Development of Chitosan Oligosaccharide-Modified Gold Nanorods

Oct 3, 2012 - In the present study, we demonstrate the synthesis and applications of multifunctional gold nanorod-based probes for specific targeting ...
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Development of Chitosan Oligosaccharide-Modified Gold Nanorods for in Vivo Targeted Delivery and Noninvasive Imaging by NIR Irradiation Shobhit Charan,†,‡,§ Kumar Sanjiv,∥ Narendra Singh,† Fan-Ching Chien,† Yi-Fan Chen,⊥ Navchtsetseg Navchaa Nergui,†,‡,§ Shih-Hsin Huang,† Chiung Wen Kuo,*,† Te-Chang Lee,⊥ and Peilin Chen*,† †

Research Center for Applied Sciences, §Taiwan International Graduate Program, Nanoscience and Technology Program, ∥TIGP Molecular Medicine Program, and ⊥Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan ‡ Department of Chemistry, National Taiwan University, Taipei 106, Taiwan S Supporting Information *

ABSTRACT: In the present study, we demonstrate the synthesis and applications of multifunctional gold nanorod-based probes for specific targeting and noninvasive imaging based on localized heating generated by gold nanorods after NIR irradiation. The structural design of the probe consists of MUA (11-mercaptoundecanoic acid)-capped gold nanorods covalently linked with low-molecular-weight chitosan oligosaccharide (Mw ∼5000) via carbodiimide (EDC) coupling agent. This surface modification is performed for complete replacement of toxic CTAB (hexadecyltrimethyl-ammonium chloride) and acid-responsive delivery of gold nanorods in acidic environment as known to be present at tumor surrounding areas. The resulting chitosan oligosaccharide-modified gold nanorods (CO-GNRs) were further conjugated with tumor targeting monoclonal antibody against EGFR (epidermal growth factor receptor) to provide localized targeting functionality owing to the overexpression of EGFR in human oral adenosquamous carcinoma cell line CAL 27. Initial in vitro and in vivo toxicity assessments indicated that CO-GNRs did not induce any significant toxicity and are thus suitable for biological applications. Furthermore, selective targeting and accumulation of CO-GNRs were observed in vitro via two-photon luminescence imaging studies in CAL 27, which was also observed through in vivo targeting studies performed via NIR (near-infrared) laser irradiation in CAL 27 xenografts of BALB/c nude mice. Hence, the CO-GNRs that we have developed are biocompatible and nontoxic and can be a potential candidate for in vivo targeted delivery, noninvasive imaging based on localized hyperthermia, and photothermal-related therapies.

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INTRODUCTION In recent years, the development of new optical materials in the near-infrared (NIR) region (700−950 nm) has attracted a great deal of research interest due to the ability of light in this spectral region to penetrate more deeply into tissues in contrast with visible light.1 In addition, the absorbance and autofluorescence from biological tissues are minimal in the NIR region. Therefore, there is great potential for the development of noninvasive in vivo applications using NIR radiation.1,2 In the past few years, a growing number of studies have reported the synthesis of NIR fluorescent dyes and other tunable nanoparticles such as quantum dots for in vivo imaging. However, NIR fluorescent dyes suffer from photobleaching, while quantum dots are known to exert a cytotoxic effect, thus limiting their applications. Hence, a nontoxic and more robust NIR material is required for in vivo imaging. Gold nanorods have recently been identified as potential candidates for in vivo imaging because of their robustness and unique optical properties in the NIR region. In principle, gold nanorods © 2012 American Chemical Society

have two plasmon resonance peaks in their absorption spectra, the longitudinal surface plasmon peak (SPR) and the transverse surface plasmon peak. This longitudinal SPR peak can be tuned to the NIR region by changing the aspect ratio and diameter of the nanorods.2 When these nanorods are excited at NIR wavelengths, they can simultaneously behave as optical contrast agents and photothermal transducers.3−5 In addition, the simple synthesis,6−8 tunability, good biocompatibility, and relative ease of conjugation with antibodies and other targeting moieties of gold nanorods make them excellent candidates for noninvasive imaging and image-guided therapies based on localized hyperthermia.3,5,9−15 However, in vivo targeted delivery of gold nanorods is the key obstacle in obtaining high-contrast images and more efficient photothermal treatment. To achieve targeted delivery using gold nanorods, many Received: March 15, 2012 Revised: October 2, 2012 Published: October 3, 2012 2173

