Photochemistry and Photobiology, 20**, **: *–*
Gold Nanorod-Assembled PEGylated Graphene-Oxide Nanocomposites for Photothermal Cancer Therapy Uuriintuya Dembereldorj†1, Seon Young Choi†2, Erdene-Ochir Ganbold1, Nam Woong Song3, Doseok Kim4, Jaebum Choo5, So Yeong Lee*2, Sehun Kim6 and Sang-Woo Joo*1 1
Department of Chemistry, Soongsil University, Seoul, Korea Laboratory of Pharmacology, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul, Korea 3 Korea Research Institute of Standards and Science, Daejeon, Korea 4 Department of Physics, Sogang University, Seoul, Korea 5 Department of Bionano Engineering, Hanyang University, Ansan, South Korea 6 Molecular-level Interface Research Center and Department of Chemistry, KAIST, Daejeon, Korea 2
Received 10 June 2013, accepted 4 November 2013, DOI: 10.1111/php.12212
ABSTRACT
Gold nanorods (AuNR) have been introduced previously in cancer diagnostics and therapy because of their strong near-infrared absorption (10). Near-infrared light-absorbing multimodal nanoparticles including AuNR are useful for cancer theranostics (11). AuNR may offer promising applications as a useful platform for targeted drug/gene delivery, cellular imaging, in vivo biosensing, biomedical diagnostics and cancer therapy (12). Photothermalization based on the strong absorption of AuNR can be an effective cancer therapy as indicated recently by Dreaden et al. (13). One of the disadvantages, however, is the high toxicity of cetyltrimethylammonium bromide (CTAB) in AuNR (14–16). Similar to AuNR, graphene oxide (GO) have also been introduced in biomedical applications (6) and photothermal therapies (7,8) because of their optical properties including near-infrared absorption. In situ fabrication methods of hybrids based on graphene materials and nanoparticles could be achieved through self-assembly processes (17). Electrostatic interactions between the oppositely charged AuNR and GO nanocomposites have been recently utilized for the colorimetric detection (18) and control of optical plasmonic resonance (19). The assembly of AuNR into GO via their electrostatic interactions is demonstrated and utilized as a novel surface-enhanced Raman scattering substrate (20). Despite previous photothermal studies of AuNR and graphenebased materials, administration of a combination therapy using these two platforms has not been reported yet. There may be several advantages of using nanocomposites of AuNR and PEG-GO: (1) high cytotoxicity of AuNR may be alleviated by encapsulating them with GO and removing CTAB, (2) both AuNR and PEGGO play an effective role in the delivery of either genes or drugs and (3) efficient bioimaging methods are applicable using both AuNR and GO. In this work, we report a photothermal cancer therapy using AuNR-PEG-GO to reduce cancer cell viabilities in the presence of light both in vitro and in vivo.
Gold nanorod-attached PEGylated graphene-oxide (AuNRPEG-GO) nanocomposites were tested for a photothermal platform both in vitro and in vivo. Cytotoxicity of AuNR was reduced after encapsulation with PEG-GO along with the removal of cetyltrimethylammonium bromide (CTAB) from AuNR by HCl treatment. Cellular internalization of the CTAB-eliminated AuNR-PEG-GO nanocomposites was examined using dark-field microscopy (DFM), confocal Raman microscopy and transmission electron microscopy (TEM). To determine the photothermal effect of the AuNR-PEG-GO nanocomposites, A431 epidermoid carcinoma cells were irradiated with Xe-lamp light (60 W cm 2) for 5 min after treatment with the AuNR-PEG-GO nanocomposites for 24 h. Cell viability significantly decreased by ~40% when the AuNR-PEGGO-encapsulated nanocomposites were irradiated with light as compared with the cells treated with only the AuNR-PEG-GO nanocomposites without any illumination. In vivo tumor experiments also indicated that HCl-treated AuNR-PEG-GO nanocomposites might efficiently reduce tumor volumes via photothermal processes. Our graphene and AuNR nanocomposites will be useful for an effective photothermal therapy.
INTRODUCTION Photothermal therapy has been proposed as a potential approach for the treatment of cancers, including that of solid tumors (1,2). Because of its unique two-dimensional physicochemical characteristics, graphene has attracted enormous research attention over the past decade (3). Recently, there has been much research activity regarding the fabrication of graphene and its potential applications (4–6). Among their numerous practical implications, graphene-based materials have recently emerged as a novel platform for effective photothermal cancer therapy (7–9).
MATERIALS AND METHODS Fabrication of CTAB-reduced AuNR-PEG-GO composites. PEG-GO molecules were synthesized from expandable graphitic flakes (Alfa Aesar) by referring to the previous studies (21–23). 6-Arm polyetheyleneglycol amine (Sunbio Inc., P6AM-15) was used to introduce PEGylation onto GO surfaces.
