Anti-EGFR Antibody Conjugation of Fucoidan ... - ACS Publications

Apr 11, 2017 - Biocompatible fucoidan (Fu) is used as the coating material for gold ... (21) Fucoidan (Fu) is a natural biopolymer obtained from marin...
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Anti-EGFR Antibody Conjugation of Fucoidan-Coated Gold Nanorods as Novel Photothermal Ablation Agents for Cancer Therapy Panchanathan Manivasagan,† Subramaniyan Bharathiraja,† Madhappan Santha Moorthy,† Yun-Ok Oh,† Kyeongeun Song,‡ Hansu Seo,‡ and Junghwan Oh*,†,‡ †

Marine-Integrated Bionics Research Center and ‡Department of Biomedical Engineering and Center for Marine-Integrated Biotechnology (BK21 Plus), Pukyong National University, Busan 48513, Republic of Korea S Supporting Information *

ABSTRACT: The development of novel photothermal ablation agents as cancer nanotheranostics has received a great deal of attention in recent decades. Biocompatible fucoidan (Fu) is used as the coating material for gold nanorods (AuNRs) and subsequently conjugated with monoclonal antibodies against epidermal growth factor receptor (antiEGFR) as novel photothermal ablation agents for cancer nanotheranostics because of their excellent biocompatibility, biodegradability, nontoxicity, water solubility, photostability, ease of surface modification, strongly enhanced absorption in near-infrared (NIR) regions, target specificity, minimal invasiveness, fast recovery, and prevention of damage to normal tissues. Anti-EGFR Fu-AuNRs have an average particle size of 96.37 ± 3.73 nm. Under 808 nm NIR laser at 2 W/cm2 for 5 min, the temperature of the solution containing anti-EGFR Fu-AuNRs (30 μg/mL) increased by 52.1 °C. The anti-EGFR FuAuNRs exhibited high efficiency for the ablation of MDA-MB-231 cells in vitro. In vivo photothermal ablation exhibited that tumor tissues fully recovered without recurrence and finally were reconstructed with normal tissues by the 808 nm NIR laser irradiation after injection of anti-EGFR Fu-AuNRs. These results suggest that the anti-EGFR Fu-AuNRs would be novel photoablation agents for future cancer nanotheranostics. KEYWORDS: gold nanorods, fucoidan, anti-EGFR antibody, photothermal therapy, cancer nanotheranostics

1. INTRODUCTION

Gold nanorods (AuNRs) have attracted great interest in biomedical fields such as drug delivery, photothermal therapy, photodynamic therapy, and photoacoustic imaging because of their excellent biocompatibility, easy synthesis, surface modification, stability, and strong absorption and scattering in NIR region.13−15 Recently, AuNRs have been recognized as excellent candidates for photothermal cancer therapy due to their photophysical property in converting NIR laser light into heat.14,16 AuNRs are synthesized by the seed-mediated growth methods in the presence of cetyltrimethylammonium bromide (CTAB).17 AuNRs are capped by CTAB bilayers and have a positive charged on the surface. The synthesized AuNRs capped with CTAB are very toxic to cells, which is not suitable for in vivo applications.18,19 To avoid the cytotoxicity effect of surface AuNRs are improved by the coating of biocompatible and nontoxic polymer such as fucoidan.20 Marine biopolymers have emerged as new candidates in the biomedical field for numerous applications such as drug delivery, tissue engineering, wound dressing, photothermal therapy, photodynamic therapy, and photoacoustic imaging.21

The development of novel nanoparticles has opened numerous new avenues for cancer nanotheranostics in the recent years.1,2 A clear trend that is imperative is developing novel alternatives that are more effective and minimally invasive in the early diagnosis and treatment of breast cancer which overcome the very limited accessibility and various side effects of presently employed methods.3,4 Photothermal therapy (PTT) has received considerable attention in recent decades, particularly in the biomedical field because of its advantages of being minimally invasive, resulting in a fast recovery, preventing damage to normal tissues, and having very few patient complications.5,6 PTT is the use of photoinduced heat to directly destroy tissue and cause irreversible cell damage by denaturing proteins and loosening cell membranes.7,8 Compared with traditional clinical therapeutic approaches including surgery, chemotherapy, and radiotherapy, PTT is an attractive alternative, being highly efficient in tumor ablation with minimal damage to healthy tissues.9,10 Therefore, more and more researchers focus on the strong near-infrared (NIR) light absorption (700−1100 nm) nanomaterials for photothermal ablation, which has a high transparency window in biological tissue, blood, and water.11,12 © 2017 American Chemical Society

Received: January 7, 2017 Accepted: April 11, 2017 Published: April 11, 2017 14633

DOI: 10.1021/acsami.7b00294 ACS Appl. Mater. Interfaces 2017, 9, 14633−14646

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ACS Applied Materials & Interfaces