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coupled plasma mass spectrometer). To test the potential of multifunctional gold nanorod-based probe, two-photon luminescence imaging was performed over CAL 27 cultured cells. Later on, in vivo studies performed via NIR laser irradiation at tumor sites in CAL 27 xenografts (CAL27 cells injected in living mice to obtain tumor) on BALB/c nude mice as the model further confirm the localized targeting as observed by elevated temperature at the tumor site. These results have confirmed that these functionalized gold nanorods can serve as a biocompatible, nontoxic, and cancer-specific probe to be utilized in targeted delivery and can be helpful in photothermalrelated therapies.

surface modification schemes have been used to replace CTAB, but they often suffer from poor biocompatibility, increased particle size, and a low capacity for loading additional moieties to enable multifunctional applications. Most of these modified particles have only improved targeting and photothermal effect in in vitro cancer cells.10,16,17 Among these modifications, PEGylated gold nanorods have shown the best performance in terms of toxicity and photothermal effect both in vitro and in vivo, but they do suffer from rapid excretion (half-life ∼1 h).18 To solve this rapid excretion problem, chitosan has been introduced recently to extend the circulation time.18 Chitosan is biodegradable, biocompatible, and water-soluble.19−23 The presence of primary amine groups also enables bioconjugation with a variety of molecules and permits the introduction of other functional groups such as carboxyl or acetylene groups. Furthermore, it maintains its structure in a neutral environment, although it becomes soluble and degrades in an acidic environment, which indicates that chitosan can act as an acidresponsive drug delivery carrier by degrading and releasing the drug in the desired environment.24 However, the high molecular weight of chitosan limits its performance in biological applications due to limited water solubility and particle− particle cross-linking.25 Therefore, low-molecular-weight chitosan or chitosan oligosaccharide (∼5000 Da) is a better choice for in vivo applications. Most of the methods reported for the in vitro/in vivo application of chitosan have used photopolymerization of acrylated chitosan18 or thiol and oleic anhydride modification of chitosan25−30 to replace the CTAB on the surface of the gold nanorods. However, these reported methods require the attachment of functionalized chitosan directly onto the surface of the gold nanorods, which may lead to aggregation and limit its performance for in vivo applications. Therefore, covalent conjugation of chitosan to the gold nanorods is highly desired to provide additional stability for in vivo applications. Hence, our aim is to develop a multifunctional probe, which utilizes the unique properties endowed by gold nanorods as photothermal transducers when excited at near-infrared wavelength and its covalent linkage with chitosan oligosaccharide for a successful acid-responsive delivery of the nanorods. The synthesized probes will be further decorated with cancer-specific antibody to enhance the localization of the probe which was subsequently used for specific targeting and noninvasive imaging based on localized hyperthermia after NIR irradiation. Herein, we report the systematic functionalization of CTABcapped gold nanorods to construct the multifunctional probe. The first step involves the simple replacement of CTAB with MUA (11-mercaptoundecanoic acid) on the nanorod surface with PEG (poly(ethylene glycol)) as an intermediate layer for stability.31 The next step involves the covalent linkage of MUAcoated gold nanorods with low-molecular-weight chitosan oligosaccharide (∼5000) using EDC chemistry32 to produce CO-GNRs (chitosan oligosaccharide-modified gold nanorods). EDC facilitates the formation of a covalent amide bond between the amine groups present on chitosan oligosaccharide and the carboxyl groups on the nanorod surface generated from the MUA treatment. The final step deals with the conjugation of CO-GNRs with monoclonal antibody against EGFR (epidermal growth factor receptor) to achieve tumor targeting functionality due to overexpression of EGFR in CAL 27 cells.33 Before in vivo tumor targeting application, we have also evaluated the toxicity of the CO-GNRs both in vitro and in vivo and quantified the cellular uptake by ICP-MS (inductively

