per well and incubated for 24 h for cell attachment. Thereafter, the media were removed and the wells were carefully washed with PBS buffer. Then, GO-DI in media at a concentration of 1 µg/mL was added to the cells and incubated for 5 min, followed by treatment with Lysotracker Green for 10 min. Thereafter, the media with the sample were removed and the cover slips were washed gently with PBS before being observed using a laser scanning confocal microscope (Leica TCS SP2; Leica Co., Wetzlar, Germany).16

In vitro cytotoxicity studies The cytotoxicity of blank GO, free DOX, free IRI, DOX/IRI cocktail, and GO-DI was studied in MCF-7, SCC-7, and MDA-MB-231 cells by using the MTT assay. For the experiments, 100 µl of cell suspension was seeded in a 96-well plate at a density of 5 × 103 cells/well, and incubated for 24 h. The samples were added to each well and the plate was incubated for another 24 h. Subsequently, 100 µl (1.25 mg/ml) of MTT-PBS mixture was added to each well and the plate was placed in an incubator for 4 h. DMSO (100 µL) was added to each well and kept for 15 min before the absorbance was measured at 570 nm using a microplate reader (Multiskan EX, Thermo Scientific, USA). Cell viability was calculated using the following formula.17-18 Cell viability % =

OD sample – OD blank x 100 OD control – OD blank

The effect of NIR laser irradiation was conducted by treating cancer cells with free dual drugs, blank GO, GO-DOX, GO-IRI, and GO-DI at drug concentrations of 1 µg/mL or equivalent to about 6 µg/mL GO for 6 h and then exposed with NIR laser (3 W/cm2) at 808 nm for 5 min. After 24 hours treatment, the cell viability was measured using above technique.



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Live/dead assay Cells were seeded in 12-well tissue culture plates at a density of 1 × 105 per well and cultured for 24 h. The cells were treated with the drug combination, blank GO, and GO-DI with the drug concentrations of 0.1, 0.2, and 1 µg/mL, and incubated for 6 h to ensure cellular uptake. Thereafter, the cells were treated with 808 nm NIR laser irradiation with a power output of 3 W/cm2 for different durations. Subsequently, the cells were stained with CalceinAM (AM), Ethidium homodimer-1 (EthD-1) in PBS at final concentrations of 2.0 µM. The stained cells were observed using inverted fluorescence microscopy (Nikon Eclipse Ti).19

Morphological changes in the nucleus observed using fluorescence microscopy Cancer cells were seeded in 6-well plates at a density of 2 × 105 cells/well. After preincubation for 24 h under normal conditions, the cells were treated with single drugs, the drug combination, and GO-DI with a drug concentration of 1 µg/ml. The well treated with NIR laser irradiation was conducted same as in in vitro cytotoxicity study. After treatment for 24 h, the cells were washed with PBS twice, and fixed with 1 ml of 4% paraformaldehyde at 4 °C for 10 min. Then, the plate was washed with PBS three times, and the cells were dyed for 10 min with Hoechst 33342 at room temperature in the dark. Thereafter, the cells were rinsed with PBS thrice and observed under an inverted fluorescence microscope (Nikon Eclipse Ti).20

Western blot analysis The apoptotic activity of cancer cells after drug treatment was evaluated by detecting relevant protein markers using western blot analysis. Cells at a density of 2 × 105 cells/well were treated with 1 µg/ml native drug or an equivalent concentration of drug entrapped in GO-DI


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for 24 h. Cells were washed three times with PBS, lysed by adding SDS sample buffer (62.5 mM Tris–HCl [pH 6.8], 2% SDS, 10% glycerol, 50 mM dithiothreitol, and 0.1% bromophenol blue), and scraped. Proteins were separated using SDS-PAGE gel and electrophoretically transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA). After blocking with 5% dry milk in Tris-Buffered Saline with 1% Tween-20 (TBST), the membrane was incubated with a specific primary antibody (1:1000 dilution in TBST) against various proteins studied or anti-β-actin antibody (sc-47778, Santa Cruz Biotechnology, Santa Cruz, CA, USA) as a loading control (1:1000 dilution in TBST). The membrane was washed three times with TBST and incubated with the secondary antibody. Proteins were detected using chemiluminescence detection reagents and an image was captured using Kodak imaging film (Kodak, USA).21

RESULTS AND DISCUSSION Preparation and characterization of GO-DI Scheme 1 illustrates the loading of DOX/IRI on GO based on π–π stacking interaction. Nanosized GO was synthesized by oxidizing graphite using the modified Hummer method.11



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Scheme 1. Schematic diagram showing the antitumor activity of dual drug-loaded graphene oxide (doxorubicin and irinotecan-loaded GO; GO-DI).

