Chitosan-Modified Stable Colloidal Gold Nanostars for the

Article pubs.acs.org/JPCC

Chitosan-Modified Stable Colloidal Gold Nanostars for the Photothermolysis of Cancer Cells Ivan Baginskiy,†,& Tsung-Ching Lai,‡,& Liang-Chien Cheng,† Yung-Chieh Chan,‡ Kuang-Yu Yang,§ Ru-Shi Liu,*,†,‡ Michael Hsiao,*,‡ Chung-Hsuan Chen,‡ Shu-Fen Hu,∥ Li-Jane Her,⊥ and Din Ping Tsai*,§,# †

Department of Chemistry and #Department of Physics, National Taiwan University, Taipei 106, Taiwan ‡ The Genomics Research Center and §Research Center for Applied Science, Academia Sinica, Taipei 115, Taiwan ∥ Department of Physics, National Taiwan Normal University, Taipei 116, Taiwan ⊥ Innovation Center, Taiwan Hopax Chemicals Manufacturing Company, Limited, Kaohsiung 831, Taiwan S Supporting Information *

ABSTRACT: The preparation and properties of plasmonic gold nanostars (Au NSs) modified with a biopolymer chitosan are reported. The colloidal stability of Au NSs at the physiological pH of 7.5 and their performance in the photothermolysis of cancer cells in vitro were compared with those of gold nanorods (Au NRs). The optical characteristics of chitosan-modified Au NSs dispersed in a medium with pH = 7.5 had higher stability than those of chitosan-capped NRs because of the slower aggregation of NSs. At pH = 7.5, the chitosan-modified Au NRs formed aggregates with highly nonuniform sizes. On the other hand, Au NSs formed small chain-like clusters, in which individual NSs were connected to one another, preferably via association of branches with central cores. It is possible that the difference in areal charge density at these parts of NSs is responsible for their preferred association. Flow cytometry analysis showed the relatively nonequivalent distribution of the chitosan-capped Au NRs across the cell line compared with that of Au NSs because of the fast and nonuniform aggregation of NRs. An in-vitro photothermolysis experiment on J5 cancer cells showed that energy fluences of 23 and 33 mJ/cm2 are necessary to cause complete death of J5 cells incubated with 4 μg/mL chitosan-capped Au NSs and NRs, respectively. When chitosan was used as a surface-capping agent, the Au NSs exhibited higher colloidal stability at the physiological pH than the NRs and lower energy fluence necessary for cell photothermolysis because of more uniform cellular uptake.

1. INTRODUCTION Studies on noble metal nanoparticles (NPs) have attracted increasing interest in the field of sensing, optoelectronics, and biomedicine because of their enhanced optical properties including strong absorption and scattering of light in the visible and near-infrared (NIR) wavelength regions related to the localized surface plasmon resonance (LSPR).1−3 In the biomedicine field, nanomaterials are selected with consideration of biocompatibility, colloidal and chemical stability, and ability to functionalize depending on the intended application.4,5 The unique combination of optical properties and chemical stability makes gold nanoparticles (Au NPs) ideal nanostructures for a wide range of biomedical applications.6−13 However, colloidal nanomaterials tend to aggregate rapidly. This underlines the need for surface-protective agents, which would prevent aggregation and enhance the properties of NPs for the intended application. A large number of surface-modifying agents have been proposed.14,15 The most commonly used are poly(ethylene glycol) derivatives, which provide high colloidal © 2013 American Chemical Society

stability as well as efficient masking of Au surface charge, which minimizes unintentional adsorption.15 Polystyrene sulfonate (PSS) is also often used because of the simple modification procedure.16 However, negatively charged polycationic compounds such as PSS cover NPs over the existing cetyltrimethylammonium bromide (CTAB) micelle capping used in NP synthesis.17 This creates danger of unintended release of toxic CTAB. The natural choice for the safe surface-capping agents is biomolecules that are innate in many types of biological organisms. Chitosan is a derivative of chitin, an abundant natural biopolymer, which underlines its advantage of biocompatibility and biodegradability. Hence, chitosan is extensively used in medicine, and chitosan NPs are studied in nanobiology as nanocarriers for drugs and functional groups.18−21 The properties of chitosan in aqueous media Received: November 14, 2012 Revised: January 10, 2013 Published: January 15, 2013 2396

dx.doi.org/10.1021/jp311271p | J. Phys. Chem. C 2013, 117, 2396−2410

The Journal of Physical Chemistry C

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strongly depend on the pH.22,23 The chitosan molecule is positively charged in acidic medium because of the protonation of its amino groups, which results in increased solubility. In a basic environment, protonated amino groups lose H+ and the chitosan molecules become neutral. Thus, the solubility of chitosan decreases. The positive charges in a weak acid provide chitosan with an ability to bind to negatively charged Au NP surfaces via an electrostatic interaction.24 In addition, the chitosan-based composites were found to have prolonged circulation in the blood (up to 72 h) as well as good accumulation in tumors via the leaky vasculature and impaired lymphatic drainage system phenomenon (enhanced permeability and retention effect).25−27 In a basic environment owing to the absence of electrostatic repulsion, chitosan-capping layers can form interpolymeric links with one another via hydrogen bonds and van der Waals forces, which results in the agglomeration of chitosan-capped Au NPs. This effect is extensively used in the directed assembly of Au NPs or production of chitosan films with embedded NPs via pH variations.28 However, in biomedicine, unintended aggregation is dangerous because controlling the NP assembly and predicting the properties of the formed aggregates are extremely difficult.29 This is true for chitosan since the physiological pH is ∼7.4. Thus, optimal NP types with minimal tendency to aggregation that exhibit the necessary properties for targeted applications must be identified when chitosan capping is intended to be used. One of the many fields where LSPR-enabled Au NPs have gained major interest is noninvasive photothermal therapy (PTT) of cancer.30 The PPT method is based on conversion of electromagnetic waves energy into local heat with the following hyperthermia or destruction of cancerous cells. NIR laser irradiation is generally used in this therapeutic approach because of the deep penetration ability in living tissues.31 Au NPs with high intrinsic absorption efficiencies can function as mediators of nonradiative photothermal conversion, which provide local destructive heating in tumors loaded with Au NPs without affecting healthy tissues. However, the optimal NP type suitable for PTT remains to be determined because the LSPR characteristics of NPs and their behavior in biological medium strongly depend on their size and shape. Au nanorods (NRs), nanoshells, and hollow NPs are recognized as the most promising plasmonic nanomaterials in the field of PTT because of their highly tunable LSPR properties.32,33 Au NRs possess the highest absorption efficiency per mass of gold and a narrow extinction band, which is tunable from the visible to the NIR wavelength range via simple adjustments in the aspect ratio of NR.33,34 In addition, highly uniform Au NRs can be synthesized on a large scale. The LSPR arises from the interaction of light with the free-electron gas at the boundary between metal NPs and a dielectric medium.1,2 This results in amplification of the electric field (E-field) near the NP surface and is evidenced by strong absorption and scattering of light. E-field enhancement is particularly high at the sharp edges and acute tips of metal NPs such as nanocubes, triangular nanoplates, and nanostars.35−40 This phenomenon is called the lightning rod effect and is particularly interesting for surface-enhanced Raman scattering (SERS) analysis,41−43 which is based on enhancement of Raman scattering signal of analyte by the optical field created by the LSPR near the metal surface. Interest in SERS applications has spurred studies on star-shaped gold NPs (Au NSs), which have multiple sharp branches and, therefore, exhibit an extra-high E-field enhancement.43−45 The strong

light-absorption efficiency of Au NSs also indicates their potential use as mediators in the noninvasive PTT of cancer cells.45−47 Although the synthesis of zero- and one-dimensional NPs (nanospheres, nanoshells, and NRs) with specific morphologies has become routine, precise control of the shape of NSs remains a big challenge. At present, no method is available to prepare uniformly shaped Au NSs. Therefore, an increasing number of synthetic routes are being developed to improve the controllability of the morphology of Au NSs.43,48 Preparation of branched nanostructures as the products of a versatile seedmediated synthesis was reported by our group.49 In this study, NSs with optimized morphologies were prepared. Surface modification of as-prepared Au NPs was performed using chitosan to improve their biocompatibility and colloidal stability in dilute solutions. The present investigation aims to conduct a comparative study of the colloidal stabilities of chitosan-capped Au NSs and NRs at pH −7.5 in nearphysiological conditions as well as their performances in photothermolysis of cancer cells in vitro using a pulse NIR laser.