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EXPERIMENTAL SECTION Materials. HAuCl4 (hydrogen tetrachloroaurate (III) hydrate), CTAB (hexadecyltrimethylammonium chloride), NaCl (sodium chloride), NaBH4 (sodium borohydride), ascorbic acid, PAA (poly(acrylic acid) , Mw ∼15 000g/mol), PSS (poly(sodium-4-styrenesulfonate), Mw ∼70 000g/mol), HCl (hydrochloric acid), EDC (carbodiimide), chitosan oligosaccharide lactate (Mw ∼5000), and anti-EGFR monoclonal antibody were purchased from Sigma-Aldrich (E2760), mPEG-SH from Nektor, Mw ca. 5000, and MTT assay kit purchased from Sigma. All chemicals were analytical grade and were used without further purification. Instrumentation. UV−visible spectra of gold nanorod solutions were measured in a UV−visible spectrophotometer (JASCO V-570) using quartz cuvettes with a 1 cm path length. For transmission electron microscopy (TEM)/EDAX, nanorod solutions were dried on a carbon-coated film and imaged at 100 kV (JEM 2010, JEOL). Zeta potential of the nanoparticles was measured using a Brookhaven Instruments BI-90 plus particle size analyzer and zeta potential analyzer. FT-IR spectra were recorded as attenuated total reflectance (ATR) on a PerkinElmer spectrum between 400 and 4000 cm−1. Preparation of the Au Seed Solution. The seed solution was prepared by mixing 9.75 mL of 0.1 M CTAB solution with 0.25 μL of 0.1 M HAuCl4. To the stirred solution, 0.6 mL icecold 0.01 M NaBH4 was added, which resulted in the formation of a bright brownish-yellow solution. Vigorous stirring of the seed solution was continued for 2 min, and then the solution was stored at 30 °C without further stirring. Preparation of the Growth Solution and Nanorods. The growth solution was prepared by mixing 9.5 mL of the CTAB solution at 0.1 M to 0.5 mL of 0.01 M HAuCl4, which resulted in the dark yellow color of the growth solution. A total of 100 μL of 0.01 M AgNO3 solution was added to the solution at 25 °C. After gentle mixing, 80 μL of 0.1 M freshly prepared ascorbic acid solution was added to the test tube. The addition of an additive such as HCl (0.1 M) just before the injection of the seed solution tuned the longitudinal surface plasmon peak from 680 to 850 nm. Further addition of Na2S allowed the red shift of the longitudinal surface plasmon peak to 1050 nm. The nanorod solutions were centrifuged twice at 7000 rpm for 20 min and redispersed in double-distilled water to remove excess CTAB molecules. Surface Modification of CTAB-Capped Gold Nanorods. MUA−Chitosan Modification. Before 11-mercaptoundecanoic acid (MUA) modification to generate carboxyl groups over CTAB-stabilized gold nanorods, we introduced PEG to provide steric stability to the nanorods. Excess PEG molecules were removed by centrifugation at 8500 rpm for 15 min, and the PEGylated rods were resuspended in H2O. To this 2174

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alone also absorbs light at the wavelength used for the protein assay (560 nm). After computation, it was found that about that ∼88% of EGFR antibodies remain attached in the CO-GNR probe even after 24 h of shaking at 100 rpm at 37 °C. Determination of Number of Antibody per Nanoparticles Using MicroBCA Assay Kit (Pierce). Protein concentrations of the antibody-conjugated nanorod solutions were measured with the bicinchoninic acid (BCA) assay (Pierce) following manufacturer’s protocol. Supernatant from the gold nanorod solution (without antibody, 200 μL gold nanorod in PBS), antibody-conjugated nanorod (5 μL antibody + 200 μL gold nanorod in PBS, wherein the supernatant contains unbound antibody) and antibody alone (5 μL antibody + 200 μL PBS to represent total antibody added) were measured and computed against a standard curve composed of serial dilution of known concentration of bovine serum albumin diluted in PBS. Aside from subtracting a reagent blank, the no-antibody gold nanorod supernatant was also subtracted from the antibody-conjugated gold nanorod supernatant measurements to eliminate background from unreacted gold nanorods. The binding efficiency was then calculated based on this forumula