As shown in Figure 1, sonication treatment of GO yielded a nanometric (200 nm) carrier, as indicated by TEM and AFM characterization, with a single or two-layered sheet, according to a previous study.22 GO aggregation is easy because of its hydrophobic and electronic surface. GO was stabilized in different media such as distilled water, PBS, and DMEM by mixing with poloxamer 188. Poloxamer consists of high portion of hydrophilic chains of polyoxyethylene which could expand stability of carrier in biological fluid. This property facilitates long blood circulation before accumulation in the tumor tissue via the enhanced permeability and retention (EPR) effect.23 In addition, poloxamer is preferred in anticancer chemotherapy due to its ability to inhibit MDR proteins and other drug efflux transporters on the surface of cancer cells, resulting in increasing the susceptibility of cancer cells to


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chemotherapeutic agents such as doxorubicin.24

Figure 1. Physical properties of doxorubicin and irinotecan-loaded graphene oxide (GO-DI): (A) Diameter of GO-DI, (B) Dispersed GO-DI in various media, (C) TEM image of GO-DI, and (D) AFM image of GO-DI. Scale bar indicates 500 nm. Doxorubicin and inrinotecan were loaded into GO in a molar ratio of 1:1.

The drugs were loaded onto GO by simply mixing the drug solution with GO. After removal of unbound DOX/IRI using centrifugal ﬁltration and repeated rinsing, loading capacity was determined indirectly by determining the amount of free drug. High EE (almost 100%) and LC (~7%) of both drugs were achieved, indicating a strong interaction between the drugs and GO (Figure 2A). Such high loading of drugs is due to the effect of π–π stacking interaction and hydrogen bonding.25



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The drug release at different pH conditions was investigated at pH 5.0 and 7.4 as shown in Figure 2B. Both drugs demonstrated a slow dissolution rate at neutral pH and significantly improved dissolution rate in the acidic condition. In particular, after 24 h, the release rate at pH 7.4 was around 40%, whereas that at pH 5.0 was 60%. Stronger hydrogen-bonding between –OH and –NH2 groups in DOX/IRI and –OH and –COOH groups on GO under the neutral condition than under the acidic condition resulted in the faster release under the acidic condition.24 On the other hand, the slightly faster release rate of IRI could be owing to a lack of –NH2 groups compared to that in DOX. The pH-sensitive release demonstrated by the carrier could be ideal for anticancer therapy, where the acidic environments in the lysosomes and late endosomes trigger a bolus drug release that may effectively kill cancer cells.26 The heat generation upon NIR irradiation was evaluated to confirm thermal energy conversion from NIR light energy. Various solutions containing GO-DI, and water only as a control, were exposed and then thermally evaluated. As shown in Figure 2C, the control showed no response to NIR irradiation for 5 min, whereas other solutions showed significant dose-dependent GO-mediated heat generation. With time, the temperature increased and reached approximately 50ºC after 5 min at concentration of 20 µg/mL.


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Figure 2. Physical properties of doxorubicin and irinotecan-loaded graphene oxide (GO-DI): (A) Drug loading capacity and entrapment efficiency, (B) In vitro drug release, (C) Photothermal capacities of GO-DI at various concentrations upon irradiation with 808-nm laser.

Cellular uptake of GO-DI The internalized fraction of GO-DI taken up by the various cells was measured using flow cytometry. As shown in Figure 3, by increasing the incubation time (0.5, 1, and 1.5 h), the intensity increased, indicating improved uptake (Figure 3A, C, E). A similar phenomenon