2. EXPERIMENTAL SECTION Chemicals. Hydrogen tetrachloroaurate(III) hydrate, trisodium citrate dehydrate (99.9%), silver nitrate (99%), ascorbic acid (AA) (99%), CTAB (99%), low molecular weight chitosan (99%; MW ≈ 17 000; deacetylation ratio approximately 70%), and fluorescein isothiocyanate (FITC) (99%) were obtained from Acros Organics and used without further purification. All water used in the study was reagent grade and produced using a Milli-Q SP ultrapure water purification system (Nihon Millipore Ltd., Japan). Preparation of Au Seeds. Aqueous trisodium citrate (1%, 0.35 mL) was added to 10 mL of 0.25 mM aqueous HAuCl4. The resulting solution was then stirred for 3 min. Afterward, 0.3 mL of ice-cold, freshly prepared 0.01 M aqueous NaBH4 was added, and the solution was stirred for 5 min. The seed solution was maintained at room temperature for ∼2 h prior to use. Preparation of Au NSs in Solution. Au NSs were synthesized using a procedure similar to that described by Chen et al.,49 with several modifications in the synthesis time and AgNO3 concentration. An aqueous solution consisting of 0.1 M CTAB, 0.25 mM HAuCl4, and 0.03, 0.04, 0.05, or 0.06 mM AgNO3 was used as the growth solution. This solution was maintained at 27 °C throughout the experiment. The Au seeds (0.1 mL) were placed in a beaker. Three volumes (1, 10, and 100 mL) of the growth solution were then mixed with 0.06 (first), 0.6 (second), and 6 mL (third) of freshly prepared AA solution (10 mM), respectively. These three colorless solutions were added stepwise to the quiescent Au seed solution at intervals of 30 s. The growth solution was maintained at 25 °C for 24−48 h and then centrifuged twice at 10 000 rpm for 10 min to remove excess CTAB. Preparation of Au NRs. AuNRs with aspect ratios of 3.5 were prepared according to a previously reported procedure.50 First, 10 mL of a solution containing 0.1 M CTAB and 0.25 mM HAuCl4 was mixed with 0.6 mL of ice-cold 0.01 M NaBH4 to prepare the Au seeds. Au seed solution was then kept at 25 °C for 2 h. Afterward, 10 mL of the growth solution containing 0.1 M CTAB, 0.007 mM AgNO3, and 0.5 mM HAuCl4 was mixed with 70 μL of 0.0788 M AA. About 12 μL of the seed solution was then added to the growth solution with vigorous stirring, and the mixture was kept at 27 °C for 3 h. The Au NRs 2397



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acquired using a Varian FTIR-640 spectrometer (Agilent Technologies, USA). UV−vis light absorption spectra of the colloidal NP solutions were obtained using a Shimadzu UV-700 spectrophotometer (Shimadzu Scientific Instruments, Japan) with a 1 cm quartz cell at room temperature. The hydrodynamic size (zeta size) distribution and potential (zeta potential) of the NPs in solution were determined at 25 °C by dynamic light scattering at 633 nm using Zetasizer 3000 (Malvern Instruments Ltd., England). Cytotoxicity Assay. Four cell lines were used. Oral mucosa fibroblasts (OMF) were cultured in a minimum essential medium (MEM, Gibco, USA). Oral epithelial (S-G) and human liver cancer (J5) cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, NY, USA). The BEAS-2b cells were maintained in a bronchial epithelial cell basal medium (BEBM, Lonza, Walkersville, MD, USA) that contained bronchial epithelial cell growth medium (BEGM) SingleQuots. All media were supplemented with 100 units/mL penicillin (Gibco, USA) and 100 mg/mL streptomycin (Gibco, USA). About 2000 cells/100 μL were seeded onto 96-well plates and incubated overnight at 37 °C in a 5% CO2 atmosphere. Afterward, 10 μL of a solution containing 0, 0.01, 0.1, 1, and 10 μL of CTAB (80 μg/mL) or chitosan (160 μg/mL) Au nanomaterial was added to the wells. Cell cultures were then incubated for 72 h. Cell viability was then determined using an AlamarBlue reagent in accordance with the procedure from the manual. Cells were incubated for a maximum of 3 h at 37 °C. Absorbance was read on a plate reader at a single wavelength of 590 nm. In-Vitro Experiments. Cell cultures were grown as a monolayer in 35 mm dishes filled with 1 mL of DMEM at 37 °C and in 5% CO2. In the confocal microscopy and cell photothermolysis experiments, the cell monolayer was grown over a glass plate (Deckglaser, Germany) that was placed at the bottom of a dish. Approximately 0.5 million to 1 million cells in the monolayer were prepared for flow cytometry and microscopic observations, whereas ∼2 million cells were prepared for laser photothermolysis to yield a gap-free monolayer. Cell Flow Cytometry. The J5 cells were incubated in 1 mL of DMEM with the FITC-labeled, chitosan-capped Au NSs and NRs each at 4 and 10 μg/mL Au concentrations. Incubation medium was then removed, and cells were rinsed with PBS solutions several times. Cells were detached using 0.5 mL of a 0.05% trypsin−EDTA solution in PBS and then incubated for 2 min. Approximately 0.5 mL of the DMEM was then added to each well. The obtained specimens were then used for the flow cytometry experiment. The emission (FITC) at 525 nm was acquired at an excitation wavelength of 488 nm using a BD FACSCanto (BD Bioscience, USA) flow cytometer. For the inflow imaging on ImageStreamX Mark II (Amnis, USA), cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and the cells were imaged using a laser at 488 and 405 nm for FITC and DAPI fluorescence, respectively. Images were acquired using a 40× objective (NA 0.75) with a 0.25 μm2 pixel area. Determination of Au NP Mass Uptake. Cells were incubated in 1 mL of DMEM with the chitosan-capped Au NSs and NRs each at concentrations of 4 and 10 μg/mL to determine the average uptake per cell of Au NPs. After incubation, the medium was removed and the cells were rinsed twice with PBS solutions. Cells were then dispersed in 0.5 mL of the 0.05% trypsin−EDTA solution in PBS. Two 10 μL