*Corresponding authors email: [email protected] (S.Y. Lee); [email protected] (S.W. Joo) † These authors contributed equally to this work. © 2013 The American Society of Photobiology
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AuNR were prepared via the previously reported method (24). In a typical procedure, 250 lL of an aqueous 0.01 M solution of gold (III) chloride trihydrate (≥99.9%, Sigma Aldrich) was added to 7.5 ml of a 0.10 M aqueous CTAB (≥99%, Sigma Aldrich) solution in a test tube to prepare the seed solution. Subsequently, 600 lL of an aqueous 0.01 M ice-cold NaBH4 (≥99%, Sigma Aldrich) solution was added, and the mixture solution was allowed to undergo rapid inversion mixing for 2 min. Then, the test tube was kept in a water bath maintained at 25°C for 2 h. The prepared seed solution was stored at room temperature for future use. We prepared gold particles at a CTAB concentration of 9.5 9 10 2 M. In a typical experiment, 4.75 mL of 0.10 M CTAB, 200 lL of 0.01 M gold (III) chloride trihydrate and 30 lL of 0.01 M AgNO3 (>99%, Sigma Aldrich) solutions were added to a test tube sequentially. The solution was subjected to gentle inversion shaking. Then, 32 lL of 0.10 M ascorbic acid (reagent grade, Sigma Aldrich) was added to the mixture solution. Finally, 10 lL of the seed solution was added, and the reaction solution was gently mixed for 10 s and left undisturbed for 3 h. HCl-treated AuNR were prepared by adding the HCl solution (10 M) to the AuNR solution until the pH reached 1.4. This solution was stirred at 65°C for 3 h and then centrifuged twice at 10 000 rpm for 30 min. After each round of centrifugation, the pellet was redispersed in deionized water. For AuNR-PEG-GO assembly formation, AuNR (55.2 (1.1) ppm, 200 lL) in water were added to 1 mL of PEG-GO solution. After overnight stirring, the dispersion solution was centrifuged at 5000 rpm for 10 min and washed with 2 mL of water. Then, the obtained AuNR-PEGGO sample was redispersed in 1 mL of water. The prepared sample was filtered by a nonpyrogenic filter (0.2 lm pore size, Sartorius Stedium Ministart, Catalog #16534) once to separate micrometer-sized composites. The absorbance of the composite solutions was measured by a Mecasys 3220 spectrophotometer in the UV-VIS-NIR region. To estimate the Au atomic percentages in the samples, inductively coupled plasma-atomic emission spectroscopy (ICP-AES) measurements were performed using a
Perkin Elmer ICP-730ES instrument. Particle sizes (number distributions) and zeta potentials of the composites were measured using an Otsuka ELS-Z analyzer. Figure 1 shows our experimental scheme. TEM, AFM and DFM/Raman characterizations. The morphologies of HCl-treated AuNR-PEG-GO nanocomposites were examined using a JEOL JEM-3010 transmission electron microscope. Atomic force microscopy (AFM) images were obtained using a Digital Instruments nanoscope. The dark-field microscopy (DFM) images with z-depth Raman spectra were obtained using a Renishaw RM 1000 Raman spectrometer with a Leica DL LM microscope equipped with a Cytovivia high-resolution adapter. Cell culture and CCK-8-based viability. A431 (epidermoid carcinoma, ATCC; CRL-2592) cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% antibiotics. The A431 cells were maintained at 37°C with 5% CO2. For the CCK-8 assay, the cells were seeded in 96-well plates at a concentration of 1 9 104 cells per well. After treatment with the composites, the cells were incubated for 24 h and subsequently irradiated using the Xe light lamp. Then, 20 lL of CCK-8 reagent (Dojindo, CK04-11, Japan) was added to each well and incubated at 37°C for 2 h. Absorbance at 450 nm was determined using a plate reader (Tecan, Infinite F50, Switzerland). The uptake of AuNR-PEG-GO nanocomposites was examined using a JEOL JEM-1010 transmission electron microscope. The A431 cells were plated into a 100-mm culture dish (SPL, Korea) at a density of 2 9 106 cells/dish containing a growth medium and incubated at 37°C. The AuNR were then added and the cells were incubated at 37°C with 5% CO2. After 24 h, the cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) and fixed with Karnovsky’s fixative. After fixation, the specimens were dehydrated in a graded series of ethanol concentrations. The samples were then embedded in a mixture of resin at 60°C. Ultrathin sections were prepared for transmission electron microscopy (TEM) measurements using a diamond knife. Karnovsky’s fixative, 0.05 M sodium carcodylated buffer, 1% osmium tetroxide, 0.5% uranyl
Figure 1. Experimental scheme for photothermal therapy. Partial removal of cetyltrimethylammonium bromide is illustrated to alleviate the toxicity problems.
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acetate, propylene oxide and Spurr’s resin were obtained from the National Instrumentation Center for Environmental Management at Seoul National University, Korea. Xenograft tumor mouse model. Six-week-old male CAnN.Cg-Foxn1nu/ CrijOri nude mice were purchased from Orientbio (Seongnam, Korea) and maintained under controlled temperature (23°C) and humidity (50%) conditions. The animals were allowed access to food and sterile water. A431 cells (0.1 mL) were injected subcutaneously at the back of the nude mice at a concentration of 1.3 9 107 cells/mL to establish a model of tumor-bearing mice. The mice were divided into three groups of Group 1 (DPBS, control), Group 2 (HCl-treated AuNR-PEG-GO composites) and Group 3 (after injection of HCl-treated AuNR-PEG-GO nanocomposites, irradiated with Xe-lamp light for 5 min). AuNR-PEG-GO nanocomposites were administered into their tumors. Tumor sizes, using a caliper, were determined using the following formula: =volume = p/6 9 ([tumor length] 9 [tumor width] 9 [tumor height]) (25). The prepared AuNR-PEG-GO nanocomposites were injected into each tumor to fill as much as 1/10th the volume of the tumor (final concentration was approximately 0.1 ppm). An in vivo irradiation experiment was performed with approximately 60 W cm 2 irradiation for 5 min using a Eurosep spot.il.5180 fiber optic illuminator (Christophe, France). All animal experiments were performed in compliance with the guidelines of the Institute of Laboratory Animal Resources, Seoul National University.
RESULTS Preparation of HCl-treated AuNR and PEG-GO Figure 2a,b show the TEM images of PEG-GO and AuNR before the assembly. PEG-GO appeared to have their submicrometer size to be eligible for the intracellular delivery platform, as recently reported (8). The crystal structure of GO molecules was checked by X-ray diffraction (XRD). The feature diffraction peak of the exfoliated GO, which appears at 10.4° (002), is observed with an interlay space (d-spacing) of 0.76 nm (23). The XRD pattern indicated the graphene-oxide pattern. The Raman spectrum of graphite shows the in-phase vibration of the graphite lattice at 1600 cm 1 (G band) and a D band at 1330 cm 1. The infrared spectrum indicated the formation of the C–O band after PEGylation onto GO molecules. The TEM images of PEG-GO molecules showed a size distribution of approximately 100 nm. The long- and short-axis lengths of AuNR molecules were measured to be 39.3 7.1 and 10.0 2.4 nm, respectively, from the TEM images. The surface charges are summarized in Table 1. Because of their electrostatic interactions, AuNR molecules can be attached efficiently onto the surfaces of PEG-GO molecules. Fig. 2c shows the absorption spectrum of the AuNRPEG-GO molecules. The absorption bands of the transverse and longitudinal modes appeared at 520 and 760 nm, respectively. These positions indicate that the aspect ratio was 4~5, which is consistent with the TEM image. The absorption spectra of the PEG-GO molecules showed substantial light absorption in the wavelength region of AuNR absorption. A hundred of the AuNR molecules can assemble on PEG-GO molecules by considering their sizes. The UV-vis absorption spectrum of the composite indicated the assembly of the AuNR molecules onto the PEG-GO surface. The UV–vis spectrum of PEG-GO molecules illustrated decreasing absorbance as the wavelength increased, whereas that of AuNR molecules exhibited the two prominent transverse and longitude modes. The relative intensities of the longitude mode with respect to that of the transversal mode for the AuNR-PEG-GO assembly decreased because of the high absorbance of the PEG-GO molecules at the wavelength region of the transverse-mode peak at 520 nm. The
Figure 2. Transmission electron microscopy images of (a) the PEG-GO molecules and (b) the HCl-treated AuNR. The scale bars are 1 lm and 20 nm, respectively. (c) UV–vis-NIR spectra of the PEG-GO molecules, AuNR and their assembled composites of AuNR-PEG-GO. Table 1. Surface potential measurements. Numbers in parenthesis are the standard deviations for repeated measurements. Zeta potential (mV) AuNR
PEG-GO
43.5 (0.2)
20.8 (2.3)
AuNR- PEG-GO 24.8 (0.6)
TEM images in Fig. 3a clearly exhibit the AuNR-loaded PEGGO molecules. ICP-AES measurements indicated that the amount of Au in AuNR was 55.2 (1.1) ppm. After the loading of AuNR molecules onto the PEG-GO molecules, the amount of Au was measured and found to be 4.7 ppm. Fabrication of PEG-GO molecules and AuNR As the surface charges of the PEG-GO molecules and AuNR were 20.8 and 43.5 mV, respectively, the driving force of the AuNR-PEG-GO nanocomposites could be considered to be electrostatic interactions. The TEM images of the AuNR-PEG-GO composites showed the loading of the AuNR onto PEG-GO molecules. The AuNR adsorbed onto the PEG-GO molecules are shown in the TEM images (Fig. 3a). We observed numerous AuNR in the GO sheets. The AFM images also supported the observation that AuNR are embedded in the PEG-GO surfaces (Fig. 3b). In addition, the AFM scanned images revealed protruded regions corresponding to the AuNR. The AuNR-PEG-GO
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Figure 3. (a) Transmission electron microscopy images of the AuNRPEG-GO composites. An enlargement of the AuNR-PEG-GO composites is shown for a better demonstration of the assembly. The scale bars are 200 nm. The inset figure with the 5 nm scale bar indicates an attached single gold nanorod. (b) AFM image of AuNR-PEG-GO composites. The black straight line in the left figure shows where the section analysis was performed, indicating a height greater than 10 nm for the AuNR attached onto the surfaces of the PEG-GO molecules. The units in the horizontal and vertical axes are lm and nm, respectively.