solution, 0.5 mL of a 20 mM solution of 11-mercaptoundecanoic acid (MUA, Sigma-Aldrich) in ethanol was added into 5 mL of the gold nanorod solution and sonicated at room temperature followed by raising the temperature till 50 °C for 2 h and then stirred mildly for 24 h at room temperature for ligand exchange process. Nanorods were then collected by centrifugation at 7000 rpm for 15 min and resuspended in PBS buffer solution. The zeta potential measured for MUA-capped gold nanorods is −35 ± 3 and particle size by DLS is found be around 82 ± 4.3.34 For covalent binding, EDC 0.04 g was simultaneously added with chitosan oligosaccharide lactate (average Mw ∼5000 dissolved in a mixture of acetic acid and water (1%)) to 5 mL of a MUA-coated gold nanorod solution. The solution was mixed, stirred overnight, centrifuged at 7000 rpm for 15 min, and resuspended in PBS (pH 3.4). The zeta potential measured for chitosan-capped gold nanorods (COGNRs) is −35 ± 3 and particle size by DLS is found be around 82 ± 4.3. PSS-Coated Gold Nanorods. To the purified CTAB capped GNR solution (1 mL) was added to 200 μL of PSS (10 mg/mL prepared in 1 mM NaCl) and NaCl (100 μL, 1 mM) simultaneously. The resulting solution was stirred vigorously for 30 minutes, and the process is repeated again for layer-bylayer approach. Surface-modified GNRs with PSS solution were centrifuged twice at 10 000 rpm to remove excess polyelectrolyte and redispersed in 1× PBS (pH = 7.4). PAA-Coated Gold Nanorods. To the purified CTAB-capped GNR solution (1 mL) was added 200 μL of PAA (10 mg/mL prepared in 6 mM NaCl) and NaCl (100 μL, 6 mM) simultaneously. The resulting solution was mixed gently for 3 h for complete polymer coating. To remove excess PAA polymer, the solution was centrifuged for 3 min at 14 000 rpm. The pellet was redispersed in 1× PBS (pH = 7.4). Pegylated Gold Nanorods. CTAB-coated gold nanorod solution was centrifuged at 14 000 rpm for 10 min, decanted, and resuspended in water to remove excess CTAB. A thiolterminated PEG solution (200 μL, 5 mM) was added to the gold nanorod solution (1 mL, 1 mM). The mixed solution was stirred for 24 h at room temperature, dialyzed for 3 days, and redispersed in 1× PBS (pH = 7.4). Preparation of anti-EGFR Conjugated CO-GNRs. The CO-GNR-capped nanorods were then mixed with an antibody solution (5 μL/mL) that was diluted in PBS and allowed to react for 30 min. The antibodies are presumed to be bound to the CO-GNRs surface by electrostatic physisorption interaction.35 The nanorods conjugated with anti-EGFR monoclonal antibodies (Sigma) were centrifuged and redispersed into PBS (pH 7.4) to form a stock solution with an optical density of approximately 0.5 at 786 nm. The anti-EGFR/nanorod conjugates were stable at 4 °C for several days. The binding efficiency was estimated by BCA assay kit (Pierce). Stability of anti-EGFR Conjugated CO-GNRs. To investigate the leakage of anti-EGFR from CO-GNR probe in 24 h, anti-EGFR conjugated CO-GNR and CO-GNR without anti-EGFR were suspended in 300 μL PBS in a 1.5 mL microtube and incubated for 24 h at 37 °C with 100 rpm shaking. After incubation, the solutions were centrifuged and the resulting supernatant was used to quantify the protein using Micro BCA assay kit (Pierce) following the manufacturer’s protocol. Protein concentration were computed against a standard curve, and values for CO-GNR without anti-EGFR were subtracted from the values for anti-EGFR conjugated COGNR to reduce the background from the GNR, since GNR

{[μg protein of total antibody added − (μg protein of unboound antibody − μg protein[background] nanorod only)]/μg protein of total antibody added]} × 100

The reaction and the assay were done more than three times with comparable results. The binding efficiency was estimated to be about 80% by BCA assay kit and 20 ± 5 antibody per nanoparticles is calculated by no. of antibodies/no. of Au atoms per nanoparticle. Cytotoxicity Assay. The cytotoxicity of the GNRs was analyzed using the CAL 27 oral adenosquamous carcinoma cell line. Cells were seeded in 96-well plates at a density of 5 × 103 cells per well and incubated for 24 h at 37 °C in a humidified incubator. CAL 27 cells were treated with the indicated concentrations (1−200 μg/mL in terms of gold nanorods) of surface-modified gold nanorods and incubated for 72 h. Cell viability was measured by MTT assay kit (Sigma) by following manufacturer’s protocol. Typically, PBS solution containing 10 μL of 5 mg/mL of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) solution was added to each well and incubated for an additional 4 h. After mixing, 90 μL of MTT solubilization solution was added to each well. The stain was aspirated and purple-colored crystals were dissolved with acidic isopropyl alcohol. The optical density (OD) of the solvent was measured at 570 nm using a microplate reader (Molecular devices Spectra Max M2e spectrophotometer). All experiments were carried out three times. Cellular Uptake. The cellular uptake of gold nanorods was investigated in terms of Au3+ concentration using ICP-MS analysis. The cells were incubated with surface modified gold nanorods at 37 °C for 4 h. After 4 h of incubation, the cells suspension was removed and the attached cells were washed with phosphate-buffered saline solution (pH 7.4) to ensure complete removal of unbound gold nanorods. Microwaveassisted digestion was adopted for 90 min to dissolve Au out of the cells using aqua regia. Later, 0.1 mL of treated solution was diluted to 50 mL volumetric flask. The signal of dissolved Au solution was measured by ICP-MS, and the concentration was 2175

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Figure 1. Schematic representation of applied strategy used for bioconjugation.