was centrifuged twice at 10 000 rpm for 5 min to remove excess polyelectrolyte and dispersed in 1× phosphate-buffered saline (PBS, pH 7.4). 2.3. Photothermal Heating Experiments. The anti-EGFR FuAuNRs suspension was diluted to various concentrations (10, 15, 20, 25, and 30 μg/mL), and the suspension (1 mL) was irradiated with an 808 nm NIR laser (Changchun New Industries Optoelectronics Technology, Changchun, China) at different power densities (0.5, 1.0, 1.5, and 2.0 W/cm2) for 5 min. The temperature was monitored and periodically confirmed by a digital thermometer with a thermocouple probe every 1 s. To analyze the photostability of anti-EGFR Fu-AuNRs (30 μg/mL), the solutions were irradiated with an 808 nm NIR laser at 2 W/cm2 for 5 min, followed by natural cooling at room temperature for 30 min without NIR laser irradiation. This cycle was repeated six times, and the irradiated solutions were observed by UV−vis spectroscopy. 2.4. Cell Culture. A human breast cancer cell line (MDA-MB-231) and a human embryonic kidney cell line (HEK 293) were cultivated in standard culture media recommended by the Korean Cell Line Bank. Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. 2.4.1. Biocompatibility and in Vitro Cytotoxicity Studies on Cells. HEK 293 and MDA-MB-231 cells were seeded in 96-well plates at a density of 1 × 104 cells/well and incubated for 24 h at 37 °C in 5% CO2 atmosphere. After incubation, the cells were treated with varying concentrations of the anti-EGFR Fu-AuNRs (5.0, 10, 15, 20, 25, 30, 50, 75, and 100 μg/mL) for 24 and 48 h to measure cytotoxicity using the MTT assay. 2.4.2. In Vitro Photothermal Therapy. To study the photothermal efficiency of anti-EGFR Fu-AuNRs, MDA-MB-231 cells were seeded in 96-well plates at a density of 1 × 104 cells/well and incubated for 24 h. After incubation, different concentrations of anti-EGFR Fu-AuNRs (10, 15, 20, 25, and 30 μg/mL) were applied to MDA-MB-231 cells and incubated for 4 h. Then, the medium was immediately removed, and 100 μL of fresh medium was added. Cells were treated without or with 808 nm laser irradiation at 2.0 W/cm2 for 5 min. After 2 h of further incubation, cell viability was analyzed by the MTT assay. MDA-MB-231 cells were seeded in 96-well plates at a density of 1 × 104 cells/well and incubated for 24 h. We set-up four groups (control cells, control cells +808 nm NIR laser, anti-EGFR Fu-AuNRs, and antiEGFR Fu-AuNRs + 808 nm NIR laser). The first group had no treatment. The second was irradiated with different power densities (0.5, 1.0, 1.5, and 2.0 W/cm2) for 5 min, and the third and fourth groups were incubated with 30 μg/mL anti-EGFR Fu-AuNRs for 4 h, without or with irradiation of different power densities (0.5, 1.0, 1.5, and 2.0 W/cm2) for 5 min, respectively. After irradiation treatment, cells were then incubated at 37 °C for 2 h, and all of the cells were stained with 0.4% (w/v) trypan blue solution in PBS for 5 min to test cell viability. Cells were observed under a light microscope (Leica Microsystems GmbH, Wetzlar, Germany) in a bright field. Dead cells accumulated the dye and were stained blue, while live cells could pump it out and remain clear. MDA-MB-231 cells were seeded in 6-well plates at a density of 2 × 105 cells/well and incubated for 24 h and set into four groups. The cells of group I were treated with 30 μg/mL anti-EGFR Fu-AuNRs at 37 °C for 4 h. Subsequently, the cells of groups II and IV were treated with 808 nm laser irradiation at 2.0 W/cm2 for 5 min. After incubating for another 2 h, all the cells were stained with acridine orange (AO) and propidium iodide (PI) to evaluate the photothermal effect of the nanoparticles on cancer cells, and fluorescent images were collected with a Leica DMI300B fluorescence microscope (Leica Microsystems, Wetzlar, Germany). Furthermore, all the cells were treated with or without anti-EGFR Fu-AuNRs (30 μg/mL) for 4 h. After washing, the cells were treated with or without 808 nm laser irradiation at 2 W/cm2 for 5 min. Subsequently, cells were incubated for an additional 2 h and they were collected, washed with PBS, dyed with fluorescein isothiocyanate Annexin V Apoptosis Detection Kit (BD Biosciences, USA) and then detected by flow cytometry (BD FACSVerse, NJ) to make identify cell death mode.

Fucoidan (Fu) is a natural biopolymer obtained from marine brown algae with a highly negative charge due to sulfate ester groups on the polysaccharide backbone.22 Fu is considered an excellent candidate for nanomedicine, with a high potential for theranostics and a wide range of bioactivities including antimicrobial, anticancer, antiviral, and antitumor.23 Important properties of Fu include biocompatibility, biodegradability, nontoxicity, water solubility, stability, ease of surface modifications, low cost, and abundance.21 The biomedical applications of synthesized AuNRs capped with CTAB are very toxic to cells.24 To avoid the toxicity, surface AuNRs are modified by the coating of nontoxic marine biomolecules such as Fu.25,26 AuNRs have been coated with Fu and have great promise for biomedical application of nanomaterials because of their excellent biocompatibility, biodegradability, nontoxicity, photostability, and strongly enhanced absorption in NIR regions.14,27 Fu-coated gold nanorods (Fu-AuNRs) were formed through an electrostatic physisorption interactions due to the positively charged −N+(CH3)3 group on the AuNRs and a negatively charged sulfate group on the Fu. Epidermal growth factor receptor (EGFR) is one of the representative targets for cancer theranostics due to EGFR being overexpressed in several epithelial cancers such as breast, lung, kidney, ovarian, and brain cancer.28,29 EGFR is the first molecular target which a monoclonal antibody (anti-EGFR) has been developed for cancer therapy.30,31 Anti-EGFR has a zwitterionic profile, that is, it is positively and negatively charged. The antibodies are bound to negatively charged FuAuNRs by electrostatic physisorption interactions.32 AuNRs coated with Fu and modified by subsequent antibody conjugation have attracted considerable attention as new candidates for cancer therapy. Anti-EGFR Fu-AuNRs were formed through electrostatic interactions due to the positively charged −N+(CH3)3 groups on the AuNRs, negatively charged sulfate groups on the Fu, and the positively charged segments of the antibodies. Combining the characteristics of these materials into the form of anti-EGFR Fu-AuNRs has opened numerous new avenues for biomedical applications because of the excellent biocompatibility, biodegradability, nontoxicity, photostability, strongly enhanced absorption in NIR regions, and target specificity. In our present study, we have developed the anti-EGFR Fu-AuNRs as novel agents for photothermal therapy.