samples were passed through syringe filters to treat cancer cells by eliminating large particles.
Figure 4. (a) Dark-field microscopic images and confocal micro-Raman spectra of the AuNR-PEG-GO composites. The spectra were taken at the position marked with the red arrow. The inset numbers are the relative z-depth positions in lm. As the three-dimensional information was necessary to estimate the localization of the nanocomposites inside the cell, we performed the z-depth-dependent Raman spectroscopy for the position marked by the arrow in the range 0–15 lm. The number “0” indicates the original starting position in which the DFM image is taken. The Raman spectra were obtained sequentially with the distance intervals of ~2 lm. The “-” sign refers to the inward direction inside the cell. (b) Uptake of AuNR-PEG-GO composites. A431 cells were exposed to AuNR-PEG-GO composites for 24 h. The white arrows indicate the location of AuNR-PEG-GO composites. (A) AuNR-PEG-GO composites internalized by the cells. (B) High-magnification transmission electron microscopy image. The scale bars in (A) and (B) are 2 and 100 nm, respectively.
Internalization of AuNR-PEG-GO composites in A431 cells To check for endocytosis, we applied DFM and Raman spectroscopy. The internalization of the 3,3′-diethylthiatricarbocyanine dye-coated AuNR in cancer cells was checked by Raman spectroscopy. This indicated that AuNR could be a useful platform using the Raman dye (Supporting Information Figure S1). For our sample of AuNR-PEG-GO composites, Raman spectroscopy could be applied to monitor the intracellular localization without using any additional Raman dyes. Similar to previous studies (12,20), we identified the location of the internalized AuNRPEG-GO composites in the DFM images. After detecting the AuNR in the A431 cells, we performed z-depth Raman spectroscopy on the internalized AuNR-PEG-GO composites. We monitored the D and G bands of the PEG-GO molecules to confirm that the AuNR-PEG-GO composites were actually endocytosed in the A431 cells. As shown in Fig. 4a, the D and G bands of the PEG-GO molecules were found at 1335 and 1610 cm 1, respectively. The AuNR-PEG-GO composites were well internalized in the mammalian cancer cells. The TEM images also indicated the internalization of the AuNR-PEG-GO composites in A431 cells, as shown in Fig. 4b.
Our DFM images may provide only the differences between nuclei and cytosols. Additional fluorescence measurements would be beneficial to understand the intracellular localization by applying the organelle markers. In vitro photothermal effect A cellular toxicity assay of the A431 cells indicated that the toxicity of the AuNR was reduced after their assembly onto the PEG-GO molecules. CCK-8 cell viability tests demonstrated that the toxicity of the HCl-treated AuNR-PEG-GO composites (0.1 ppm) was relatively low (Fig. 5a). As shown in Fig. 5b, cell viability decreased in a concentration-dependent manner after 24 h of incubation. We found that cell viability was not significantly affected at concentrations up to 0.3 ppm, whereas it significantly reduced at a concentration range from 1 to 2 ppm. Cell viability was not significantly reduced by irradiation using a Xe-lamp under the given experimental conditions (Fig. 5c). To determine the photothermal effect of HCl-treated AuNR-PEGGO composites, the A431 cells were irradiated with Xe-lamp
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Figure 5. (a) In vitro cytotoxic assays of the HCl-treated AuNR-PEG-GO composites. Because of the decrease in the surface potential values of AuNRPEG-GO composites, the toxicity of AuNR could be reduced assembly on PEG-GO molecules. Removal of the cetyltrimethylammonium bromide layers in AuNR by HCl treatment further reduced cytotoxicity. (b) Decrease in cell viability in a concentration-dependent manner. (c) No significant reduction in cell viability after Xe-lamp irradiation for up to 20 min. (d) Composite materials demonstrating a synergistic photothermal action. Cell viability was significantly decreased by ~40% in the irradiated HCl-treated AuNR-PEG-GO–incubated cells compared with those treated with only HCl-treated AuNR-PEG-GO composites in the absence of light exposure. The “*” indicates P < 0.05. The irradiation of the cells was performed under ambient conditions.
light for 5 min after treatment with HCl-treated AuNR-PEG-GO composites. As shown in Fig. 5d, cell viability of the irradiated HCl-treated AuNR-PEG-GO–incubated cells significantly decreased by ~40% compared with either the cells treated with only free HCl-treated AuNR-PEG-GO composites or those not exposed to the light. These results suggest that HCl-treated AuNR-PEG-GO composites have a photothermal effect on A431 carcinoma cells. These results also indicate that the photothermal effect on these carcinoma cells was an in vitro synergistic effect of the light and the endocytosis of the composite because only irradiation for 5 min or endocytosis of the composite did not reduce cell viability as significantly compared with their combined treatment. In vivo photothermal effect To investigate the photothermal effect of HCl-treated AuNRPEG-GO composites in vivo, we prepared xenograft models of the nude nice. The A431 tumor-bearing mice were divided into three groups. As shown in Fig. 6, the tumor size in mice treated
with NRs and light (n = 4) was smaller than that in the control group (treated with DPBS, n = 6) or in the NR group (treated with only AuNR-PEG-GO composites, n = 7). We also measured the change in body weight during the treatments (Fig. 6b). We found no significant change in body weight among the groups. Fig. 6c shows the changes in relative tumor volume during the treatment. The DPBS-treated mice demonstrated rapid increase in tumor volume. Slight reduction in tumor volumes in the absence of light may be because of the toxic CTAB molecules remaining in the tumor areas (26). However, in the mice treated with the composite and Xe-lamp light for 5 min, tumor volume reduced more significantly. Our results reveal that the AuNR-PEG-GO composites have a potential photothermal effect on cancer in vivo as well.