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was observed when dose was changed from 5 µg/mL to 10 µg/mL after incubation for 1 h. However, in comparison with the uptake of free drug at same concentration and exposure time, the uptake of carrier is less, which could be explained by the high solubility of drug resulting in high diffusion rate through cell membrane. This difference in the uptake of free drug and carrier is diminished in resistant cell-line, demonstrating the potential of the carrier in overcoming drug efflux.27 In vitro imaging studies is an important tool in studying the localization of nanoparticles inside the cell. Consistent with cytometry data, the carriers are taken up inside the cells very quickly and distributed almost homogenously inside the cells (Figure 4). Some studies have mentioned that clathrin-mediated endocytosis is the main pathway for penetration of GO into the lysosomal region, wherein the cargo was released and transferred to the nucleus.28-29


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Figure 3. Flow cytometric analysis of doxorubicin and irinotecan-loaded graphene oxide (GO-DI) uptake into cancer cells (A, B) SCC-7, (C, D) MCF-7, and (E, F) MDA-MB-231 in a time-dependent (left channel) and dose-dependent manner (right channel); Time-dependent uptake: control (black), GO-DI for 0.5 h (light blue), GO-DI for 1 h (green), GO-DI for 1.5 h (blue), and free DOX (red), and dose-dependent uptake (right channel): control (black), GODI at 5 µg/mL (blue), and GO-DI at 10 µg/mL (red).



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Figure 4. CLSM images of SCC-7, MCF-7, and MDA-MB-231 cells incubated with doxorubicin (DOX)-loaded graphene oxide (GO). Scale bars: 30 µm.

In vitro cytotoxicity studies The dose-dependent cytotoxicity of DOX, IRI, combined DI, and GO-DI was characterized in SCC-7, MCF-7, and MDA-MB-231 cell lines (Figure 5). All samples showed toxicity in a dose-dependent manner whereas the blank GO was found to be almost safe up to a concentration of ~60 µg/mL (equivalent to 10 µg/mL drug in GO-DI) with cell survival being more than 80%.30 At a molar ratio of 1:1, the performance of combined DOX+IRI was better than that of single drug. The combination index, which was calculated by Calcusyn® software, was less than 1, demonstrating the synergistic effect of dual drugs on all cell-lines.31 In comparison with free dual drugs, GO-DI is less toxic to sensitive cell-lines such as SCC-7 and MCF-7. Because in sensitive cells, drugs can easily bypass the cell membrane and express their activity. However, GO-DI inhibited the growth of resistant cell


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line MDA-MB-231 greater than free dual drugs at low concentration (≤1 µg/mL). The incorporation of poloxamer 188 to carrier could inhibit drug efflux to maintain high drug concentration inside the cell and kill more cell.24

Figure 5. In vitro viability of cancer cells treated with free doxorubicin (DOX), irinotecan (IRI), combination of DOX+IRI, plain graphene oxide (GO), and GO-DI for 24 h at different concentrations: 0.01, 0.1, 1, and 10 µg/mL. Pure DMEM served as the control. (A, B) SCC-7 cells, (C, D) MCF-7 cells, and (E, F) MDA-MB-231 cells, (A, C, E) Combination index (CI) for all cell-lines. Errors bars indicate the standard deviation of three separate experiments.



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To evaluate the efficiency of GO-DI as a chemo-thermal therapy vesicle, its phototherapeutic effect on three cancer cells was evaluated by MTT assay. Cancer cells were treated with free dual drugs, blank GO, GO-DOX, GO-IRI, and GO-DI at drug concentrations of 1 µg/mL or equivalent to about 6 µg/mL GO for 6 h and then irradiated with NIR laser (3 W/cm2) at 808 nm for 5 min, followed by viability measurements at 24 h. As shown in Figure 6, GO-DI in response to NIR irradiation exhibited a greater anticancer effect on all cell-lines compared with combination of free drug, blank GO, or GO containing single drug. In addition, the significant difference between treatments with/without NIR laser exposure groups indicated the role of heat on synergistic effect.


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Figure 6. In vitro viability of cancer cells treated with combination of free doxorubicin and irinotecan (DOX+IRI), plain graphene oxide (GO), GO-DOX, GO-IRI, and GO-DI with/without NIR laser exposure, as determined by MTT assay. (A) SCC-7, (B) MCF-7, and (C) MDA-MB-231. Errors bars indicate the standard deviation of three separate experiments. *Statistical significance at P < 0.05