were then centrifuged twice at 10 000 rpm for 10 min to remove excess CTAB. Surface Modification Using Chitosan. The Au NSs and NRs were modified with chitosan as follows: 0.1 g of chitosan (Sigma-Aldrich, MW 17 000, deacetylation rate 70%) was dissolved in 10 mL of 1% acetic acid. The resulting solution was sonicated for 1 h. The solution was then added dropwise into 100 mL of the Au NPs solution. The mixture was vigorously stirred for 4 h and then set aside for another 4 h. The Au NS and Au NR solutions were centrifuged twice at 7000 and 9000 rpm for 7 min, respectively, to remove excess electrolytes. Surface Modification Using FITC-Conjuncted Chitosan. FITC-labeled chitosan was prepared using a procedure similar to the one described in the literature.51 FITC (10 mg) in 10 mL of dehydrated methanol was added to 10 mL of a 1% chitosan solution. After the reaction proceeded for 3 h in the dark, the FITC-labeled chitosan (FITC−CS) was precipitated by increasing the pH to 10 via addition of 0.1 M NaOH. The unreacted FITC was washed with distilled water and then separated by repetitive centrifugation with subsequent redispersion and sonication until no fluorescence was detected in the supernatant. Au NP surfaces were modified with the FITClabeled chitosan following a procedure similar to that for modification of Au NPs using pure chitosan. Electrical Charge Density and Resistive Heating Simulations. In the simulations, the commercial Comsol Multiphysics software was used to calculate the field distributions, which are based on the finite element method (FEM), to compare the resistive heating properties of Au NSs and NRs. The three-dimensional figures of the NPs were carefully constructed in accordance to their transmission electron microscopy (TEM) images. In addition, the permittivity of Au NPs was determined by the Drude−Lorentz model at a plasmon frequency ωp = 8.997 eV and a damping constant Γp = 0.14 eV in the optical frequency region. On the other hand, the refractive index of the surrounding solution is 1.33 (water). The incident source is an x(y)-polarized plane wave that propagates along the z axis. The wavelength was selected using the value that corresponds with the calculated spectral absorbance peaks from the calculations, namely, 700 and 810 nm for NSs and NRs, respectively. Furthermore, the field distributions were obtained using a lateral section perpendicular to the wave-propagating direction to facilitate the observations. Characterization. TEM was performed to characterize the overall morphology of the samples. TEM images were captured using a JEM-2010 (JEOL, Japan) electron microscope. Highresolution transmission electron microscope (HRTEM) images and electron diffraction patterns were obtained using a JEOL JEM-2100F electron microscope. The specimens were obtained by placing several drops of the colloidal solution onto a carboncovered copper grid and evaporating the solution in air at room temperature. Prior to specimen preparation, the colloidal solution was sonicated for 1 min to promote dispersion of particles on the copper grid. Synchrotron radiation X-ray diffraction patterns were acquired on a beamline 01C2 at the Taiwan National Synchrotron Radiation Research Center (NSRRC) using a wavelength of 0.774907 Å. The Au concentration in the colloidal solution was determined by inductively coupled plasma mass spectrometry (ICP MS) using a Thermo X-Series II spectrometer (Thermo Fisher Scientific Inc., USA). The transmittance Fourier transform infrared (FTIR) spectra of the Au NPs dispersed in KBr pellets were 2398



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probes were then collected from the dispersions and mixed with 10 μL of trypan blue. The cells in 10 μL of the medium were then counted using an automatic cell counter (Invitrogen, USA) and visually observed using a microscope. The remaining cell dispersions were dissolved in 1 mL of aqua regia (HCl:HNO3 = 3:1) for 1 h, diluted 50 times, and then analyzed by inductively coupled plasma mass spectrometry (ICP-MS) using an X-Series II spectrometer. Confocal Microscopy and Photothermal Cell Ablation Experiments. The uptake of the FITC-chitosan-capped Au NSs was determined using a Leica TCS-SP5-SMD confocal microscope (Leica Microsystems GmbH, Germany). The J5 cells were treated in fresh DMEM with Au NSs (16 μg/mL) capped with FITC-modifyed chitosan for 12 h incubation. On the next day, cells were stained with 5 μg/mL LysotTacker Red DND-99 and 50 μg/mL Hoschst 33342 (Invitrogen, USA) for 30 min as given in the user instruction. Stained cells were then observed by confocal microscopy. In the cell photothermolysis experiment, cell monolayers were incubated for 12 h in 1 mL of DMEM with the chitosan-capped Au NP solutions at 10 and 4 μg/mL of Au mass concentrations. After incubation, the culture medium was replaced to remove excess Au NPs. Target cells were irradiated with a 3.11 W NIR femtosecond (fs) pulse laser at a wavelength of 765 nm and an estimated maximum power in the pulse of approximately 18, 26, 41, 60, 90, 105, 140, and 170 mW (measured using a Newport Instrumentsoptical power meter model 842 PE). The full-width at half-maximum (fwhm) of the laser pulse was determined as 88 fs, and the period between pulses was 20.5 ns. The scanned area was a square with a length of 500 μm in the sides that contained around 518 ± 31 cells. The time per scan was 1.314 s. After irradiation, the cells were incubated for an additional 2 h. The DMEM was then removed, and 0.5 mL of trypan blue was added to stain the cells with membrane permeability. Dead cells (necrotic) with leaky membranes appeared blue in the microscope images. To determine the viability of J5 cells upon laser treatment the amount of cells with consistent membranes was counted manually relative to the total number of cells within the scanned area using an optical microscope. The experiment on laser scanning was conducted at 25 °C. An oil-immersion objective lens with a numerical aperture (NA) of 0.7 was used. Energy fluences were calculated in accordance with a previously reported method.52,53 A 512 × 512 pixel area was scanned at a rate of 1.314 s. The exposure time of each pixel per scan was 5.01 μs. The focal spot area was calculated as πd2/4, where d is the fwhm of the beam waist and was calculated from the formula d = 0.61λ/NA = 0.67. In this condition, the total exposure time for Au NPs was estimated as (focal spot area/ pixel area) ×5.01 μs = 0.832 μs per scan. In this study, the scanning of different excited power densities at maximum in pulse, namely, 0.2, 0.31, 0.46, 0.67, 1.01, 1.18, 1.57, and 1.92 kW/cm2, at a focal spot area was applied to the specimens. The number of scans was either 30 or 60. Energy fluences were calculated from the product of the power density, number of scans, and total exposure time per scan.

Figure 1. TEM images of CTAB-encapsulated branched Au NPs that were obtained from a growth solution that contained 0.04 mM AgNO3 within a growth period of (a) 24 and (b) 48 h; (c) HRTEM images of branched Au NPs grown for 42 h; (d) lattice fringes on a single branch tip; (e) synchrotron XRD pattern of Au NP (λ = 0.774907 Å).

Scheme 1. Stages of Au NP Growth and Modification with Chitosan

synthetic process. Au NPs with spherical morphologies were initially used as seeds in the Au growth solution. As the Au seeds grew, the silver cation complexes were adsorbed on the Au surface and functioned as growth inhibitors.49 Hence, after a growth time of 24 h (Figure 1a), irregular plate-like Au NPs with few short branches were observed because the capping ability of the silver ions results in the blocking of most of the Au NP surface.49 Thus, the remaining unblocked parts on the surface of irregular Au NPs expanded in a specific direction, which eventually formed branches. The diameter of the Au NP cores increased only slightly between a growth time of 24 and 48 h. Continuous growth predominantly occurred on the corners of the irregular plate-like Au NPs and in a particular direction, which results in branching (Figure 1b and 1c). Silver capping is effective on specific facets of initial Au crystallites and results in growth of branches rather than expansion of the core. HRTEM was performed to characterize branched Au NPs in detail and identify their crystalline boundaries (Figure 1c). Visible boundaries reveal that a branched Au NP consists of

3. RESULTS AND DISCUSSION TEM images (Figure 1) of the products prepared in the presence of silver ions at a growth time of 24−48 h indicate that the final products were branched particles with 3−6 branches. Longer growth periods resulted in more branches per particle. The average size of the NP core is 30−40 nm, and the branches can grow up to 50 nm. Scheme 1 presents the overall 2399



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absorption band for CTAB, which indicates that CTAB was not present after chitosan treatment. Figure 2 shows UV−vis spectra of the as-prepared branched AuNPs in various synthesis conditions. Shorter and longer