DISCUSSION The AuNR-PEG-GO composites may have advantages with respect to either drug loading or gene delivery because of the
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(a)
(b)
(c)
Figure 6. In vivo tumor growth reduction. (a) Representative tumor tissue images of nude mice. The scale bar is 10 mm. (b) Mean body weights of different groups after treatment. (c) Tumor growth curves of different groups after treatment. The tumor volumes were normalized to their initial sizes. All data are represented as mean SE. All the AuNR were treated with HCl to reduce the cetyltrimethylammonium bromide layers. The tumors were irradiated with Xe-lamp light with an optical fiber unit every other day.
binary nature of the composite. We have incorporated doxorubicin (DOX) drugs on PEG-GO surfaces by means of p–p interactions (23). Due to the fact that DOX cannot directly bind onto Au surfaces via a chemical linkage, the composite nature of the AuNR-PEG-GO sample has several benefits in terms of assembling anticancer drugs consisting of aromatic rings. In addition, the positive charge of the composites is expected to bind easily to negatively charged oligonucleotides to lead to an efficient gene delivery system. As AuNR have been used for gene delivery, AuNR-PEG-GO composites can be used in assembling either DNA or RNA molecules for therapeutic purposes. The large GO sheets in lm size range could be eliminated after filtration. Data using other cell lines, such as A549 and MCF-7/ADR cells, exhibited similar behaviors to those of A431 cells. A431 skin carcinoma cells were chosen for efficient
xenografted growth and in vivo photothermal test under our experimental conditions. The composite properties of AuNR-PEG-GO will also be helpful in introducing appropriate functional groups to target specific cancer cells. It is possible that folate can be assembled with the carboxylate group on the surface of PEG-GO molecules to target folate-positive cancer cells (27). The AuNR surfaces can also be a good platform through which proteins and other molecules can be linked to target specific cancer cells after inserting the appropriate cross-linkers (28). Our future research direction is to develop a more versatile tool for effective cancer therapy by taking advantage of dual platforms offered by nanocomposites. It should also be noted that the Raman peaks of the D and G modes of the PEG-GO molecules could also be observed by means of confocal micro-Raman spectroscopy. Our methods
Photochemistry and Photobiology have advantages in imaging of internalized graphene-based AuNR-PEG-GO cargos inside cells, as we were unable to find carbon-based materials only by dark-field microscopy and Raman spectroscopy without AuNR. Indeed, AuNR may provide useful information on the internal location of the AuNR-PEG-GO composites inside mammalian cells. On the other hand, we could not observe the graphene structures inside cancer cells only by the TEM method. By identifying the AuNR, we could estimate the intracellular locations of carbon-based AuNR-PEG-GO composites. The assembly of AuNR-PEG-GO composites could thus provide a convenient method for imaging the cells, as only PEGGO molecules are not easily discernible inside cancer cells. As illustrated in Fig. 5c,d, the AuNR-PEG-GO composites played a significant role in reducing cancer cell viability compared with the Xe-lamp-irradiated sample. In vitro and in vivo photothermal cancer therapy was performed by treating the A431 carcinoma cells with the AuNR-assembled PEG-GO composites. A cell viability assay was also performed to determine the cytotoxicity of AuNR-PEG-GO composites. In vitro toxicity test was performed to determine the optical irradiation conditions of the photothermal therapy using AuNR-PEG-GO composites. By irradiating the Xe-lamp light on the nanocomposites, photothermal therapy was achieved with the AuNR assembled onto the PEG-GO molecules. In vivo tumor growth measurements also indicated that the AuNR-assembled PEG-GO composites with irradiation might efficiently reduce the tumor volume. We also performed a light control experiment using xenograft mice and found significant reduction in tumor volume as shown in Fig. 6c. This reduction is presumably because of direct exposure of the mice skin to light in the in vivo model. In the in vivo experiment, direct exposure of the skin to light resulted in temperature increase, whereas the cell culture media solution prevented direct exposure to the light in the in vitro experiment. Despite this performance in vivo, our in vitro data clearly indicate the potential photothermal effects of AuNR-PEG-GO nanocomposites. We plan to find optimal conditions for in vivo experiments. In conclusion, we demonstrated AuNR-PEG-GO nanocomposites to be the photothermal platform using A431 cells. Our graphene– AuNR nanocomposites would be a potentially useful platform for future possibilities of photothermal therapy. Acknowledgements—The National Research Foundation of Korea supported this work through grant numbers R11-2008-0061852 and K20904000004-12A0500-00410. The Nano Material Technology Development Program also supported this work, through the National Research Foundation of Korea, funded by the Ministry of Education, Science, and Technology (grant number 2012035286). This research was also partially supported by the Agency for Defense Development through Chemical and Biological Defense Research Center and the Development of Characterization Techniques for Nano-materials Safety Project of KRCF. This work was supported by the National Research Foundation (NRF) grant funded by the Korea Government (MEST) nos. 20110017435 and the Korea government [MSIP] [no. 20090083525].
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. (a) DFM images of AuNRs in the other A549 cancer cells. AuNRs appeared to be more conspicuous than the case of A431 cells. (b) Surface-enhanced Raman spectra of NIR dye
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of 3,3′-diethylthiatricarbocyanine (DPPC) at 633 and 785 nm. (c) DFM and Raman spectra of the NIR dye DPPC on AuNRs in cancer cells. The yellowish colored AuNRs are clearly discernible in the DFM images.
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25. Tomayko, M. M. and C. P. Reynolds (1989) Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother. Pharmacol. 24, 148–154. 26. Choi, W. I., J. Y. Kim, C. Kang, C. C. Byeon, Y. H. Kim and G. Tae (2011) Tumor regression in vivo by photothermal therapy based on gold-nanorod-loaded, functional nanocarriers. ACS Nano 5, 1995–2003. 27. Zhang, L., J. Xia, Q. Zhao, L. Liu and Z. Zhang (2010) Functional graphene oxide as a nanocarrier for controlled loading and targeted delivery of mixed anticancer drugs. Small 22, 537–544. 28. Park, J.-H., J. Park, U. Dembereldorj, K. Cho, K. Lee, S. I. Yang, S. Y. Lee and S.-W. Joo (2011) Raman detection of localized transferrin-coated gold nanoparticles inside a single cell. Anal. Bioanal. Chem. 401, 1635–1643.