2. MATERIALS AND METHODS 2.1. Synthesis of AuNRs. AuNRs were synthesized based on the seed-mediated growth mechanism according to the previous literature with some modifications.33 Gold seed solution was prepared by adding 5 mL of 0.2 M CTAB to 5 mL of 0.005 M HAuCl4 in a scintillation vial; then, 0.6 mL of ice-cold 0.01 M NaBH4 was added to the stirred solution under vigorous stirring for 2 min. The brownish-yellow color of the gold seed solution was observed, denoting the formation of gold seeds. The growth mixture was synthesized by mixing 250 mL of CTAB at 0.2 M with 250 mL of 0.001 M HAuCl4 and 12 mL of 4 mM AgNO3 After gentle mixing, 3.5 mL of 0.0788 M freshly prepared ascorbic acid solution was added, and the solution changed from dark yellow to transparent. Then, 0.6 mL of gold seed solution was added to the growth mixture at 27 °C, indicating in the formation of a dark red solution within 30 min. Excess CTAB in the AuNR solutions was removed with four rounds of centrifugation and water washing at 16 000 rpm for 20 min. 2.2. Fucoidan-Coated AuNRs. Next, 1 mL of purified CTAB capped AuNRs was added to 0.3 mL of Fu (1 mg/mL) while stirring at room temperature for 24 h. Surface-modified AuNRs with Fu solution 14634

DOI: 10.1021/acsami.7b00294 ACS Appl. Mater. Interfaces 2017, 9, 14633−14646

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ACS Applied Materials & Interfaces Scheme 1. Conjugation of Fu-AuNRs and Anti-EGFR Antibodies and Mechanisms as Cancer Therapy

a

Schematic illustration of Fu-AuNRs and subsequent conjugation with anti-EGFR antibody. bPossible mechanisms of the biocompatible anti-EGFR Fu-AuNRs as novel photothermal ablation agents for cancer therapy. 2.5. In Vivo Photothermal Therapy. Tumor-bearing mice were intravenously injected with 100 μL of 2 mg/mL anti-EGFR Fu-AuNRs when the tumor volume reached ∼120 mm3. The mice were randomly assigned into four groups (n = 5): (a) PBS injection; (b) PBS + 808 nm NIR laser; (c) anti-EGFR Fu-AuNRs; and (d) anti-EGFR FuAuNRs + 808 nm NIR laser. The control and treatment mice were injected with 100 μL of PBS and anti-EGFR Fu-AuNRs, respectively. After 1 h, the mice with tumors of the groups b and d were irradiated with an 808 nm laser at 2 W/cm2 for 5 min. Temperature change under laser irradiation in the tumor region was recorded by a FLIR i5 infrared (IR) camera (Flir Systems Inc., Portland, OR). Tumor growth and mice weight were measured in the following days. The tumor size was measured with calipers every day and calculated as follows:

Tumor volume =

(Tumor length) × (Tumor width)2 2

gold (Au) was measured by inductively coupled plasma mass spectrometry (ICP-MS, Nexion 300D, PerkinElmer, USA).

3. RESULTS AND DISCUSSION Fu is a natural biopolymer with a highly negative charge on the polysaccharide backbone. Fu has attracted special interest in recent years because of its excellent biocompatibility, biodegradability, low cost, and nontoxic nature. Gold nanorods (AuNRs) are capped by CTAB bilayers and positively charged on the surface. AuNRs have received considerable attention in the recent years because of their excellent biocompatibility, stability, and strong absorption and scattering in the NIR regions. The biomedical applications of synthesized AuNRs are very limited due to the cytotoxic effect of CTAB. To avoid the cytotoxicity, AuNRs are modified by the coating of nontoxic biomolecules such as Fu. Fu-AuNRs were formed through electrostatic interactions due to the positively charged −N+(CH3)3 group on the AuNRs and a negatively charged sulfate group on the Fu. EGFR is a good target for cancer therapy. The antibodies are bound to negatively charged Fu-

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2.6. Biodistribution of Nanoparticles in Tumor and Organs. The mice were sacrificed at 24 h and 20 days postinjection of antiEGFR Fu-AuNRs; major organs and tumor tissues were harvested from all the mice and weighed. Major organs and tumor tissues were set into aqua regia solution and heated overnight at 80 °C. Subsequently, after further heating at 130 °C for 2 h, the amount of 14635

DOI: 10.1021/acsami.7b00294 ACS Appl. Mater. Interfaces 2017, 9, 14633−14646

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Figure 1. (a) UV−vis−NIR absorption spectra of AuNRs, Fu-AuNRs, and anti-EGFR Fu-AuNRs. (b) XRD patterns of AuNRs and Fu-AuNRs. (c) FTIR spectra of AuNRs and Fu-AuNRs. (d) FETEM image of Fu-AuNRs and anti-EGFR Fu-AuNRs. (e) Hydrodynamic diameters of AuNRs, FuAuNRs, and anti-EGFR Fu-AuNRs for the DLS measurements. (f) Zeta potentials of AuNRs, Fu-AuNRs, and anti-EGFR Fu-AuNRs.