of CO-GNRs, blood was drawn from the heart, and animals were sacrificed to obtain the organs for histopathology studies. Blood parameters were analyzed with an Abbott Cell-Dyn 3700 machine, and biochemical parameters were analyzed with a Fuji Dri-Chem Clinical Chemistry Analyzer FDC 3500. Biodistribution and in Vivo Imaging. To induce a solid tumor, CAL 27 cells (10 × 106 in 100 μL PBS) were injected subcutaneously into both the left and right rear flank areas of male BALB/c nude mice with ages from 6 to 7 weeks. When the tumors grew to approximately 50 mm3 in diameter, antiEFGR-conjugated CO-GNRs (100 μg in terms of GNR amount) suspended in 0.85% saline solution (100 μL) were either injected intravenously through the tail vein (4 mg/kg) or directly at the xenograft site. Saline solution was used as control. Biodistribution of CO-GNRs and anti-EGFR conjugated CO-GNRs was performed after 24 h after intravenous injection. The major organs including tumors were lysed with HNO3 and the amount of Au was calculated by ICP-MS. Next, the thermal images were obtained by irradiating the mice by NIR light which were collected by FLIR A320 infrared camera at the indicated time after intravenous or direct injection. Statistical Analysis. All the statistical analysis was carried out using two-tailed Student’s t test, with P < 0.05 considered to be statistically different.

calculated using the calibration method. ICP-MS (inductively coupled plasma mass spectrometer) (Thermo−II) was used with direct nebulization in normal mode using optimized conditions consisting of an Xs skimmer cone with a platinum sampler cone. The extraction voltage was typically −573 V, the Rf power 1250 W, focus voltage was 17.5 V, and the nebulizer gas flow rate was 0.91 L/min. Dwell times were 10 ms for Au; 100 sweeps were required per replicate and 10 replicates per sample. Formula used for Au NR per cell Wt. of single Au atoms/Wt. of single Au NR = Number of cells

Western Blot. Protein was extracted with RIPA buffer with protease inhibitors (150 mM NaCl, 50 mM Tris pH 7.2, 0.1% SDS, 1% Triton X-100, 1% deoxycholate). Later, 30 μg protein lysates from either CAL27 or from mouse liver were loaded per lane of 8% SDS-PAGE gel, transferred in PVDF membrane (Millipore), followed by incubating overnight with antibody, 1:3000 dilution in 1% nonfat milk in 0.05% PBST. Upper half of the blot with antibody was run against EGFR (Cell Signaling), and the lower half with β-actin (Abcam). The blot was then washed with 1× PBST for 30 min, incubated with HRP-labeled antibody against rabbit (Abcam) for 1 h at RT and again washed for 30 min. The washed blot was then visualized by incubating it for 1 min with ECL substrate (T-Pro Lumifast Plus Chemiluminsescent substrate). Toxicity Evaluation of CO-GNRs in BALB/C Mice. Use of the mouse model for toxicity studies was approved by and conducted according to the guidelines of the Academia Sinica Institutional Animal Care and Utilization Committee. Male 6week-old BALB/c nude mice were obtained from the breeding stock of the Institute of Biomedical Sciences (IBMS), Academia Sinica, Taipei, Taiwan. The animals were housed in a specific pathogen-free environment under controlled conditions of light and humidity and were given sterilized food and water ad libitum. Hematological and biochemical parameters and the histopathology of the liver, kidney, lung, and spleen in mice treated with CO-GNRs (100 μg in terms of GNR amount) were examined at the Pathology Core of the Institute of Biomedical Sciences, Academia Sinica, and Taiwan Mouse Clinic. After the seventh day following the intravenous injection

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RESULTS AND DISCUSSION Characterization of CTAB-Capped Gold Nanorods. We first synthesized the tunable CTAB-stabilized gold nanorods by a modified additive-controlled method,36 which were later characterized by TEM for various aspect ratio, elemental analysis using EDS and FT-IR (characterization details are provided in the Supporting Information Figures S1 and S2). The gold nanorods used in the in vitro and in vivo experiments had a length of 47 ± 3 nm and a diameter of 11 ± 1.8 nm (an aspect ratio of 4.2 as measured from the TEM image shown in Figure 2a) and exhibit longitudinal SPR absorption at 780 nm as shown in Figure 2b. Surface Modification of CTAB-Capped Gold Nanorods. Due to the toxicity issues associated with the CTABcapped gold nanorods, by convention, its surface has to be further modified for its possible use in biological application. We have used 11-mercapto-undecanoic acid (MUA) mod2176