Gold Nanorod-Assembled PEGylated Graphene-Oxide Nanocomposites for Photothermal Cancer Therapy Uuriintuya Dembereldorj†1, Seon Young Choi†2, Erdene-Ochir Ganbold1, Nam Woong Song3, Doseok Kim4, Jaebum Choo5, So Yeong Lee*2, Sehun Kim6 and Sang-Woo Joo*1 1
Department of Chemistry, Soongsil University, Seoul, Korea Laboratory of Pharmacology, College of Veterinary Medicine and Research Institute for Veterinary Science, Seoul National University, Seoul, Korea 3 Korea Research Institute of Standards and Science, Daejeon, Korea 4 Department of Physics, Sogang University, Seoul, Korea 5 Department of Bionano Engineering, Hanyang University, Ansan, South Korea 6 Molecular-level Interface Research Center and Department of Chemistry, KAIST, Daejeon, Korea 2
Received 10 June 2013, accepted 4 November 2013, DOI: 10.1111/php.12212
ABSTRACT
Gold nanorods (AuNR) have been introduced previously in cancer diagnostics and therapy because of their strong near-infrared absorption (10). Near-infrared light-absorbing multimodal nanoparticles including AuNR are useful for cancer theranostics (11). AuNR may offer promising applications as a useful platform for targeted drug/gene delivery, cellular imaging, in vivo biosensing, biomedical diagnostics and cancer therapy (12). Photothermalization based on the strong absorption of AuNR can be an effective cancer therapy as indicated recently by Dreaden et al. (13). One of the disadvantages, however, is the high toxicity of cetyltrimethylammonium bromide (CTAB) in AuNR (14–16). Similar to AuNR, graphene oxide (GO) have also been introduced in biomedical applications (6) and photothermal therapies (7,8) because of their optical properties including near-infrared absorption. In situ fabrication methods of hybrids based on graphene materials and nanoparticles could be achieved through self-assembly processes (17). Electrostatic interactions between the oppositely charged AuNR and GO nanocomposites have been recently utilized for the colorimetric detection (18) and control of optical plasmonic resonance (19). The assembly of AuNR into GO via their electrostatic interactions is demonstrated and utilized as a novel surface-enhanced Raman scattering substrate (20). Despite previous photothermal studies of AuNR and graphenebased materials, administration of a combination therapy using these two platforms has not been reported yet. There may be several advantages of using nanocomposites of AuNR and PEG-GO: (1) high cytotoxicity of AuNR may be alleviated by encapsulating them with GO and removing CTAB, (2) both AuNR and PEGGO play an effective role in the delivery of either genes or drugs and (3) efficient bioimaging methods are applicable using both AuNR and GO. In this work, we report a photothermal cancer therapy using AuNR-PEG-GO to reduce cancer cell viabilities in the presence of light both in vitro and in vivo.
Gold nanorod-attached PEGylated graphene-oxide (AuNRPEG-GO) nanocomposites were tested for a photothermal platform both in vitro and in vivo. Cytotoxicity of AuNR was reduced after encapsulation with PEG-GO along with the removal of cetyltrimethylammonium bromide (CTAB) from AuNR by HCl treatment. Cellular internalization of the CTAB-eliminated AuNR-PEG-GO nanocomposites was examined using dark-field microscopy (DFM), confocal Raman microscopy and transmission electron microscopy (TEM). To determine the photothermal effect of the AuNR-PEG-GO nanocomposites, A431 epidermoid carcinoma cells were irradiated with Xe-lamp light (60 W cm 2) for 5 min after treatment with the AuNR-PEG-GO nanocomposites for 24 h. Cell viability significantly decreased by ~40% when the AuNR-PEGGO-encapsulated nanocomposites were irradiated with light as compared with the cells treated with only the AuNR-PEG-GO nanocomposites without any illumination. In vivo tumor experiments also indicated that HCl-treated AuNR-PEG-GO nanocomposites might efficiently reduce tumor volumes via photothermal processes. Our graphene and AuNR nanocomposites will be useful for an effective photothermal therapy.
INTRODUCTION Photothermal therapy has been proposed as a potential approach for the treatment of cancers, including that of solid tumors (1,2). Because of its unique two-dimensional physicochemical characteristics, graphene has attracted enormous research attention over the past decade (3). Recently, there has been much research activity regarding the fabrication of graphene and its potential applications (4–6). Among their numerous practical implications, graphene-based materials have recently emerged as a novel platform for effective photothermal cancer therapy (7–9).
MATERIALS AND METHODS Fabrication of CTAB-reduced AuNR-PEG-GO composites. PEG-GO molecules were synthesized from expandable graphitic flakes (Alfa Aesar) by referring to the previous studies (21–23). 6-Arm polyetheyleneglycol amine (Sunbio Inc., P6AM-15) was used to introduce PEGylation onto GO surfaces.