AuNRs by electrostatic physisorption interactions. The final step deals with the conjugation of Fu-AuNRs with an antibody against EGFR to reach tumor targeting functionality owing to overexpression of EGFR in MDA-MB-231 cells. Combining the characteristics of these materials into the form of anti-EGFR Fu-AuNRs has opened numerous new avenues for biomedical application because of its excellent biocompatibility, biodegradability, nontoxicity, stability, strongly enhanced absorption in NIR regions, and target specificity. Furthermore, we have successfully synthesized anti-EGFR Fu-AuNRs for photothermal therapy (Scheme 1 and Figure S1). 3.1. Characterization. The dark red color was formed within 30 min, resulting in the formation of AuNRs. The AuNRs, Fu-AuNRs, and anti-EGFR Fu-AuNRs solutions were observed by UV−vis spectroscopy. They exhibited a longi-

tudinal surface plasmon resonance (SPR) absorption at 842 nm, which confirmed strong absorption scattering in the NIR region (700−1100 nm) (Figure 1a). The XRD patterns of AuNRs and Fu-AuNRs were shown in the Figure 1b. The XRD pattern of AuNRs and Fu-AuNRs showed intense peaks corresponding to (111), (200), (220), and (311) Bragg reflection. Furthermore, the XRD pattern was compared with the Joint Committee on Powder Diffraction Standards (JCPDS) Powder Diffraction File (PDF) No. 04-0784 (2015) and confirmed the formation of Fu-AuNRs with the facecentered cubic (fcc) crystal structure, which is also consistent with the earlier reports.34,35 Figure 1c shows the FTIR spectra of AuNRs and Fu-AuNRs. The AuNRs exhibited characteristic peaks at ∼1483 cm−1 (CTAB). The FTIR spectra also confirmed the surface coating of AuNRs with Fu, where the 14636

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Figure 2. Energy-dispersive X-ray spectrum of AuNRs (a) and Fu-AuNRs (b). UV−vis−NIR absorption spectra of Fu-AuNRs: (c) 6 months stability study, (d) under various pH conditions, and (e) at different concentrations of NaCl.

strong peak at ∼1483 cm−1 disappeared and the characteristic peaks of the Fu spectrum at 1160−1260 and 840 cm−1 were associated with the SO asymmetric stretching and C−O−S stretching of the sulfate groups measured in the spectrum of Fu-AuNRs. FETEM was used to observe AuNRs, Fu-AuNRs, and antiEGFR Fu-AuNRs. The AuNRs can be clearly seen from the FETEM images (Figure S2). In the FETEM micrograph shown in Figure 1d for Fu-AuNRs, there is a gray shell encompassing each AuNR, indicating AuNRs coated with Fu. However, the image of Fu-AuNRs after anti-EGFR conjugation is quite obscure, because of the large size and nonconductivity of antibodies. The SAED patterns of the AuNRs and Fu-AuNRs were investigated by FETEM. The SAED patterns were observed and the obtained Debye−Sherrer rings are shown in Figure S3, which are denoted to (111), (200), (220), and (311) planes and supported by the XRD analysis. The DLS histogram of AuNRs, Fu-AuNRs, and anti-EGFR Fu-AuNRs have an average particle size of 58.57 ± 3.21, 84.36 ± 2.57, and 96.37 ± 3.73 nm, respectively (Figure 1e). The zeta potential (ZP) of the AuNRs, Fu-AuNRs, and anti-EGFR Fu-AuNRs were determined to be +38.54, −56.43, and −31.56 mV, respectively (Figure 1f). The ZP revealed the positive, negative, and

zwitterionic charges of AuNRs, Fu-AuNRs, and anti-EGFR FuAuNRs, respectively. Nanoparticles with a ZP > 30 mV (positive or negative) have been shown to be stable in suspension, as the surface charge prevents aggregation of the particles. Additional experimental evidence is provided by EDX analysis. The AuNRs and Fu-AuNRs had a noticable characteristic peak assigned to gold (Au) metal in EDX spectrum, also evidence of the successful formation of AuNRs (Cu peak is from the support grid) (Figure 2a,b). The stability of FuAuNRs is one of the most important characteristics for biomedical applications. The stability of Fu-AuNRs solutions was investigated under various conditions such as different months, various pH levels, and different concentrations of NaCl (Figure 2c−e). There was no observable change in the UV−vis spectrum of the Fu-AuNRs solution under various conditions, which suggests a highly stable nature. The size of Fu-AuNRs after 6 months was 83.68 ± 3.02 nm as measured by DLS. To check the dispersion stability, anti-EGFR Fu-AuNRs were dispersed in deionized water (DW), PBS solution, and DMEM containing 10% fetal bovine serum (FBS). UV−vis spectra of all the samples were taken after 30 min and 1, 3, 5, and 7 days of incubation at 25 °C (Figure 3a−e). There was no observable change in the UV−vis spectrum of the anti-EGFR Fu-AuNRs 14637

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Figure 3. UV−vis−NIR absorbance spectra of anti-EGFR Fu-AuNRs of dispersion stability in deionized water (DW), phosphate-buffered saline (PBS), and cell culture medium (DMEM without phenol red) containing 10% fetal bovine serum (FBS) for 30 min (a), 1 day (b), 3 days (c), 5 days (d), and 7 days (e). (f) UV−vis−NIR absorbance spectrum of different concentrations of anti-EGFR Fu-AuNRs.