numerous single Au crystallites. The boundaries are in the middle of the branches, which indicates the occurrence of preferential growth along the boundary of two crystallites. This result suggests that Au NPs initially grew in the form of irregular hexagonal or pentagonal multicrystalline structures (Figure 1a), which function as initial intermediate products in the growth of branched Au NPs. If the AgNO3 concentration in the growth solution is 0.04 mM and the synthesis period does not exceed ∼42 h, then most of the formed Au NPs have twodimensional (2D) star-like structures with 3−5 branches (Figure 1c). When the growth time is increased to 48 h or when the AgNO3concentration is 0.05 mM, more irregular three-dimensional (3D) Au NSs with more than 6 branches are formed (Figure 1b). The HRTEM image of a single branch (Figure 1d) shows lattice fringes with an average interfacet distance of ∼0.23 nm, which corresponds to the space between the ⟨111⟩ planes of the Au face-centered cubic (fcc) structure. This result indicates preferential growth on the ⟨111⟩ facets, which is consistent with previous reports on Au NP synthesis in the presence of Ag+ cations.49,54 The crystalline structure of Au NPs was analyzed using synchrotron XRD (Figure 1e). The diffraction peaks of an as-prepared sample coincide with those of a standard Au (ICSD 58393). On the basis of the Scherrer equation, the primary crystallite size was estimated at 16.2 nm from the fwhm of the ⟨111⟩ reflex,55 which is considerably smaller than the particle sizes observed by TEM. These results confirm that each of the Au NSs is multicrystalline, i.e., constructed from several single crystals. Following formation of branched Au NPs, chitosan was used to replace CTAB as a stabilizer on the Au surface for subsequent application. The CTAB-protected Au NPs form a highly stable suspension. However, relatively weak electrostatic bonds between CTA+ and the Au surface result in a CTAB capping layer that is vulnerable to the medium, to thermal treatments, or even to simple dilutions if an excess CTAB is not added. Dilution of both the as-prepared Au NRs and NSs with an Au mass concentration of 80 μg/mL to target 10 and 4 μg/mL results in immediate precipitation of Au NPs because the CTAB content in solution is reduced below its critical micellar concentration and cannot retain stable and continuous capping micelles around NPs.56 Thus, the CTAB on the surface of Au NPs was replaced by the biopolymer chitosan to increase the biocompatibility and stability of NPs in solution. FTIR spectra (Figure S1, Supporting Information) show replacement of CTAB by chitosan on the surface of Au. The characteristic peaks of chitosan are at 3449 cm−1 for ν(OH) (stretching deformation), 1084 cm−1 for ν(C−O−C), 1597 cm−1 for −NH2 bending, and 1650 cm−1 for ν(CO) or ν(C−N). The sharp band at 1386 cm−1 corresponds to formation of a −CH3 group. Comparison with the FTIR spectra of chitosan in Figure S1, Supporting Information, shows that all peaks in the FTIR spectra of the chitosan-modified Au NPs can be indexed to the functional groups of chitosan. The intensity of the peak at 1525 cm−1, which corresponds to an −NH2 group, is considerably reduced, and the peak shifts to higher energy because the interaction between NH2 and the heavy Au atom changes the vibration mode of NH2 and affects its conformation. Figures S1c and S1d (Supporting Information) show the FTIR spectra of the CTAB-modified Au NPs and pure CTAB. These spectra show a strong and sharp characteristic band at 2918 cm−1, which corresponds to the stretching vibration of −CH2 groups and can be compared with those of the chitosan-modified Au NPs.57 FTIR spectra of the chitosan-modified Au NPs show no

Figure 2. UV−vis SPR spectra of branched Au NPs obtained (a) from a solution that contained 0.04 mM AgNO3 in a growth period of 24, 36, 42, and 48 h, (b) from a solution that contained 0.03 mM AgNO3, 0.05 mM AgNO3, and 0.06 mM AgNO3 for 42 h of growth time; (c) deconvolution of absorbance spectra of Au NSs sample grown for 24 h; (d) deconvolution of absorbance spectraof Au NSs sample grown for 48 h.

wavelength LSPR extinction bands appear because of the high anisotropy of the NS LSPR properties. By analogy with the Au NR spectra, shorter wavelengths contributions are assigned to transverse LSPR whereas longer wavelengths are assigned to longitudinal LSPR.33,34,41 The changes in the extinction band intensities and peak positions (Figure 2a and 2b) correspond to the morphological evolution of the branched Au NPs with growth time and variation in the silver content, respectively. The contribution of the longitudinal LSPR to the extinction spectra increases in intensity and shifts to a considerably longer wavelength (red shift) following the increase in the amount and length of the branches, respectively. Extinction spectra of the CTAB-coated Au NPs can be deconvoluted in at least three different bands, namely, T, T′, and L, as shown in Figure 2c and 2d for growth times of 24 and 48 h, respectively. The simulated distributions of the electric energy density of both NSs and NRs are shown in Figure 3a−d. The NS figure is constructed from the HRTEM image from Figure 1c. For simplification, the multifacet branches and central core were approximated with conic and spherical shapes, respectively. For each structure, the x- and y-polarized incidence cases were calculated to determine the various modes of LSPR. Figure 3e−h shows the distributions of the time-average resistive heating of the particles excited by these LSPR modes during polarized illumination. For the star-shaped NPs, the strongest field enhancement is achieved around the branch vertices when the longitudinal axis of the branches is in maximum alignment with the polarization direction (in the XY plane). In addition, the greater enhancement of the E-field is found at more acute vertices (Figure 3a and 3b), which is consistent with previous reports on experimental and theoretical E-field enhancement of Au NSs.35−40 A significantly greater areal density of free 2400



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simulated based on the equation that describes the dissipative electric energy qe37 qe =

1 ε0ωIm ε(ω)|E|2 2

(1)

where ε0 is the permittivity of vacuum, Im ε (ω) is the imaginary part of the permittivity of Au, ω is the frequency of the incident wave, and E is the electric field amplitude. Figure 3e and 3f and 3g and 3h shows the distributions of the time-average resistive heating of Au NSs and Au NRs, respectively. The simulated results of the star-shaped NPs indicate that dissipative heating can be mostly generated in two regions. One region is close to the branch vertices, whereas the other is corners at the intersection between the branches and the Au core structures (Figure 3e and 3f). For the vertices aligned with the E-field, a strong field enhancement can be induced based on the lightning rod effect. The enhanced nearfield accelerates the free electrons in the NPs, which then interact with phonons, which results in heat generation. The other mechanism is due to the surface roughness in the intersection between the branch and the Au core structure. The oscillated electrons are scattered by surface defects and generate heat. The simulated results for the NRs show that the strongest resistive heating was obtained at the middle of the structure (Figure 3g). These results indicate that the conductive charges are mostly accumulated at the edges and the highest enhanced field distribute outside the charge-accumulated regions. Thus, the strongest contributed electrical field is at the middle of the NRs, at which most of the heat is generated. In addition, a comparison of Figure 3g and 3h indicates that the resistive heating is significantly stronger when the particles are aligned with the E-field. These results are consistent with the discussed results obtained from calculation of the electric energy density distribution. The simulation shows the strong resistive heating ability of Au NSs under NIR irradiation because of the multiple sharp morphological features like branch tips and branch−core intersections. However, the nonuniform shapes of the NSs result in a broad LSPR absorption efficiency distribution across a relatively wide wavelength range. The broadening of the extinction spectra of Au NSs is disadvantageous in comparison with Au NRs, which possess a highly narrow longitudinal absorption distribution in the NIR region. Figure 3j shows the simulated extinction spectra profiles of the modeled Au NSs when an electric field was applied in the X, Y, and Z directions, that is, all NS branches are in the XY plane. A comparison with the TEM results shows that the broad, shorter wavelength T and longer wavelength L bands in Figure 2c and 2d can be attributed to the different calculated LSPR bands of Au NSs shown in Figure 3j. The presented simulation shows that when the electric field polarization is parallel to one of the longitudinal branch axes (Figure 3b), a longer wavelength extinction band Y with a calculated resonant wavelength of 700 nm is obtained. Hence, L denotes the longitudinal LSPR extinction on the Au NP branches. If the Efield is not perfectly aligned with any of the long branch axes (Figure 3a), a shorter wavelength band X centered at 670 nm appears. Therefore, T should be attributed to the transverse LSPR bands of the Au NP branches, which correspond to the E-field polarization that is perpendicular to the branch axes in the XY plane (Figure 3i). However, the simulated spectra deviate from those of real samples (Figure 2a). For example, the simulated bands are relatively narrow compared with the

Figure 3. Simulated distributions of electric energy density of a gold nanostar when the E-field is applied in the (a) X direction and (b) Y direction, a gold nanorod when the E-field is applied in the (c) X direction and (d) Y direction; distribution of time-average resistive heating produced by interaction of light with surface plasmons of a gold nanostar when the E-field is applied in the (e) X direction and (f) Y direction, a gold nanorod when the E-field is applied in the (g) X direction and (h) Y direction; (i) simulation conditions for a twodimensional nanostar; (j) simulated extinction spectra of a gold nanostar for an E-field polarization in the X, Y, and Z directions.