*Corresponding authors email: [email protected] (S.Y. Lee); [email protected] (S.W. Joo) † These authors contributed equally to this work. © 2013 The American Society of Photobiology
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AuNR were prepared via the previously reported method (24). In a typical procedure, 250 lL of an aqueous 0.01 M solution of gold (III) chloride trihydrate (≥99.9%, Sigma Aldrich) was added to 7.5 ml of a 0.10 M aqueous CTAB (≥99%, Sigma Aldrich) solution in a test tube to prepare the seed solution. Subsequently, 600 lL of an aqueous 0.01 M ice-cold NaBH4 (≥99%, Sigma Aldrich) solution was added, and the mixture solution was allowed to undergo rapid inversion mixing for 2 min. Then, the test tube was kept in a water bath maintained at 25°C for 2 h. The prepared seed solution was stored at room temperature for future use. We prepared gold particles at a CTAB concentration of 9.5 9 10 2 M. In a typical experiment, 4.75 mL of 0.10 M CTAB, 200 lL of 0.01 M gold (III) chloride trihydrate and 30 lL of 0.01 M AgNO3 (>99%, Sigma Aldrich) solutions were added to a test tube sequentially. The solution was subjected to gentle inversion shaking. Then, 32 lL of 0.10 M ascorbic acid (reagent grade, Sigma Aldrich) was added to the mixture solution. Finally, 10 lL of the seed solution was added, and the reaction solution was gently mixed for 10 s and left undisturbed for 3 h. HCl-treated AuNR were prepared by adding the HCl solution (10 M) to the AuNR solution until the pH reached 1.4. This solution was stirred at 65°C for 3 h and then centrifuged twice at 10 000 rpm for 30 min. After each round of centrifugation, the pellet was redispersed in deionized water. For AuNR-PEG-GO assembly formation, AuNR (55.2 (1.1) ppm, 200 lL) in water were added to 1 mL of PEG-GO solution. After overnight stirring, the dispersion solution was centrifuged at 5000 rpm for 10 min and washed with 2 mL of water. Then, the obtained AuNR-PEGGO sample was redispersed in 1 mL of water. The prepared sample was filtered by a nonpyrogenic filter (0.2 lm pore size, Sartorius Stedium Ministart, Catalog #16534) once to separate micrometer-sized composites. The absorbance of the composite solutions was measured by a Mecasys 3220 spectrophotometer in the UV-VIS-NIR region. To estimate the Au atomic percentages in the samples, inductively coupled plasma-atomic emission spectroscopy (ICP-AES) measurements were performed using a
Perkin Elmer ICP-730ES instrument. Particle sizes (number distributions) and zeta potentials of the composites were measured using an Otsuka ELS-Z analyzer. Figure 1 shows our experimental scheme. TEM, AFM and DFM/Raman characterizations. The morphologies of HCl-treated AuNR-PEG-GO nanocomposites were examined using a JEOL JEM-3010 transmission electron microscope. Atomic force microscopy (AFM) images were obtained using a Digital Instruments nanoscope. The dark-field microscopy (DFM) images with z-depth Raman spectra were obtained using a Renishaw RM 1000 Raman spectrometer with a Leica DL LM microscope equipped with a Cytovivia high-resolution adapter. Cell culture and CCK-8-based viability. A431 (epidermoid carcinoma, ATCC; CRL-2592) cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% antibiotics. The A431 cells were maintained at 37°C with 5% CO2. For the CCK-8 assay, the cells were seeded in 96-well plates at a concentration of 1 9 104 cells per well. After treatment with the composites, the cells were incubated for 24 h and subsequently irradiated using the Xe light lamp. Then, 20 lL of CCK-8 reagent (Dojindo, CK04-11, Japan) was added to each well and incubated at 37°C for 2 h. Absorbance at 450 nm was determined using a plate reader (Tecan, Infinite F50, Switzerland). The uptake of AuNR-PEG-GO nanocomposites was examined using a JEOL JEM-1010 transmission electron microscope. The A431 cells were plated into a 100-mm culture dish (SPL, Korea) at a density of 2 9 106 cells/dish containing a growth medium and incubated at 37°C. The AuNR were then added and the cells were incubated at 37°C with 5% CO2. After 24 h, the cells were washed with Dulbecco’s phosphate-buffered saline (DPBS) and fixed with Karnovsky’s fixative. After fixation, the specimens were dehydrated in a graded series of ethanol concentrations. The samples were then embedded in a mixture of resin at 60°C. Ultrathin sections were prepared for transmission electron microscopy (TEM) measurements using a diamond knife. Karnovsky’s fixative, 0.05 M sodium carcodylated buffer, 1% osmium tetroxide, 0.5% uranyl
Figure 1. Experimental scheme for photothermal therapy. Partial removal of cetyltrimethylammonium bromide is illustrated to alleviate the toxicity problems.
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acetate, propylene oxide and Spurr’s resin were obtained from the National Instrumentation Center for Environmental Management at Seoul National University, Korea. Xenograft tumor mouse model. Six-week-old male CAnN.Cg-Foxn1nu/ CrijOri nude mice were purchased from Orientbio (Seongnam, Korea) and maintained under controlled temperature (23°C) and humidity (50%) conditions. The animals were allowed access to food and sterile water. A431 cells (0.1 mL) were injected subcutaneously at the back of the nude mice at a concentration of 1.3 9 107 cells/mL to establish a model of tumor-bearing mice. The mice were divided into three groups of Group 1 (DPBS, control), Group 2 (HCl-treated AuNR-PEG-GO composites) and Group 3 (after injection of HCl-treated AuNR-PEG-GO nanocomposites, irradiated with Xe-lamp light for 5 min). AuNR-PEG-GO nanocomposites were administered into their tumors. Tumor sizes, using a caliper, were determined using the following formula: =volume = p/6 9 ([tumor length] 9 [tumor width] 9 [tumor height]) (25). The prepared AuNR-PEG-GO nanocomposites were injected into each tumor to fill as much as 1/10th the volume of the tumor (final concentration was approximately 0.1 ppm). An in vivo irradiation experiment was performed with approximately 60 W cm 2 irradiation for 5 min using a Eurosep spot.il.5180 fiber optic illuminator (Christophe, France). All animal experiments were performed in compliance with the guidelines of the Institute of Laboratory Animal Resources, Seoul National University.