AuNRs with different concentrations to 808 nm laser irradiation for 5 min at different power densities (0.5, 1.0, 1.5, and 2.0 W/cm2). The results indicated that temperature elevation was dependent upon anti-EGFR Fu-AuNRs and 808 nm laser power densities (Figure 4a−d). The aqueous solution containing 30 μg/mL anti-EGFR Fu-AuNRs shows a maximum temperature increase from 28.5 to 52.1 °C for 5 min at 2 W/ cm2, while pure water without anti-EGFR Fu-AuNRs shows a negligible temperature change from 28.2 to 31.1 °C. When the temperate increases to 50 °C for 4−6 min, protein denaturation as well as DNA damage happens, and irreversible cellular damage occurs promptly.36 The aqueous solution containing 30 μg/mL anti-EGFR Fu-AuNRs were exposed to 808 nm laser irradiation at different laser power densities of 0.5, 1.0, 1.5, and 2.0 W/cm2 for 5 min, resulting in a temperature elevation of 34.0, 40.3, 43.8, and 52.1 °C, respectively (Figures 4e and S5). In addition, IR thermography also indicated that the temper-

solution under various aqueous solutions, which suggested good colloidal stability. Furthermore, the size and particle size distribution of anti-EGFR Fu-AuNRs suspended in these solutions were relatively stable without any obvious changes, indicating the colloidal stability of anti-EGFR Fu-AuNRs. The size of anti-EGFR Fu-AuNRs in PBS solution before and after 7 days was 96.37 ± 3.73 and 95.85 ± 2.54 nm as measured by DLS. 3.2. Photothermal Heating Experiments. Anti-EGFR Fu-AuNRs solutions with different concentrations (10, 15, 20, 25, and 30 μg/mL) were observed by UV−vis spectroscopy (Figures 3f and S4). The aqueous solution of anti-EGFR FuAuNRs was scanned in the range of 300−1100 nm. The strong NIR absorption of anti-EGFR Fu-AuNRs motivated us to investigate their potential application as photothermal ablation agents. Temperature elevation of anti-EGFR Fu-AuNRs was investigated by exposing an aqueous solution of anti-EGFR Fu14638

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Figure 4. Temperature elevation of aqueous solution anti-EGFR Fu-AuNRs at different concentrations (0, 10, 15, 20, 25, and 30 μg/mL) as a function of irradiation time by 808 nm laser at different laser poser densities: 0.5 W/cm2 (a), 1.0 W/cm2 (b), 1.5 W/cm2 (c), and 2.0 W/cm2 (d). (e) Temperature elevation of aqueous solution at a concentration of 30 μg/mL anti-EGFR Fu-AuNRs under 808 nm laser irradiation at different power densities (0.5, 1.0, 1.5, and 2.0 W/cm2) for 5 min. (f) NIR thermographic image of anti-EGFR Fu-AuNRs (30 μg/mL) aqueous solution under exposure to an 808 nm NIR laser at 2.0 W/cm2 for 5 min.

ature in 35 mm cell culture plate could quickly reach to 52.1 °C in the presence of anti-EGFR Fu-AuNRs upon 808 nm laser irradiation at 2.0 W/cm2 for 5 min (Figure 4f). These results suggested the anti-EGFR Fu-AuNRs as novel agents for photothermal therapy. To investigate the NIR photothermal stability of anti-EGFR Fu-AuNRs, 6 cycles of laser on/off were performed, and the aqueous solution containing 30 μg/mL anti-EGFR Fu-AuNRs were exposed to 808 nm laser irradiation at 2.0 W/cm2 for 5 min (laser on), followed by cooling down to room temperature without 808 nm laser irradiation for 30 min (laser off). After 6 cycles of laser on/off irradiation, there was no notable decrease in temperature elevation during the experiment (Figure 5a). Furthermore, the UV−vis spectrum of anti-EGFR Fu-AuNRs

exhibited no obvious decrease in the absorbance at 808 nm NIR laser irradiation after 6 cycles of laser on/off (Figure 5b), suggesting good photothermal stability after a long period of 808 nm laser irradiation, which could be advantageous for cancer therapy. In addition, the FETEM micrograph of antiEGFR Fu-AuNRs was relatively stable without any observable changes after 6 cycles of laser on/off, which further confirmed the stable nature of anti-EGFR Fu-AuNRs (Figure S6). The size of anti-EGFR Fu-AuNRs after 6 cycles of laser on/off was 94.77 ± 3.28 nm as measured by DLS. 3.3. Biocompatibility and in Vitro Cytotoxicity Studies on Cells. The biocompatibility of anti-EGFR Fu-AuNRs is essential for their biomedical applications. The biocompatibility study was for 24 and 48 h on HEK 293 cells treated with 14639

DOI: 10.1021/acsami.7b00294 ACS Appl. Mater. Interfaces 2017, 9, 14633−14646

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Figure 5. (a) Temperature elevation of aqueous solution at a concentration of 30 μg/mL anti-EGFR Fu-AuNRs during for 6 successive cycles of an on/off NIR laser irradiation (2.0 W/cm2). (b) UV−vis−NIR spectra of anti-EGFR Fu-AuNRs solution before and after 808 nm NIR laser irradiation at 2.0 W/cm2 for 6 successive cycles of an on/off laser irradiation. (c) Biocompatibility study of anti-EGFR Fu-AuNRs against HEK 293 cells for 24 and 48 h. Data is expressed as mean ± SD of the three experiments. (d) MTT assay results confirming the in vitro cytotoxicity effect of anti-EGFR Fu-AuNRs against MDA-MB-231 cells for 24 and 48 h. Data is expressed as mean ± SD of the three experiments. (e) Cell viability of anti-EGFR FuAuNRs treated MDA-MB-231 cells with or without 808 nm NIR laser irradiation at 2.0 W/cm2 for 5 min. Data is expressed as mean ± SD of the three experiments (*, significant p < 0.05; **, highly significant p < 0.01). (f) Cell viability of anti-EGFR Fu-AuNRs (30 μg/mL) treated MDA-MB231 cells with or without 808 nm NIR laser irradiation of different laser power densities (0.5, 1.0, 1.5, and 2.0 W/cm2) for 5 min. Data is expressed as mean ± SD of the three experiments (*, significant p < 0.05; **, highly significant p < 0.01).