electrons was observed for sharper vertices, which indicates excitation of a considerably stronger electrostatic field. This phenomenon is called the lightning rod effect, in which an increase in the vertex angle results in aggregation of free electrons and a shift of the LSPR toward shorter wavelengths (blue shift) as the overall extinction efficiency decreases.35 Therefore, a greater electric field enhancement should occur at sharper corners of metal nanostructures such as cubes, triangular nanoplates, and branched structures with high tip curvatures. Moreover, sharper vertices should induce a red shift of the LSPR wavelength.35 The sharp tips on the NS branches that are not aligned with the E-field polarization direction (in the XY plane) also cause substantial field enhancements (Figure 3b) because of plasmonic coupling with other strongly excited parts of NS.35,41,42 Areas of high free electron density are also located at sharp vertices in intersections between the branches and the central core or between closely situated branches. Meanwhile, two different particle-aligned cases are shown for the Au NRs: one with the main axis parallel to the excited Efield (longitudinal plasmon) and the other perpendicular to the E-field (transverse plasmon). A comparison of Figure 3c and 3d shows that the particles aligned with the E-field polarization contribute a greater field enhancement. This result is also consistent with the previously mentioned lightning rod effect and coincides with previous reports.58 In a similar manner, when an E-field polarization is perpendicular to all branch axes of the NS (Z direction in Figure 3i), the field enhancement is significantly smaller than when the E-field is aligned with the XY plane. By using the calculated contributed E-field intensities as well as the imaginary part of Au permittivity, the distributions of the time-average resistive heating can be 2401



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the Ag+ content in solution. Au NSs that were obtained with a growth time of 42 h and an Ag+ concentration of 0.04−0.05 mM have a longitudinal absorption band that completely covers the NIR light transparency window of living tissues within 630−950 nm.59,60 Increasing the synthesis time to 48 h shifts the NIR absorption maximum above the NIR transparency limit of 950 nm, where the light absorption of water is substantially higher. Figure 4 presents the normalized absorbance spectra of asprepared branched Au NSs capped with CTAB and after being

observed ones. The main reasons for this disagreement are the approximations performed for the simulated electric energy density distributions and, more importantly, the nonuniform shapes of the as-prepared NSs. As the growth time increased from 24 to 48 h, the fitted L peak attributed to the longitudinal LSPR band shifts from 853 to 995 nm, which corresponds to the increase in the length of the branches as shown in the corresponding TEM images (Figure 1a and 1b) because the SPR effect is strongly size dependent. The fitted T peaks at 604 and 627 nm in Figure 2c and 2d represent the shifts in the positions of the transverse LSPR bands of the branches. Relatively broad shapes of T and L bands suggest various lengths of transverse and longitudinal axes because the branch sizes are nonuniform. The T band is only slightly red shifted by ∼23 nm, whereas the red shift of the L band is almost ∼150 nm when the synthesis time was increased from 24 to 48 h. This result suggests that the anisotropic growth of branches occurs preferably along the longitudinal axes. According to the lightning rod effect, more acute vertices yield a longer wavelength LSPR extinction.35 The much slower growth of the transverse axes compared with longitudinal ones results in the decrease of branch vertices angles, which is the primary reason for a substantial red shift of the longitudinal LSPR band with increasing synthesis time. Moreover, formation of branches on the Au NPs in the proposed conditions is slow and time-dependent because the gold concentration in the solution gradually diminishes. This fact can be exploited to tune the optical properties of Au NSs. The narrow band T′ of the transverse region that is centered at around ∼610 nm is assigned to the extinction that corresponds to the E-field polarization perpendicular to the NSs plane (Z direction in Figure 3i). The relative intensity of T′ decreases with increasing growth time and AgNO3 concentration because of the increasing share of three-dimensional NRs according to TEM investigation. Growth of branches outside the XY plane (as depicted in Figure 3i) results in the appearance of an additional longitudinal axis in the Z direction as well as a gradual red shift of the corresponding LSPR contribution from T′ to L. The morphology of Au NSs and their absorbance spectra are also highly sensitive to the silver cation content in the growth solution. At a growth time of 42 h, the relative intensity of the longitudinal part of the extinction spectra initially increases until the Ag+ concentration reaches 0.05 mM, as displayed in Figure 2b. These results are attributed to the increase in the number of branches and in the appearance of three-dimensional NSs. The relative intensity of the longitudinal band declines when the growth solution contains 0.06 mM AgNO3, which indicates that a further increase in the Ag+ content above 0.05 mM blocks formation of new branches. Despite the substantial growth of the relative intensity of the L band, which is obviously due to the increase in the average amount of elongated branches per NPs, the longitudinal LSPR wavelength remains within the relatively narrow range from ∼932 to 943 nm and does not suffer a noticeable shift when the silver content in the growth solution increases from 0.03 to 0.06 mM (Figure 2b). In accordance with the lightning rod effect,35,37 the vertex angle on the tips and the average length of branches do not significantly change within the investigated variation of the silver concentration for the same growth time period. However, the amount of branches per particle increases when the Ag+ concentration is increased from 0.03 to 0.05 mM. The desired morphological (2D or 3D NSs) and optical properties can be obtained by setting both the growth time and

Figure 4. Normalized extinction spectra of water solutions of Au NSs capped with CTAB and chitosan.

modified with chitosan. The normalized spectra of chitosancapped Au NSs demonstrate a slight blue shift in the transverse LSPR extinction band by ∼5 nm from that of the CTABcapped NPs. The LSPR absorption properties of Au NPs are known to be strongly affected by the local dielectric function of the surrounding medium.17,61 Another reason for this phenomenon could be the slight plasmon coupling that results from association of chitosan-capped Au NPs. The interaction of two or more surface plasmons of closely situated metal NP can lead to the appearance of a completely new plasmon field that also results in a change in the extinction band intensity and wavelength.36 Thus, the variations in the intensity and shape of the absorbance spectra of Au NPs provide a simple way to evaluate the stability of Au NPs dispersion in solution. The polymer chitosan and its derivatives were previously used for both the stabilization of Au NPs in solution and the controlled assembly via chitosan linkers.22,62 Hydrodynamic diameter measurements show that the average sizes (zeta average) of CTAB- and chitosan-modified Au NSs in DI water are 26.1 and 36.7 nm, respectively. The increased hydrodynamic diameter for chitosan-capped NSs shows a higher degree of agglomeration, which is likely due to hydrogen bonding and van der Waals interaction between chitosan molecules that envelop Au NPs. In a DI-water solution, this interaction is compensated by the electrostatic repulsion between charged capping layers of Au NSs. The chitosan molecules are able to bind hydrogen cations in an acidic medium and detach them in a basic solution because of the −NH2 groups. Therefore, the properties of the chitosan capping layer such as surface potential strongly depend on the composition and pH of the medium. Water-soluble chitosan-capped NPs are stabilized better in weak acids.22 The solution of as-prepared chitosan-capped Au NPs in DI water has a slightly acidic reaction with a pH of 5.9. Hence, Au NPs remain dispersed because protonated chitosan layers carry a positive charge with a zeta potential of +36.4 mV. Thus, chitosan-capped NSs (gold mass concentration of 10 μg/mL) are relatively stable within a temperature range from 4 to 37 °C 2402



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Figure 5. Time-dependent evolution of extinction spectra of dispersion of chitosan-capped (a) Au NRs and (b) Au NSs with gold mass concentrations of 10 μg/mL treated at 37 °C in a PBS buffer with pH = 7.5 (spectra of Au NRs and Au NSs in DI water are also presented); (c) TEM image of Au NR aggregates after 4 h in PBS; (d) TEM image of Au NS aggregates after 4 h in PBS (inset, HRTEM image of two-NS aggregate); time-dependent evolution of extinction spectra of a dispersion of (e) chitosan-capped Au NRs and (f) Au NSs with gold mass concentrations of 10 μg/mL treated at 37 °C in DMEM medium.