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Figure 2. (a) TEM image of 780 nm gold nanorods. (b) UV−vis−NIR extinction spectra of surface-modified and CTAB-capped gold nanorods with a 780 nm peak. (c) Zeta potential measurements of various surface-modified gold nanorods before (in PBS) and after treatment with serum. (d) Particle size measurement by DLS before (in PBS) and after treatment with serum for 8 h. n = 3 for DLS and zeta potential measurements.

ification to generate carboxylic groups over CTAB-stabilized gold nanorods, which was later covalently linked with chitosan and used for further experiments (see Supporting Information Figures S3, S4, and S5). The chemical mechanism of the reaction of coupling between MUA and chitosan involves activating the carboxyl group of MUA with EDC to form an unstable reactive o-acylisourea ester and subsequently allowed to react with the amine groups present over the surface of chitosan to form a stable amide bond (Figure 1). Aside from surface modification with low-molecular-weight chitosan oligosaccharide (Mw ∼5000), we have also used various polyelectrolytes such as PSS (poly(sodium-4-styrenesulfonate), PAA (poly(acrylic acid)), and biocompatible/biodegradable polymers, i.e., mPEG-SH (poly(ethylene glycol)) to replace cytotoxic CTAB for biological experiments. Surface modification with PSS, PAA, PEG, and chitosan oligosaccharide produced a negligible shift within 10 nm in the longitudinal SPR peak (Figure 2b), indicating that these surface modifications did not significantly change the optical properties of the nanorods. Figure 2c shows the zeta potential measurement to evaluate the stability for all surface-modified gold nanorods in PBS and after treatment with culture medium containing 10% FBS (fetal bovine serum) for 8 h at pH = 7.4. The zeta (ζ) potential of gold nanorods serves as a criterion for inspecting surface charge changes on the nanoparticles, as well as a measure of their stability followed by various surface coating. In addition, the size determination by dynamic light

scattering (DLS) showed increased particle size after treatment with cell culture media containing 10% fetal bovine serum (FBS) in Figure 2d. This may be due to possible binding of serum proteins over the nanorod surface, as it can bind to many different surface charges due to its differently charged domains.37 However, for biological applications, biocompatibility and cytotoxicity are still the primary concerns. Cytotoxicity. To test the biocompatibility, we evaluated in vitro cell viability at different concentration of various surfacemodified gold nanorods by using MTT Assay using CAL 27 oral cancer cells at a seeding density of 5 × 103 cells/well. Figure 3 displays the result of the cytotoxicity measurement 72 h after the addition of the surface-functionalized gold nanorods at various concentrations. CTAB-stabilized gold nanorods exert a concentration-dependent loss of viability (between 50% and 96%), whereas no significant cytotoxicity was observed for all other surface-modified gold nanorods. By comparison of all the surface-modified nanorods tested in this study, both PEG and chitosan modification exhibited the least toxicity. However, we chose to use chitosan in the subsequent experiments due to the inherent limitation of rapid excretion of PEG (half-life ∼1 h).18 Cellular Uptake. Furthermore, the cellular uptake of gold nanorods was quantified by inductively coupled plasma mass spectrometry (ICP-MS) 4 h after the addition of 0.1 μg gold nanorod solution. The results demonstrate a clear effect of the surface modification on the uptake of gold nanorods by CAL 27 cells (Figure 4a). CTAB-coated gold nanorods exhibited the 2177