anti-EGFR Fu-AuNRs for 24 and 48 h. They exhibited slightly reduced viability in a dose- and time-dependent manner; no significant cytotoxicity was noticed after 24 and 48 h incubation with any concentration of anti-EGFR Fu-AuNRs, indicating that anti-EGFR Fu-AuNRs have low cytotoxicity and excellent biocompatibility, resulting in their suitability for cancer treatment. 3.3.1. In Vitro Photothermal Therapy. To investigate the anti-EGFR Fu-AuNRs as photoablation agents, the in vitro photothermal efficiency on MDA-MB-231 cells was further studied using a quantitative MTT assay. MDA-MB-231 cells

different concentrations of anti-EGFR Fu-AuNRs (5.0, 10, 15, 20, 25, 30, 50, 75, and 100 μg/mL; Figure 5c). The MTT results exhibited more than 95% cell viability even at a high concentration of 100 μg/mL, suggesting that anti-EGFR FuAuNRs have excellent biocompatibility, biodegradability, and nontoxicity, resulting in their suitability for in vivo experiments. For the successful application of nanoparticles in the biomedical field, it is most important to check their potential cytotoxicity. The cytotoxicity effect of anti-EGFR Fu-AuNRs were evaluated on MDA-MB-231 cells using MTT assay. Figure 5d shows the cell viability against different concentrations of 14640

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Figure 6. (a) Morphological alterations in MDA-MB-231 cells treated with or without anti-EGFR Fu-AuNRs under 808 nm NIR laser irradiation at 2.0 W/cm2 for 5 min (20× magnification). (b) Optical images stained by trypan blue. The optical image of MDA-MB-231 cells treated with or without anti-EGFR Fu-AuNRs under 808 nm NIR laser irradiation at 2.0 W/cm2 for 5 min (20× magnification). (c) MDA-MB-231 cells stained by AO (live: green) and PI (dead: red). Merged fluorescence microscope images of MDA-MB-231 cells after treatment with or without anti-EGFR FuAuNRs (30 μg/mL) under 808 nm NIR laser irradiation at 2.0 W/cm2 for 5 min (20× magnification). (d) Merged fluorescence microscope images of MDA-MB-231 cells after treatment with or without anti-EGFR Fu-AuNRs (30 μg/mL) under 808 nm NIR laser irradiation of different laser power densities (0.5, 1.0, 1.5, and 2.0 W/cm2) for 5 min (20× magnification).

anti-EGFR Fu-AuNRs were treated with or without 808 nm laser irradiation at 2 W/cm2. The morphology changes were observed in MDA-MB-231 treated with 30 μg/mL anti-EGFR Fu-AuNRs and 808 nm laser irradiation at 2 W/cm2 for 5 min compared with the corresponding control experimental samples without 808 nm laser irradiation (Figure 6a). The morphological changes such as cell blebbing, cell shape alterations, membrane integrity loss, protein denaturation, and chromatin condensation were observed for the cells in the presence of anti-EGFR Fu-AuNRs and 808 nm laser irradiation at 2 W/cm2 for 5 min, while no visible change in cell morphology was observed for the control cells. We next evaluated the photothermal efficiency of anti-EGFR Fu-AuNRs with or without 808 nm laser irradiation against MDA-MB-231 cells using trypan blue staining. As seen from the optical microscopic images in Figure 6b, there were no observable changes in the control groups of MDA-MB-231 with or without 808 nm NIR laser irradiation at 2 W/cm2 for 5 min. Furthermore, negligible cell ablation was noticed for cells incubated with anti-EGFR Fu-

were incubated with different concentrations of anti-EGFR FuAuNRs (10, 15, 20, 25, and 30 μg/mL) for 4 h and then irradiated by the 808 nm NIR laser for 5 min (Figure 5e), the results of which indicated that photothermal efficiency is dependent on the concentration of anti-EGFR Fu-AuNRs and 808 nm laser power density. The results showed a significant decrease to 35% cell viability even at a high concentration of 30 μg/mL anti-EGFR Fu-AuNRs and 808 nm laser irradiation for 5 min compared with corresponding control experimental samples without 808 nm laser irradiation. In addition, the cells treated with 30 μg/mL anti-EGFR Fu-AuNRs were exposed to 808 nm laser irradiation at different laser power densities of 0.5, 1.0, 1.5, and 2.0 W/cm2 for 5 min. If the power density was increased to 2.0 W/cm2, then most of the cancer cells were killed, and no cell survived with a solution of 30 μg/mL antiEGFR Fu-AuNRs (Figure 5f), indicating anti-EGFR Fu-AuNRs as a novel agent for photothermal therapy. To visually evaluate the photothermal efficiency of the antiEGFR Fu-AuNRs, the MDA-MB-231 cells in the presence of 14641

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Figure 7. (a) Flow cytometry analysis of MDA-MB-231 cells after treatment with or without anti-EGFR Fu-AuNRs (30 μg/mL) under 808 nm NIR laser irradiation at 2.0 W/cm2 for 5 min. (b) Statistical data of percentage of apoptotic and necrotic cells with different treatments (*, significant p < 0.05; **, highly significant p < 0.01). (c) NIR thermographic images captured after 5 min of 808 nm NIR laser irradiation with intravenously injected of PBS and anti-EGFR Fu-AuNRs. (d) Temperature monitoring in the tumors of PBS-treated mice and anti-EGFR Fu-AuNRs-treated mice during 808 nm NIR laser irradiation at 2.0 W/cm2 for 5 min. (e) Quantitative measurement of tumor volume in mice after different treatment. Data is expressed as mean ± SD of the three experiments.