reaction proceeds at substantially different rates for rod- and star-shaped nanoparticles (Figure 5a and 5b). Chitosan molecules contain NH2 groups that function as a weak base, which bind H+ cations in acidic media and detaches them in basic solution. The average zeta potential of chitosan-capped Au NSs in DI water is +36.4 mV at a pH of 5.9. However, this value sharply decreases to about +3 mV in PBS because of the deprotonation of amino groups in chitosan molecules. The increase in pH to 7.5 (PBS buffer) results in degradation of the positive charge of the capping layer, and as a consequence, the electrostatic repulsion forces between nanoparticles decrease to a point where they cannot sustain a stable dispersion. As a result, chitosan-coated NPs become aggregated, which is confirmed by a remarkable shift and broadening of extinction bands. In the Au NRs solution, the extinction spectra shifts from ∼765 (in a water solution) to ∼870 nm immediately upon dissolution in PBS with a sharp drop in peak intensity (Figure 5a). The longitudinal LSPR band of chitosan-capped Au NRs in PBS is significantly broadened. The hydrodynamic diameter measurement results for Au NRs upon dissolution in PBS at

when dispersed in DI water. After a long period of time, NPs precipitate but can be easily redispersed with simple shaking or sonication. Figure 5 displays the time-dependent evolution of the extinction bands of chitosan-capped Au NRs and NSs dispersions (10 μg/mL) at 37 °C in a phosphate buffer saline (PBS) and DMEM medium with pH = 7.5. Figure 5a and 5b shows spectra for Au NRs and NSs in PBS, respectively. Au NRs with an aspect ratio of ∼3.5 were prepared according to the method described by El-Sayed et al.50 and used for comparison purposes. The water dispersion of Au NRs has an extremely narrow and strong longitudinal LSPR absorption band at 765 nm because of the size and shape uniformity of the NRs. An initial intensity at the peak of the longitudinal band for the chitosan-capped NRs solution in water is about 2.5 times higher than that of the NSs solution, which, as noted before, was the superposition of three relatively broad bands. However, the absorbance spectra of both Au NRs and NSs experienced an immediate and drastic change when PBS solution was used instead of DI water because of aggregation. However, the 2403



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first show an average size of 147 nm with a relatively narrow distribution (Figure S2a, Supporting Information). This value is equal to about three times the size of NRs along the longitudinal axis. The instrument is unable to provide correct measurements of hydrodynamic size after prolonged treatments in PBS at 37 °C because of the high polydispersity of particles in the Au NR solution sample. This originates from a very broad size distribution of formed aggregates and from the decrease in the effective particle concentration in the suspension. The longitudinal LSPR band of the Au NR solution becomes asymmetric during PBS treatment (Figure 5a), which also indicates that the obtained NRs aggregates had a wide size and shape distribution.58 In Figure 5b, a substantial drop in the extinction intensity for the Au NS solution in PBS was confirmed only after ∼1 h (at 37 °C), which implies a slower aggregation of the as-prepared gold NSs compared with NRs. Immediately upon dissolution the average hydrodynamic size of Au NSs in PBS is 106 and 174 nm after 1 h of treatment at 37 °C. The histograms of hydrodynamic size distributions are shown in Figures S2b and S2c, Supporting Information. The TEM images of chitosan-capped gold NRs and NSs after 4 h of treatment in PBS are displayed in Figure 5c and 5d, respectively, and in Figure S3, Supporting Information. The Au NRs form clusters that comprise tens or even hundreds of randomly associated particles. This clustering leads to a decrease in the effective concentration of particles in the solution as well as the complex interaction of surface plasmons of NRs that results in a substantial change in its absorbance characteristics (Figure 5a). The star-shaped nanoparticles predominantly form chain-like aggregates comprising a relatively small amount of NPs compared with Au NRs. A high-resolution TEM image (inset in Figure 5d) illustrates the main aggregation route of chitosan-capped Au NSs. Branch tips with a high curvature preferably associate with the lowcurvature surfaces of NS, which are ordinarily the central cores or flat parts of the branches. This result can be explained by the electrostatic interaction between different parts of star-shaped NPs. As noted before, the zeta potential of chitosan-capped Au NPs drastically decreases to around +3 mV at a pH of 7.5 in PBS from +36.4 mV in DI water. The measured average potential in PBS refers to the aggregates of several NSs, which suggests that the chitosan capping layer of individual Au NSs could be even smaller. A diminished positive charge of the chitosan layer decreases its ability to mask a negative charge located at the surface of Au NSs. The areas of a stronger LSPR E-field enhancement upon interaction with light of resonant wavelength (polarization in the XY plane as displayed in Figures 3a and 3b) reflect parts of NSs with a higher aerial electron density. On the basis of the FEM simulation results, the highest charge density is concentrated around the acute branch tips (lightning rod effect). The similar electric field distributions across the Au NSs occur because of an electrostatic interaction of the charged surface of Au NSs with electrolyte medium. Moreover, the aerial free electron density is directly proportional to the surface area of a specific part of the Au NP and is inversely proportional to the corresponding volume.35 The much larger specific area to volume ratio at the sharp vertices results in a greater electric field at the branch tips. Thus, a higher surface charge density at the vertices should cause a stronger electrostatic repulsion between the tips of the branches. Hence, acute tips preferably associate with lowcurvature cores or flat parts of the NPs that possess a much lower charge density at the surface. In an ideal scenario, one

chitosan-capped NSs can associate with two other NSs via the connection to their branches by different sides of the central core (opposite sides of the XY plane of a NSs in Figure 3i), which results in chain-like structures. This restriction on the mutual orientation of NSs also makes the agglomeration process slower compared with NRs. Au NRs have a much smaller surface charge density gradient along the longitudinal axis (Figures 3c). Hence, the aggregation of chitosan-capped Au NR in PBS predominantly occurs in a random way (Figure 5c). Chain-like aggregation was also observed for Au NPs prepared via direct reduction of NAuCl4 by chitosan.63 The tail-tomiddle assembly into stair-like chains as a result of the mutual orientation of Au NRs that were modified by N-methyl-2pyrrolidone was also reported recently.58 The observed chainlike aggregation of NSs could be also the consequence of the low probability of contact between branch tips of different NSs because the as-prepared Au NSs have a relatively big size (∼70−90 nm in diameter). Therefore, ultrasmall (∼30−40 nm) branched gold NPs were prepared via HEPES reduction of HAuCl4 according to Xie et al.64 to check if chain-like aggregation is induced by a branched structure or is a consequence of the large size of the as-prepared NSs. After modification with chitosan, the concentrated NP solution was dissolved in PBS to produce a gold mass concentration of 10 μg/mL. This solution was kept for 4 h at 37 °C. TEM results (Figure S3, Supporting Information) also reveal a chain-like aggregation similar to Au NSs that were investigated in the present study but comprised a larger amount of nanoparticles. Moreover, the smaller Au NSs produce highly branched chains that can associate further in ring-like structures, probably because of the high ratio of three-dimensional NSs produced by the HEPES reduction method as opposed to two-dimensional Au NSs that were investigated in the present study. Chitosancapped Au NSs with sizes within 30−40 nm also experienced a much slower intensity degradation of the extinction band in the PBS solution compared with Au NRs, as displayed in Figure S4, Supporting Information. Figure 5e and 5f displays the time-dependent evolution of extinction bands for chitosan-capped gold NRs and NSs, respectively, that were dispersed in a protein-rich DMEM solution (10% serum), which was used as a cell culture medium for the in-vitro study. In DMEM, the change in the spectral characteristics of chitosan-capped Au NPs is not as sharp as that in PBS despite the same pH value. In the case of Au NSs, an insignificant red shift was observed in the band maximum position and almost no degradation was observed in the intensity of the extinction spectra even after 24 h at 37 °C. This result suggests a stable dispersion. On the other hand, hydrodynamic diameter measurement results for the chitosancapped Au NS dispersion in DMEM clearly show a slight shift of the zeta size distribution toward larger sizes as time progressed (Figures S2d and S2e, Supporting Information). Moreover, the zeta size histogram shows an additional peak within a range of 500−800 nm after 4 h, which is a clear sign of aggregation. However, in DMEM, this process appears to be much slower than in the PBS solution. The amino groups in chitosan molecules can create hydrogen bonds with the proteins in DMEM.20,21 A strong electrostatic interaction is unlikely due to a substantially decreased charge of the chitosan capping layer at pH = 7.5. Relatively large protein molecules can form linkers between chitosan-capped gold NPs. At the same time, these protein molecules can function as separating layers that prevent the coalescence of chitosan layers that 2404



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Figure 6. Cell viability as a function of the Au concentration placed in a single well capped with (a) CTAB and (b) chitosan. After incubation with maximum concentration of Au for 72 h, image of (c) a blank J5 cell culture (incubated without Au NSs), (d) J5 cell culture treated with chitosancoated Au NSs, and (e) J5 cell culture treated with CTAB-coated Au NSs.