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In vitro photothermal efficacy In order to determine the phototherapy efficacy of GO-DI, the live/dead assay was employed with calcein-AM and propidium iodide (PI) fluorescence dyes to identify living cells (green) and dead cells (red). The effect of irradiation time, formulation concentration, as well as the presence or absence of NIR exposure on all cell lines was evaluated. Owing to NIR irradiation, most cells stained red, while the cells in the area not exposed to NIR laser irradiation stained green with calcein AM, which indicated selective ablation of cells exposed to NIR irradiation. Moreover, the fluorescence image (Figure 7A) shows much more red from cells incubated with GO-DI than that for the ones incubate with PBS or blank GO, which demonstrated the stronger phototherapy efficiency. The effects of irradiation time and formulation concentration on photothermal ablation of cancer cells were also evaluated. The diameter and intensity of red-fluorescing area increased with the duration of exposure to NIR irradiation and range of concentration. These results suggest that the activity was time- and dose-dependent and is in good agreement with those of the MTT assay.


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Figure 7. In vitro photothermal ablation of tumor cells using a Live/Dead staining method. (A) Effect of various samples with or without NIR laser exposure, (B) Effect of exposure time, (C) Effect of graphene oxide (GO) concentration. Live cells stained green by calceinAM and dead cells stained red by ethidium homodimer.

The in vitro cytotoxicity and thermal ablation studies clearly demonstrated that only thermal therapy is not enough for perfect treatment due to limited exposure time. Thus, chemotherapy should be applied to ensure the desired outcome. For that purpose, apoptosisrelated cellular- and molecular-level details were investigated. In this study, Hoechst 33324 staining of nuclei was observed after treatments with free single drug, dual drugs, and GO-DI. As shown in Figure 8, both the nuclei and the cytoplasm of the control cells homogeneously stained while those of the cells treated with the other treatments exhibited chromatin



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condensation and formation of apoptosis bodies, which are typical features of drug-induced apoptosis.32 The combination of drugs induced more cell apoptosis than that induced by a single drug as well as by GO-DI, and with NIR laser irradiation, the number of apoptotic cells increased.

Figure 8. Cell apoptosis observed using Hoechst 33342 staining. Cancer cells were treated with free doxorubicin (DOX), irinotecan (IRI), combination of DOX+IRI, and GO-DI with and without NIR laser exposure.

Western blotting assay For a deeper investigation relevant to factors of tumor cell apoptosis after treatment, western blot analysis of p53, p27, and p21 proteins from the tumor cells was performed (Figure 9). The augmentation of p53 protein expression can be attributed to the promotion of cell apoptosis.33 On another hand, the free drugs and the formulation induced the expression of the cyclin-dependent kinase inhibitors p21 and p27, which are well-known regulators of cell cycle progression in the G1 and S phases.34-35 Importantly, the efficacy of monotherapy was lower than that of the combination treatment.


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Figure 9. Protein quantification of p53, p27, and p21 expression in SCC-7, MCF-7, and MDA-MB231 cells by western blotting after 24 h of treatment at equivalent drug concentration of 1 µg/mL. 1. Control, 2. Free irinotecan (IRI), 3. Free doxorubicin (DOX), 4. doxorubicin and irinotecan-loaded graphene oxide (GO-DI), 5. Combination of DOX+IRI.

CONCLUSIONS In conclusion, for the first time, GO-DI was successfully formulated, which can combine photothermal therapy with dual chemotherapies in a system. GO-DI demonstrated a high loading efficiency and exhibited pH-responsive DOX/IRI release. The in vitro cell experiments indicated that the synergistic therapy was highly efficient for cancer therapy, showing significant suppression of cell viability. The cell nucleus showed significant morphological changes and apoptosis upon carrier exposure, especially under NIR light irradiation. GO-DI induced cell apoptosis through up-regulation of p53, p27, and p21 due to the efficient combination of the drugs as well as drugs and GO under the NIR exposure condition. Our results suggested that GO-DI composite vesicles could be a powerful tool for drug delivery to achieve improved therapeutic efficacy and overcome drug resistance in combined chemo-photothermal therapy.



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ACKNOWLEDGMENTS This research was supported by the National Research Foundation of Korea (NRF) grant funded

by

the

Korea

government

(MSIP)

(No.

2015R1A2A2A01004118,

2015R1A2A2A04004806). This work was also supported by the Medical Research Center Program (2015R1A5A2009124) through the NRF funded by MSIP.


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Abstract graphic


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Development of a Graphene Oxide Nanocarrier for Dual-Drug Chemo

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