suggesting that almost all of the cells were dead. In addition, we noted the in vitro photothermal efficiency of anti-EGFR FuAuNRs (30 μg/mL) exposed to 808 nm laser irradiation at different laser power densities of 0.5, 1.0, 1.5, and 2.0 W/cm2 for 5 min. The merged fluorescence images (AO/PI) of control, control plus laser (0.5, 1.0, 1.5, and 2.0 W/cm2), antiEGFR Fu-AuNRs, and anti-EGFR Fu-AuNRs plus laser (0.5, 1.0, and 1.5 W/cm2) showed green fluorescence, suggesting the negligible numbers of dead cells. The merged fluorescence images (AO/PI) of 808 nm NIR laser irradiation and antiEGFR Fu-AuNRs showed red fluorescence, suggesting that almost all of the cells were dead (Figure 6d). The photothermal efficiency was further quantified by flow cytometry (Figure 7a). MDA-MB-231 cells were treated with anti-EGFR Fu-AuNRs for 4 h with or without 808 nm NIR laser irradiation and then labeled with Annexin V and PI.

AuNRs without 808 nm NIR laser irradiation. In contrast, the MDA-MB-231 cells incubated with 30 μg/mL anti-EGFR FuAuNRs were stained blue after 808 nm laser irradiation at 2 W/ cm2 for 5 min, thus creating a significant photothermal efficiency for cancer cells. The photothermal efficiency of cancer cells was also confirmed by fluorescence microscopy. The cells were stained with acridine orange (AO) and propidium iodide (PI) to identify live (green) and dead/late apoptotic cells (red), respectively (Figure 6c). Strong green fluorescence was observed in MDA-MB-231 cells treated by control, control plus laser, and anti-EGFR Fu-AuNRs, and a negligible number of red fluorescence were noticed. In contrast, the majority of MDA-MB-231 cells were able to be ablated and showed red fluorescence after treatment with 30 μg/mL anti-EGFR FuAuNRs under 808 nm laser irradiation at 2 W/cm2 for 5 min, 14642

DOI: 10.1021/acsami.7b00294 ACS Appl. Mater. Interfaces 2017, 9, 14633−14646

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ACS Applied Materials & Interfaces

Figure 8. (a) Representative photographs of tumors in mice taken before treatments (day 0) and 2, 5, 10, 15, and 20 days after treatments of antiEGFR Fu-AuNRs under 808 nm NIR laser irradiation at 2.0 W/cm2 for 5 min. (b) Body weight of mice at different treatments indicated in 20 days. Data is expressed as mean ± SD of the three experiments. (c) Final tumor weight was acquired after sacrifice of mice. Data is expressed as mean ± SD of the three experiments (*, significant p < 0.05; **, highly significant p < 0.01). (d) Digital photographs of relevant tumors originated from each group in mice. (e) Biodistribution of nanoparticles (Au concentration % ID/g) in tumor tissues and organs at 24 h after the intravenous injection. Data is expressed as mean ± SD of the three experiments.

photothermal efficiency of anti-EGFR Fu-AuNRs, the temperature change of the tumor area was recorded by a FLIR i5 IR camera (Figure 7c). After laser irradiation (808 nm, 2 W/cm2, 5 min), the temperature of the tumor area intravenously injected with anti-EGFR Fu-AuNRs rapidly reached 65.2 °C, which could exceed irreversible tumor tissue damage. In comparison, the maximum temperature of tumor area intravenously injected with PBS for the same laser irradiation was only about 38.1 °C, which is insufficient to irreversibly damage tumor tissues (Figure 7d). The photothermal efficiency was estimated by measuring tumor volume of all the groups. Tumors grew quickly over time in control groups (PBS, PBS + 808 nm NIR laser, and antiEGFR Fu-AuNRs). No significant difference was seen among these groups in final tumor sizes. In contrast, a significant decrease was observed in tumor volume for the (anti-EGFR Fu-

Double-stained cells were defined as late apoptotic/necrotic cells. The results showed that photothermal efficiency significantly induced the number of cells in late apoptosis/ necrosis (95%) compared with that in control groups (5%) (Figure 7b). The results evidently illustrated that 808 nm NIR laser irradiation could obviously promote the efficiency of antiEGFR Fu-AuNRs on apoptosis and cell death. 3.4. In Vivo Photothermal therapy. We studied the in vivo photothermal effect of anti-EGFR Fu-AuNRs in tumorbearing mice. When the tumor volume reached ∼120 mm3, mice were intravenously injected with PBS or anti-EGFR FuAuNRs for 24 h and then the mice were irradiated with 808 nm laser at 2 W/cm2 for 5 min. The tumor-bearing mice were randomly assigned into four groups: (a) PBS; (b) PBS + 808 nm NIR laser; (c) anti-EGFR Fu-AuNRs; and (d) anti-EGFR Fu-AuNRs + 808 nm NIR laser. To verify the in vivo 14643

DOI: 10.1021/acsami.7b00294 ACS Appl. Mater. Interfaces 2017, 9, 14633−14646

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ACS Applied Materials & Interfaces

Figure 9. Representative H&E stained images of major organs including heart, kidney, spleen, lung, and liver collected from different groups of mice 20 days after laser irradiation.