Figure 7. Confocal microscopic images that indicate overlap of (a) bright field with green FITC fluorescence, (b) bright field with red Lysotracker DND-99 fluorescence, (c) overlap of bright field with FITC, Lysotracker, and blue Hoschst-33342 fluorescences, (d−f) respective images of fluorescence only for the J5 cell culture incubated for 12 h with 16 μg/mL of FITC-labeled chitosan-capped Au NSs; (g−i) confocal microscopic images of untreated J5 cells (incubated without Au NSs). Scale bar is 20 μm.

h) means that the aggregation process of Au NSs has a high reversibility. This result suggests a very week bonding via protein molecules. For the Au NRs solution in DMEM, the longitudinal band peak wavelength is red shifted just by ∼12 nm from its initial position in DI water (compared with ∼105 nm in PBS) and the maximum intensity decreases by ∼30% after 24 h at 37 °C. However, the evolution of the extinction

envelop NPs and the strong interaction between the surface plasmons of neighboring particles. Therefore, the shifts of the extinction bands of Au NPs dispersions in DMEM shown in Figure 5e and 5f are insufficient compared with those of PBS solutions (Figure 5a and 5b) despite agglomeration. The fact that the intensity of the extinction band of Au NSs in DMEM does not noticeably decrease within the investigated period (24 2405



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Figure 8. (a) Mass uptake of chitosan-capped Au NSs and Au NRs by J5 cells incubated for 12 h with 10 and 4 μg/mL Au NP concentrations in an incubation DMEM medium; flow cytometry FITC fluorescence histograms of (b) blank J5 cells incubated for 12 h with (c) a 10 μg/mL solution of FITC-chitosan-capped Au NRs, (c) a 10 μg/mL solution of FITC-chitosan-capped Au NSs, (d) a 4 μg/mL solution of FITC-chitosan-capped Au NRs, (e) a 4 μg/mL solution of FITC-chitosan-capped Au NSs.

spectra of chitosan-capped gold NRs in DMEM appears to be only partially reversible even upon sonication (Figure 5e). Thus, chitosan-capped Au NPs can retain at least a partial dispersion in a high-serum DMEM medium for a prolonged period of time (up to 24 h) despite a basic medium with a pH of 7.5. An in-vitro cytotoxicity analysis was further performed by incubating several cell lines (BEAS-2b, OMF, S-G, J5) with chitosan- and CTAB-modified branched Au NSs for 72 h, as displayed in Figure 6. CTAB-modified Au NPs show an obvious cytotoxicity toward all cell types when their concentration exceeds 0.08 μg/mL. This result is consistent with previous findings on the bioeffect of CTAB-capped NPs.16,17 The viability of BEAS cells declines to less than 60% and then to ∼0% as the concentration of CTAB-capped NPs increases to 0.8 and 8 μg/mL, respectively. The cell lines used in the study have different tolerances to the CTAB-modified NPs. The viability of the four cell lines that were incubated with 8 μg/mL of CTAB-modified Au NPs were all ∼0%, which indicates a high cytotoxicity that is disadvantageous to bioapplications such as thermal therapy and drug delivery. Concentrations of CTAB-modified Au NP that are less than 0.8 μg/mL have no effect on viability except for BEAS-2b cells.

This effect may be the result of the negligible uptake of CTABcapped Au NSs at very low concentrations in solution.53 Another potential reason for this phenomenon is the substitution of weakly bonded CTAB on the surface of NPs by various species in the culture medium. By contrast, the chitosan-modified Au NSs exhibit no significant cytotoxicity toward any cell line. Figure 6c−e presents photographs of the untreated J5 cells and J5 cells that were incubated with 16 μg/ mL of chitosan-modified Au NPs for 72 h and 8 μg/mL of CTAB-capped Au NPs. Cells that were incubated with CTABcapped Au NSs all have a spherical shape, which indicates their death. The state of the cells that were treated with chitosancapped NPs remains mostly unchanged after 72 h, which confirms the biocompatibility of chitosan-modified branched Au NPs. Cytotoxicity assay results shows that the cytotoxicity of Au NSs observed in Figure 6a is related to CTAB coating. However, the BEAS-2b cell line had a viability below 100% when the concentration of chitosan-capped Au NPs was at its highest value (16 μg/mL). This result suggests a concentration limit in the safe uptake of chitosan-capped Au NSs, which also depends on the cell type. Internalization of Au NP in large amounts may have an effect other than toxicity on the viability of cells or may disrupt important intracellular processes.65 The 2406



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Figure 9. J5 cell monolayers stained with a trypan blue dye pulsed laser scanning with energy flunces: cells incubated with 10 μg/mL of chitosancapped Au NSs (a) 33, (b) 17, (c) 23, (d) 12, and (e) 15 mJ/cm2; cells incubated with 10 μg/mL of chitosan-capped Au NRs (f) 33, (g) 17, (h) 23, (i) 12, and (j) 15 mJ/cm2; cells incubated with 4 μg/mL of chitosan-capped Au NSs (k) 33, (l) 17, (m) 23, (n) 12, and (o) 15 mJ/cm2; cells incubated with 4 μg/mL of chitosan-capped Au NRs (p) 33, (r) 17, (s) 23, (t) 12, and (u) 15 mJ/cm2.

the fluorescence hystogram of the control J5 cells. Figure 8c−f shows the fluorescence distributions of the J5 cells that were incubated for 12 h with different contents of FITC-chitosanmodified Au NSs and NRs. A shift in the overall distribution toward a higher fluorescence in reference to the control sample indicates the internalization of fluorescent nanoparticles. The histograms for the J5 cells that were incubated with Au NRs (Figure 8c and 8e) appear to be noticeably broader compared with those of cells treated with Au NSs (Figure 8d and 8f). An experimental therapeutical approach on in-vitro cell photothermolysis was carried out on a monolayer of J5 cancer cells using a femtosecond laser source with a wavelength of 765 nm, which corresponds to the maximum absorption efficiency of as-prepared Au NRs in a water solution. After laser treatment, cell samples were incubated for two more hours and stained with trypan blue to indicate dead cells. In this study, an energy fluence of 86 mJ/cm2 was found to be enough to cause a substantial effect on the control J5 cells (without Au NPs), as shown Figure S6 in the Supporting Information. In the literature, the energy fluence that causes Hela cell mortality is given as 113 mJ/cm2.52 Figure 9a−e and 9f−j shows the effects of pulse laser scanning (area of 500 μm × 500 μm) on the cells that were incubated with 10 μg/mL of chitosan-capped gold NSs and NRs, respectively. The average amount of cells in the area is 518 ± 31. The photothermal impact on cells is relatively strong for both investigated types of NPs because of the high concentration of Au NPs. In cell samples treated with chitosancapped Au NSs solution starting from an energy fluence of ∼12 mJ/cm2 (0.46 kW/cm2 power density and 30 scans), almost ∼100% percent of cells are destroyed as seen in Figure 9e. In cell samples incubated with 10 μg/mL of chitosan-capped Au NRs (Figure 9g−j), only an energy fluence of ∼17 mJ/cm2