RESULTS Preparation of HCl-treated AuNR and PEG-GO Figure 2a,b show the TEM images of PEG-GO and AuNR before the assembly. PEG-GO appeared to have their submicrometer size to be eligible for the intracellular delivery platform, as recently reported (8). The crystal structure of GO molecules was checked by X-ray diffraction (XRD). The feature diffraction peak of the exfoliated GO, which appears at 10.4° (002), is observed with an interlay space (d-spacing) of 0.76 nm (23). The XRD pattern indicated the graphene-oxide pattern. The Raman spectrum of graphite shows the in-phase vibration of the graphite lattice at 1600 cm 1 (G band) and a D band at 1330 cm 1. The infrared spectrum indicated the formation of the C–O band after PEGylation onto GO molecules. The TEM images of PEG-GO molecules showed a size distribution of approximately 100 nm. The long- and short-axis lengths of AuNR molecules were measured to be 39.3 7.1 and 10.0 2.4 nm, respectively, from the TEM images. The surface charges are summarized in Table 1. Because of their electrostatic interactions, AuNR molecules can be attached efficiently onto the surfaces of PEG-GO molecules. Fig. 2c shows the absorption spectrum of the AuNRPEG-GO molecules. The absorption bands of the transverse and longitudinal modes appeared at 520 and 760 nm, respectively. These positions indicate that the aspect ratio was 4~5, which is consistent with the TEM image. The absorption spectra of the PEG-GO molecules showed substantial light absorption in the wavelength region of AuNR absorption. A hundred of the AuNR molecules can assemble on PEG-GO molecules by considering their sizes. The UV-vis absorption spectrum of the composite indicated the assembly of the AuNR molecules onto the PEG-GO surface. The UV–vis spectrum of PEG-GO molecules illustrated decreasing absorbance as the wavelength increased, whereas that of AuNR molecules exhibited the two prominent transverse and longitude modes. The relative intensities of the longitude mode with respect to that of the transversal mode for the AuNR-PEG-GO assembly decreased because of the high absorbance of the PEG-GO molecules at the wavelength region of the transverse-mode peak at 520 nm. The
Figure 2. Transmission electron microscopy images of (a) the PEG-GO molecules and (b) the HCl-treated AuNR. The scale bars are 1 lm and 20 nm, respectively. (c) UV–vis-NIR spectra of the PEG-GO molecules, AuNR and their assembled composites of AuNR-PEG-GO. Table 1. Surface potential measurements. Numbers in parenthesis are the standard deviations for repeated measurements. Zeta potential (mV) AuNR
PEG-GO
43.5 (0.2)
20.8 (2.3)
AuNR- PEG-GO 24.8 (0.6)
TEM images in Fig. 3a clearly exhibit the AuNR-loaded PEGGO molecules. ICP-AES measurements indicated that the amount of Au in AuNR was 55.2 (1.1) ppm. After the loading of AuNR molecules onto the PEG-GO molecules, the amount of Au was measured and found to be 4.7 ppm. Fabrication of PEG-GO molecules and AuNR As the surface charges of the PEG-GO molecules and AuNR were 20.8 and 43.5 mV, respectively, the driving force of the AuNR-PEG-GO nanocomposites could be considered to be electrostatic interactions. The TEM images of the AuNR-PEG-GO composites showed the loading of the AuNR onto PEG-GO molecules. The AuNR adsorbed onto the PEG-GO molecules are shown in the TEM images (Fig. 3a). We observed numerous AuNR in the GO sheets. The AFM images also supported the observation that AuNR are embedded in the PEG-GO surfaces (Fig. 3b). In addition, the AFM scanned images revealed protruded regions corresponding to the AuNR. The AuNR-PEG-GO
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Figure 3. (a) Transmission electron microscopy images of the AuNRPEG-GO composites. An enlargement of the AuNR-PEG-GO composites is shown for a better demonstration of the assembly. The scale bars are 200 nm. The inset figure with the 5 nm scale bar indicates an attached single gold nanorod. (b) AFM image of AuNR-PEG-GO composites. The black straight line in the left figure shows where the section analysis was performed, indicating a height greater than 10 nm for the AuNR attached onto the surfaces of the PEG-GO molecules. The units in the horizontal and vertical axes are lm and nm, respectively.
samples were passed through syringe filters to treat cancer cells by eliminating large particles.
Figure 4. (a) Dark-field microscopic images and confocal micro-Raman spectra of the AuNR-PEG-GO composites. The spectra were taken at the position marked with the red arrow. The inset numbers are the relative z-depth positions in lm. As the three-dimensional information was necessary to estimate the localization of the nanocomposites inside the cell, we performed the z-depth-dependent Raman spectroscopy for the position marked by the arrow in the range 0–15 lm. The number “0” indicates the original starting position in which the DFM image is taken. The Raman spectra were obtained sequentially with the distance intervals of ~2 lm. The “-” sign refers to the inward direction inside the cell. (b) Uptake of AuNR-PEG-GO composites. A431 cells were exposed to AuNR-PEG-GO composites for 24 h. The white arrows indicate the location of AuNR-PEG-GO composites. (A) AuNR-PEG-GO composites internalized by the cells. (B) High-magnification transmission electron microscopy image. The scale bars in (A) and (B) are 2 and 100 nm, respectively.
Internalization of AuNR-PEG-GO composites in A431 cells To check for endocytosis, we applied DFM and Raman spectroscopy. The internalization of the 3,3′-diethylthiatricarbocyanine dye-coated AuNR in cancer cells was checked by Raman spectroscopy. This indicated that AuNR could be a useful platform using the Raman dye (Supporting Information Figure S1). For our sample of AuNR-PEG-GO composites, Raman spectroscopy could be applied to monitor the intracellular localization without using any additional Raman dyes. Similar to previous studies (12,20), we identified the location of the internalized AuNRPEG-GO composites in the DFM images. After detecting the AuNR in the A431 cells, we performed z-depth Raman spectroscopy on the internalized AuNR-PEG-GO composites. We monitored the D and G bands of the PEG-GO molecules to confirm that the AuNR-PEG-GO composites were actually endocytosed in the A431 cells. As shown in Fig. 4a, the D and G bands of the PEG-GO molecules were found at 1335 and 1610 cm 1, respectively. The AuNR-PEG-GO composites were well internalized in the mammalian cancer cells. The TEM images also indicated the internalization of the AuNR-PEG-GO composites in A431 cells, as shown in Fig. 4b.
Our DFM images may provide only the differences between nuclei and cytosols. Additional fluorescence measurements would be beneficial to understand the intracellular localization by applying the organelle markers. In vitro photothermal effect A cellular toxicity assay of the A431 cells indicated that the toxicity of the AuNR was reduced after their assembly onto the PEG-GO molecules. CCK-8 cell viability tests demonstrated that the toxicity of the HCl-treated AuNR-PEG-GO composites (0.1 ppm) was relatively low (Fig. 5a). As shown in Fig. 5b, cell viability decreased in a concentration-dependent manner after 24 h of incubation. We found that cell viability was not significantly affected at concentrations up to 0.3 ppm, whereas it significantly reduced at a concentration range from 1 to 2 ppm. Cell viability was not significantly reduced by irradiation using a Xe-lamp under the given experimental conditions (Fig. 5c). To determine the photothermal effect of HCl-treated AuNR-PEGGO composites, the A431 cells were irradiated with Xe-lamp
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Figure 5. (a) In vitro cytotoxic assays of the HCl-treated AuNR-PEG-GO composites. Because of the decrease in the surface potential values of AuNRPEG-GO composites, the toxicity of AuNR could be reduced assembly on PEG-GO molecules. Removal of the cetyltrimethylammonium bromide layers in AuNR by HCl treatment further reduced cytotoxicity. (b) Decrease in cell viability in a concentration-dependent manner. (c) No significant reduction in cell viability after Xe-lamp irradiation for up to 20 min. (d) Composite materials demonstrating a synergistic photothermal action. Cell viability was significantly decreased by ~40% in the irradiated HCl-treated AuNR-PEG-GO–incubated cells compared with those treated with only HCl-treated AuNR-PEG-GO composites in the absence of light exposure. The “*” indicates P < 0.05. The irradiation of the cells was performed under ambient conditions.