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lowest uptake, while the gold nanorods modified with chitosan exhibited the highest uptake among all the other surface modifications. With both viability and cell uptake taken into account, chitosan appeared to be the best choice for surface modification, and thus, chitosan was used for the antibody conjugation for further experiments. Zeta potential of antiEGFR conjugated CO-GNRs was found to be −18 ± 2.86 mV, and size distribution by DLS measurement was observed around 104.5 ± 5.28 nm.38 The UV−visible measurement showed the red shift in the longitudinal surface plasmon peak after conjugation of anti-EGFR with CO-GNRs indicating successful bioconjugation39 (Supporting Information S6). AntiEGFR-conjugated CO-GNRs showed an enhanced cellular uptake compared to CO-GNRs as indicated statistically. In addition, the distribution of CTAB-stabilized gold nanorods and anti-EGFR conjugated CO-GNRs was also visualized by transmission electron microscopy (TEM), which clearly showed that the nanorods were trapped inside the vesicles of the cells (Figure 4b,c, respectively). Two-Photon Luminescence Imaging. Gold nanorods are known to exhibit strong two-photon fluorescence. Therefore, the distribution of gold nanorods among CAL 27 cells were further investigated with two-photon luminescence imaging using a Ti:sapphire laser (100 fs, 80 MHz laser pulse and laser power of 3.6mW) with an excitation wavelength of 780 nm.9 Consistent with the cellular uptake measured by ICP-MS, antiEGFR-conjugated CO-GNRs were observed to be enriched inside the cells compared to CO-GNRs alone (Figure 5). Furthermore, no two-photon signal was observed from control cells without gold nanorods. To check the specificity of the CO-GNRs, we used Chinese hamster ovary cells (CHO cells) because of its nonexpression of epidermal growth factor receptor (EGFR), which makes them an ideal negative control in our investigation for targeted delivery of gold nanorods. The results clearly indicate that CHO cells which do not express EGFR receptors showed negligible cellular uptake with antiEGFR conjugated CO-GNRs in contrast with Cal27 cells, thus confirming the specific targeting potential of CO-GNRs. Furthermore, we also used IgG-modified COGNRs as a negative control to demonstrate the selectivity of anti-EGFRconjugated COGNRs in cal27 cells (Supporting Information Figure S7). In Vivo Toxicity. Based on the in vitro toxicity and cellular uptake results, the in vivo toxicity of gold nanrods (i.e., COGNRs and anti-EGFR conjugated COGNRs) was also analyzed. To test the in vivo toxicity, an intravenous tail vein injection containing (100 μg in terms of GNR amount) suspended in PBS (100 μL) at pH 7.4 was administered in the BALB/c nude mice model. The toxicity was evaluated 7 days after injection using hematological, clinical biochemistry and histological analysis. Both surface-modified gold nanorods showed no significant difference in WBC (white blood cell) count in comparison with the control group. However, a slight toxic effect on the bone marrow was observed indicated by a slight reduction in RBC (red blood cell) count and Hgb (hemoglobin) concentration (Supporting Information Table S1). The rest of the blood parameters of the surface-modified gold nanorod treated mice are shown in Table S2 (Supporting Information) and presented no significant changes. The biochemical profile of mice treated with surface-modified gold nanorods (CO-GNRs and anti-EGFR conjugated COGNRs) was also examined in this study. In general, drugs administered to the body of an animal pass through the liver for metabolism.

Figure 3. MTT proliferation assay of CAL 27 cells treated at different concentrations of surface-modified gold nanorods.

Figure 4. (a) Cellular uptake of CTAB and surface-modified gold nanorods as measured by ICP-MS (n = 3). *P value obtained by comparison with CTAB; error bars represent 1 standard deviation. (b) TEM images of CTAB-stabilized gold nanorods and (c) anti-EGFR conjugated CO-GNRs; Scale bar: 2 μm and 100 nm for inset.

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Figure 5. Two-photon luminescence and merged image with DIC of CO-GNRs with and without anti-EGFR conjugation in Cal 27, CHO cells were used as a negative control for CO-GNRs. Cells were incubated with 0.1 μg gold nanorods for 4 h. Scale bar, 10 μm.

Figure 6. Biodistribution of gold nanorods in nude BALB/c mice at 24 h after intravenous (tail vein) injection. The gold amounts in tissue samples were measured by ICP-MS (n = 3).

However, no significant changes in the AST (aspartate aminotransferase), ALT (alanine transaminase), CRE (creatinine), BUN (blood urine nitrogen), or GLU (glucose) levels were observed in COGNRs in contrast with slightly higher values observed in AST and ALT levels of anti-EGFR conjugated COGNRs (Supporting Information Table S3). Furthermore, histopathological assessment of major organs (kidney, heart, spleen, lung, and liver) was also conducted 7 days after the administration of both the gold nanorod groups

(SI Figure S8). Only the liver exhibited a significant difference between the control and CO-GNR, anti-EGFR conjugated COGNR cases. The liver section shows a few minor spots of vacuolar degeneration, a reversible type of change. This reversible change observed in the liver was well-correlated with the increase in the AST level. However, all the measured factors were within the normal range, and no statistically significant difference was observed in renal function parameters such as blood urea nitrogen and creatine levels. These results 2179

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Figure 7. NIR thermal imaging of tumor in CAL 27 xenografts after (a) 1 min laser irradiation (780 nm, 3 W/cm2), (b) 2 min laser irradiation 24 h after tail vein injection, and (c) 30 s laser irradiation 1 h after direct injection at tumor site. Control mice were administered via saline.