AuNRs + laser) group (Figure 7e). The potential in vivo toxicity of anti-EGFR Fu-AuNRs was measured by body weight loss of each group. After 20 days of treatment, there was no observable weight loss in any of the four groups, suggesting no significant systemic toxicity to the treated mice (Figure 8b). For mice treated with anti-EGFR Fu-AuNRs plus NIR laser irradiation, while no tumor recurrence was seen in the tumor site with a black scar after 20 days, the mice completely recovered and were finally reconstructed with normal tissues (Figures 8a, S7, and S8). All the treatment groups of mice were sacrificed, and tumor tissues were dissected and weighed after 20 days as shown in Figure 8c. The mean tumor weight in the treatment group was notably smaller than that of control groups. All of the tumors from the treatment group were successfully decreased, while the tumor size in the control groups kept increasing. This is further confirmed by the tumor photograph for each group in Figure 8d. These results confirmed that anti-EGFR Fu-AuNRs under irradiation could realize completed tumor destruction without tumor recurrence during the experimental period. The biodistribution of anti-EGFR Fu-AuNRs was evaluated at 24 h and at 20 days postinjection by performing an ICP-MS analysis of Au element accumulated in the heart, kidney, spleen, lung, liver, and tumor. Figure 8e shows the biodistribution of antiEGFR Fu-AuNRs in the tumor and organs at 24 h after the intravenous injection, which exhibited that the Au accumulated in the liver, spleen, and tumors at much higher levels than those in the heart, kidney, and lung. The highest accumulation of Au in the liver was over 14.47% ID/g (injected dose/g), followed by the spleen (11.68% ID/g). Particularly, the content of Au in the tumor was over 7.34% ID/g. Figure S9 shows the biodistribution of anti-EGFR Fu-AuNRs in the tumor and organs at 20 days postinjection, with the Au accumulated in the liver and spleen at much higher levels than those in the heart, kidney, lung, and tumors. The maximum accumulation of Au in the liver was over 8.52% ID/g, followed by the spleen (5.48% ID/g). Therefore, the bioaccumulation levels observed by us of Au in tumor and organs are not likely to be dangerous.

Evaluation of Au element within feces and urine suggests that the primary route of clearance was biliary excretion. It is interesting to note that majority of Au element was removed through biliary excretion within first 24 h.37 Tumors of PBS and anti-EGFR Fu-AuNRs-injected mice were immediately collected after laser irradiation. Histological examination was performed after hematoxylin and eosin (H&E). As expected, distinct signs of cell damage, such as cell shrinkage, nuclear damage, and loss of contact, were observed in anti-EGFR Fu-AuNRs-treated tumors exposed to laser irradiation. Furthermore, the tumors of PBS and antiEGFR Fu-AuNRs-treated mice were collected at 3 and 7 days after laser irradiation, and the mice were sliced and stained with H&E for histology analysis. As shown in Figure S10, there were no obvious histological changes in the anti-EGFR Fu-AuNRs treated mice, which exhibited no obvious toxic effect of antiEGFR Fu-AuNRs-treated mice. After 7 days, the tumor tissues gradually disappeared, showed no tumor recurrence, and finally reconstructed with normal tissues. The tumor-bearing mice completely recovered after anti-EGFR Fu-AuNRs injection as well as NIR laser irradiation, and the tumor almost disappeared after 20 days. To further assess the photothermal efficiency and the in vivo toxicity of anti-EGFR Fu-AuNRs, major organs (heart, kidney, spleen, lung, and liver) of the mice were sliced and stained with H&E for histology analysis. As shown in Figure 9, no obvious histopathological abnormalities were noticed in heart, kidney, spleen, lung, and liver in the anti-EGFR Fu-AuNRs treated mice, which exhibited no obvious toxic effect of anti-EGFR FuAuNRs on the major organs of mice. However, the tumor tissues had disappeared after anti-EGFR Fu-AuNRs injection under laser irradiation. These results suggested that no significant organ damage in the anti-EGFR Fu-AuNRs treatment group was noticed, indicating that the biocompatibility of anti-EGFR Fu-AuNRs is more suitable for in vivo photothermal therapy. 14644

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4. CONCLUSIONS Anti-EGFR Fu-AuNRs have successfully been developed as novel photothermal ablation agents for cancer therapy. AntiEGFR Fu-AuNRs have opened numerous new avenues for biomedical applications because of their excellent biocompatibility, biodegradability, low cost, nontoxicity, photostability, strongly enhanced absorption in NIR regions, target specificity, and ability of converting NIR light into heat, which makes them suitable as theranostic agents for photothermal ablation of cancer cells in vitro and in vivo. More interestingly, mice with tumors irradiated with an 808 nm laser with a suitable energy after anti-EGFR Fu-AuNRs injection completely recovered, showed no tumor recurrence, and finally were reconstructed with normal tissues. To our knowledge, no earlier studies described the use of anti-EGFR Fu-AuNRs as agents for photothermal ablation of cancer. This work highlights the great potential of anti-EGFR Fu-AuNRs as novel photoablation agents for future clinical cancer therapy.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b00294. Schematic diagram showing the anti-EGFR Fu-AuNRs, characterization of FE-TEM and SAED, linear relationship between the absorbance at 808 nm wavelength, linear fitting of temperature elevation of anti-EGFR FuAuNRs, photographs of tumors in mice, biodistribution of nanoparticles, and histology staining (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +82-51-629-5771. Fax: +8251-629-5779. ORCID

Junghwan Oh: 0000-0002-5837-0958 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by a grant from Marine Biotechnology Program (20150220) funded by the Ministry of Oceans and Fisheries, Republic of Korea.

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