light for 5 min after treatment with HCl-treated AuNR-PEG-GO composites. As shown in Fig. 5d, cell viability of the irradiated HCl-treated AuNR-PEG-GO–incubated cells significantly decreased by ~40% compared with either the cells treated with only free HCl-treated AuNR-PEG-GO composites or those not exposed to the light. These results suggest that HCl-treated AuNR-PEG-GO composites have a photothermal effect on A431 carcinoma cells. These results also indicate that the photothermal effect on these carcinoma cells was an in vitro synergistic effect of the light and the endocytosis of the composite because only irradiation for 5 min or endocytosis of the composite did not reduce cell viability as significantly compared with their combined treatment. In vivo photothermal effect To investigate the photothermal effect of HCl-treated AuNRPEG-GO composites in vivo, we prepared xenograft models of the nude nice. The A431 tumor-bearing mice were divided into three groups. As shown in Fig. 6, the tumor size in mice treated
with NRs and light (n = 4) was smaller than that in the control group (treated with DPBS, n = 6) or in the NR group (treated with only AuNR-PEG-GO composites, n = 7). We also measured the change in body weight during the treatments (Fig. 6b). We found no significant change in body weight among the groups. Fig. 6c shows the changes in relative tumor volume during the treatment. The DPBS-treated mice demonstrated rapid increase in tumor volume. Slight reduction in tumor volumes in the absence of light may be because of the toxic CTAB molecules remaining in the tumor areas (26). However, in the mice treated with the composite and Xe-lamp light for 5 min, tumor volume reduced more significantly. Our results reveal that the AuNR-PEG-GO composites have a potential photothermal effect on cancer in vivo as well.
DISCUSSION The AuNR-PEG-GO composites may have advantages with respect to either drug loading or gene delivery because of the
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(a)
(b)
(c)
Figure 6. In vivo tumor growth reduction. (a) Representative tumor tissue images of nude mice. The scale bar is 10 mm. (b) Mean body weights of different groups after treatment. (c) Tumor growth curves of different groups after treatment. The tumor volumes were normalized to their initial sizes. All data are represented as mean SE. All the AuNR were treated with HCl to reduce the cetyltrimethylammonium bromide layers. The tumors were irradiated with Xe-lamp light with an optical fiber unit every other day.
binary nature of the composite. We have incorporated doxorubicin (DOX) drugs on PEG-GO surfaces by means of p–p interactions (23). Due to the fact that DOX cannot directly bind onto Au surfaces via a chemical linkage, the composite nature of the AuNR-PEG-GO sample has several benefits in terms of assembling anticancer drugs consisting of aromatic rings. In addition, the positive charge of the composites is expected to bind easily to negatively charged oligonucleotides to lead to an efficient gene delivery system. As AuNR have been used for gene delivery, AuNR-PEG-GO composites can be used in assembling either DNA or RNA molecules for therapeutic purposes. The large GO sheets in lm size range could be eliminated after filtration. Data using other cell lines, such as A549 and MCF-7/ADR cells, exhibited similar behaviors to those of A431 cells. A431 skin carcinoma cells were chosen for efficient
xenografted growth and in vivo photothermal test under our experimental conditions. The composite properties of AuNR-PEG-GO will also be helpful in introducing appropriate functional groups to target specific cancer cells. It is possible that folate can be assembled with the carboxylate group on the surface of PEG-GO molecules to target folate-positive cancer cells (27). The AuNR surfaces can also be a good platform through which proteins and other molecules can be linked to target specific cancer cells after inserting the appropriate cross-linkers (28). Our future research direction is to develop a more versatile tool for effective cancer therapy by taking advantage of dual platforms offered by nanocomposites. It should also be noted that the Raman peaks of the D and G modes of the PEG-GO molecules could also be observed by means of confocal micro-Raman spectroscopy. Our methods
Photochemistry and Photobiology have advantages in imaging of internalized graphene-based AuNR-PEG-GO cargos inside cells, as we were unable to find carbon-based materials only by dark-field microscopy and Raman spectroscopy without AuNR. Indeed, AuNR may provide useful information on the internal location of the AuNR-PEG-GO composites inside mammalian cells. On the other hand, we could not observe the graphene structures inside cancer cells only by the TEM method. By identifying the AuNR, we could estimate the intracellular locations of carbon-based AuNR-PEG-GO composites. The assembly of AuNR-PEG-GO composites could thus provide a convenient method for imaging the cells, as only PEGGO molecules are not easily discernible inside cancer cells. As illustrated in Fig. 5c,d, the AuNR-PEG-GO composites played a significant role in reducing cancer cell viability compared with the Xe-lamp-irradiated sample. In vitro and in vivo photothermal cancer therapy was performed by treating the A431 carcinoma cells with the AuNR-assembled PEG-GO composites. A cell viability assay was also performed to determine the cytotoxicity of AuNR-PEG-GO composites. In vitro toxicity test was performed to determine the optical irradiation conditions of the photothermal therapy using AuNR-PEG-GO composites. By irradiating the Xe-lamp light on the nanocomposites, photothermal therapy was achieved with the AuNR assembled onto the PEG-GO molecules. In vivo tumor growth measurements also indicated that the AuNR-assembled PEG-GO composites with irradiation might efficiently reduce the tumor volume. We also performed a light control experiment using xenograft mice and found significant reduction in tumor volume as shown in Fig. 6c. This reduction is presumably because of direct exposure of the mice skin to light in the in vivo model. In the in vivo experiment, direct exposure of the skin to light resulted in temperature increase, whereas the cell culture media solution prevented direct exposure to the light in the in vitro experiment. Despite this performance in vivo, our in vitro data clearly indicate the potential photothermal effects of AuNR-PEG-GO nanocomposites. We plan to find optimal conditions for in vivo experiments. In conclusion, we demonstrated AuNR-PEG-GO nanocomposites to be the photothermal platform using A431 cells. Our graphene– AuNR nanocomposites would be a potentially useful platform for future possibilities of photothermal therapy. Acknowledgements—The National Research Foundation of Korea supported this work through grant numbers R11-2008-0061852 and K20904000004-12A0500-00410. The Nano Material Technology Development Program also supported this work, through the National Research Foundation of Korea, funded by the Ministry of Education, Science, and Technology (grant number 2012035286). This research was also partially supported by the Agency for Defense Development through Chemical and Biological Defense Research Center and the Development of Characterization Techniques for Nano-materials Safety Project of KRCF. This work was supported by the National Research Foundation (NRF) grant funded by the Korea Government (MEST) nos. 20110017435 and the Korea government [MSIP] [no. 20090083525].
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. (a) DFM images of AuNRs in the other A549 cancer cells. AuNRs appeared to be more conspicuous than the case of A431 cells. (b) Surface-enhanced Raman spectra of NIR dye
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of 3,3′-diethylthiatricarbocyanine (DPPC) at 633 and 785 nm. (c) DFM and Raman spectra of the NIR dye DPPC on AuNRs in cancer cells. The yellowish colored AuNRs are clearly discernible in the DFM images.
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