Figure S10). Knowing that the anti-EGFR conjugated COGNRs (100 μg in terms of GNR amount suspended in 100 μL of PBS, pH = 7.4) concentrated in the tumor site and that the gold nanorod exhibited large plasmonic resonance in the NIR region, we then conducted selective NIR laser irradiation in an animal model with solid tumors. Anti-EGFR conjugated COGNRs were intravenously injected, and 24 h after administration, the mice were exposed to NIR laser irradiation (780 nm, 3 W/cm2, 2 mm beam diameter) for 2 min. As shown in Figure 7, the elevation in temperature at the tumor site during laser exposure confirmed targeted delivery of CO-GNR. AntiEGFR conjugated CO-GNRs (100 μg in terms of GNR amount suspended in 100 μL of PBS, pH = 7.4) were also injected directly into the tumor site to observe any thermal changes associated with this different injection protocol. One hour after administration, laser irradiation at the tumor site yielded a temperature increase up to ∼71 °C in only 30 s compared with intravenous injection, which took 2 min at the same power density (in vitro heating profile of GNR in culture media with increasing laser irradiation time experiment was also carried out

suggested that there was no significant toxicity of the surfacemodified gold nanorods. Based on the hematological, clinical biochemistry analysis and histopathological examination, both CO-GNRs and anti-EGFR conjugated COGNRs do not exert statistically significant toxicity at the concentration (100 μg in terms of GNR amount) tested here. Biodistribution and Photothermal Imaging. To further investigate the localization of CO-GNRs in an animal model with solid tumors, the anti-EGFR conjugated CO-GNRs were intravenously injected (100 μg in terms of GNR amount suspended in 100 μL of PBS, pH = 7.4) into the tail vein of BALB/c nude mice bearing bilateral CAL 27 tumors. Major organs including tumors were excised 24 h after intravenous injection to determine the biodistribution of the gold nanorods. ICP-MS was used to quantify the amount of gold nanorods in various organs. Figure 6 clearly shows the higher uptake of antiEGFR conjugated CO-GNRs in the tumor and low liver uptake, indicating efficient targeting in contrast with CO-GNRs alone (also confirmed by Western blot of comparison between overexpression of EGFR in CAL 27 and BALB/c mice liver; see 2180

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MY3, NSC 101-212-M-001-011, and Academia Sinica Research Project on Nanoscience and Technology.

to compare in vivo results; see Figure S9). This observation could be explained by the decreased amount of gold nanorods reaching the tumor site for systemic injection compared with direct injection. Although EGFR was used to enhance the specificity of the delivery to the tumor site, the circulating blood delivers the CO-GNRs to other body parts, and EGFR is present in other organs although at lower levels than in the tumor. Nonetheless, the evidence still shows the enrichment in the tumor and the specific delivery of the CO-GNRs to the tumor site.

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CONCLUSION In conclusion, we have demonstrated the systematic functionalization and characterization of multifunctional gold nanorodbased probes for specific targeting and noninvasive imaging applications. Our results suggest that anti-EGFR-conjugated CO-GNRs showed no significant toxicity either in vitro or in vivo. Additionally, cellular uptake and biodistribution were analyzed quantitatively for Au content with ICP-MS, which revealed successful localization of anti-EGFR-conjugated COGNRs in the tumor area. More importantly, two photon optical images clearly demonstrate the internalization and selectivity of anti-EGFR-conjugated CO-GNRs for CAL 27 human tongue oral cancer cells overexpressing EGFR. This observation was perfectly correlated with the localized temperature shift at the tumor site after irradiation with NIR laser of the BALB/c mice with CAL27 xenografts. Based on these results, functionalized CO-GNRs can be a promising probes for biosensing, drug delivery, noninvasive imaging based on localized hyperthermia generated by gold nanorods and other photothermal-based applications.

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ASSOCIATED CONTENT

S Supporting Information *

Additional figures and tables as described in the text. This material is available free of charge via the Internet at http:// pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. Author Contributions

S. Charan synthesized multifunctional gold nanorod-based probes and performed experiments, analyzed data and wrote the paper. S. Charan and K. Sanjiv prepared BALB/c nude mice bearing CAL27 xenografts and prepared cultured cells. K. Sanjiv and Yi-Fan Chen perform in vivo toxicity. S. Charan and F. C. Chien performed experiment for two-photon luminescence imaging and NIR based in vivo studies. N. N. Nergui helped during synthesis and surface modification of gold nanorods. S. H. Huang prepared TEM samples of cell sectioning. N. Singh and C. W. Kuo supervised the experiments for surface modification and cellular uptake studies. T. C. Lee provided reagents and materials for in vitro and in vivo experiments. C. W. Kuo and P. Chen designed experiments, analyzed data, and wrote the paper. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is supported, in part, by National Science Council, Taiwan, under contract NSC 100-2113-M-001-0272181

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