uptake and localization of chitosan-capped Au NSs by J5 cancer cells was verified by a three-dimensional analysis of the fluorescence distribution via confocal microscopy. Cells were treated with a modified green-fluorescent FITC dye-labeled chitosan Au NSs with a concentration of 16 μg/mL. Blue fluorescent Hoschst 33342 was added to mark positions of nuclei in J5 cells, and LysoTracker Red DND-99 was used to determine whether internalized Au NSs are localized in lysosomes. Figure 7 shows the bright-field images, FITC, LysoTracker Red DND-99, and Hoschst 33342 fluorescence images and their overlaps with Hoschst 33342-fluorescence for the J5 cells incubated with FITC-labeled Au NSs in comparison with untreated J5 cells. The intracellular staining result showed that the green-fluorescent spots of Au NSs were generaly merged with lysosomal markers (Figure 7c and 7f). The yellow spots indicate merging of FITC fluorescence with lysosomal probe in cytosol. The positions of Au NSs are remarkably colocalized with red-fluorescent LysoTracker. A three-dimensional analysis (Figure S5, Supporting Information) showed over a 58% colocalization ratio. It suggests that Au NSs in J5 cell would accumulate in cellular organelle, lysomomes. Uptake of NPs essentially depends on the concentration of Au NPs in the medium and treatment time. Cellular uptakes of chitosan-capped Au NSs and Au NRs (measured by gold mass) are relatively close to each other when the incubation time is ∼12 h, as shown in Figure 8a, where data for 4 and 10 μg/mL of gold concentrations in incubation medium are presented. However, the mechanism and kinetics of chitosan-modified Au NPs internalization by cells were not a subject of detailed investigation in this study. The flow cytometry results of the J5 cells that were incubated with fluorescent FITC-chitosancapped Au NPs are presented in Figure 8c−f. Figure 8b displays 2407



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(0.67 kW/cm2 power density and 30 scans) results in ∼100% cell death as shown in Figure 9f. However, when the mass concentration of gold is ∼10 μg/mL, the photothermal impact mediated by Au NPs from laser power density over 0.46 kW/ cm2 is strong enough to cause a very intense cavitation and bubble formation in cells (Figure S7, Supporting Information), and the size of cavities can reach up to tens of micrometers. According to Chen et al.,52 bubble formation proceeds around big clusters of Au NPs because of intense evaporation. The problem in this case is that vapor bubbles are also efficient scattering centers and absorbers of NIR irradiation.66 Hence, they quickly grow in size until the laser scanning is halted. Therefore, a gold NP concentration was reduced to 4 μg/mL to eliminate the evaporation effect. The results are presented in Figure 9k−o and 9p−u for cells treated with chitosan-capped Au NSs and Au NRs, respectively. Laser irradiation starts to cause photothermolysis of the cells that were incubated with 4 μg/mL of Au NPs at a power density of 0.31 kW/cm2 after 60 scans, which corresponds to an energy fluence of 15 mJ/cm2 for both Au NSs and NRs in Figure 9o and 9u, respectively. However, at this level of laser power density only a small fraction of cells is killed (less than 50% for the sample treated with Au NRs). An increase in the power density leads to a smaller amount of scans needed to achieve similar result. At 0.46 kW/cm2, up to 80% of the cells are killed after 30 scans, which corresponds to an energy fluence of 12 mJ/cm2. An energy fluence of 23 mJ/cm2 (power density 0.46 kW/cm2 and 60 scans) results in an almost complete photothermolysis of cells that were incubated with 4 μg/mL of chitosan-capped Au NSs as shown in Figure 9m. A similar result for cells that were incubated with 4 μg/mL of Au NRs can be achieved when the energy fluence reaches 33 mJ/cm2 (at a power density of 0.67 kW/cm2 and 60 scans). For Au NSs-treated samples, 6%, 5%, 9%, 8%, and 27% of cells retain a consistent membrane after laser scanning with energy fluences of 33, 23, 17, 12, and 15 mJ/cm2, respectively. On the other hand, for samples incubated with Au NRs, 5%, 12%, 16%, 19%, and 52% of the cells retain a consistent membrane after irradiation with energy fluences of 33, 23, 17, 12, and 15 mJ/cm2, respectively. In samples treated with Au NRs, a higher amount of cells generally remains undamaged in comparison with those incubated with Au NSs. This result is the opposite of what was expected from the extinction measurement data for Au NSs and NRs at a wavelength of 765 nm. Au NRs have about a 150% higher extinction efficiency per mass unit of gold compared with NSs (Figure 5a and 5b). This contradiction has several explanations. First, the absorption maximum wavelength could shift from 765 nm measured in the water solution because of the aggregation of chitosan-capped NRs. However, as shown before (Figure 5e) in a protein-rich medium, the shape and position of the extinction spectra do not significantly change. Second, Figure 5e and 5f shows that the agglomeration of chitosan-capped Au NRs (unlike that of Au NSs) in a DMEM medium still causes a noticeable degradation in the absorbance intensity in the NIR region probably because the extinction in the cross-section of the sample decreases. In addition, the absorption efficiency is optimal when the longitudinal axis of the rod-shaped particle is aligned with the E-field polarization during laser irradiation, which is impossible to achieve for all NRs in the sample because aggregation restricts an independent orientation of NRs in the clusters. Cells incubated with either Au NRs or Au NSs have a nearly equal lowest energy fluence of 12−15 mJ/cm2 when laser

irradiation starts to trigger cell death. This result means that in the as-performed experiment, no substantial difference was observed in the photothermolysis efficiency between two kinds of investigated NPs to claim a significant advantage of any type from the obtained data. However, every specimen after laser scanning contains a specific amount of cells with consistent membranes whose amount decreases with increasing applied laser energy fluence. The most obvious reason for incomplete photothermolysis is that the survived cells had internalized a smaller amount of Au NPs or, in other words, as a result of the nonuniform distribution of gold across cells in the specimen. Figure 8c and 8e reveals a substantial broadening of histograms of the FITC fluorescence distribution versus cell count for specimens that were incubated with Au NRs modified by FITC-labeled chitosan compared with the histograms of control cell samples and samples treated with Au NSs (Figure 8d and 8f). The overlays of normalized histograms for gold concentrations of 10 and 4 μg/mL are presented in Figure S8, Supporting Information. Broader histograms obtained from samples treated with Au NRs mean that FITC fluorescence has a more nonequivalent distribution across the cell line. This result suggests that the distribution of FITC-chitosan-capped Au NRs among cells is more nonuniform compared with that of Au NSs. The examples of in-flow images of the J5 cells that were incubated with Au NPs and modified by FITC-labeled chitosan are presented in Figure S9, Supporting Information. Very large FITC fluorescence areas that mark positions of Au NRs clusters can be spotted in some cells treated with FITCchitosan Au NRs, whereas other cells show very little or no fluorescence at all. The highly nonuniform aggregation of the chitosan-capped Au NRs and as a consequence of nonequivalent mass distribution of gold NRs across cell lines are the most likely reasons for a greater amount of surviving J5 cells in the specimens that were incubated with Au NRs compared with those treated with Au NSs after laser irradiation. A difference in the aggregation of chitosan-capped Au NSs and NRs in incubation wells can also be observed in the optical microscope as displayed in Figures S10, Supporting Information. Rare but relatively large agglomerates (black spots) can be seen in images taken using an optical microscope in cell samples after 4 h of incubation in a DMEM culture medium containing chitosan-capped Au NRs. In this case, large aggregate formation proceeds mostly in the vicinity of cell membranes. In the samples treated with both 4 and 10 μg/mL of chitosan-capped Au NSs for 4 h a high amount of much smaller aggregates with a relatively even distribution over the dish bottom and cell layer can be observed. A nonuniform distribution of Au NRs across the cell line results in a strong photothermal effect on cells that internalized larger aggregates, which leads to membrane destruction that can be detected via trypan blue staining. Cells with a smaller amount of internalized Au NRs can remain unaffected at specific energy fluences.

4. CONCLUSION In summary, Au NSs with sizes between 70 and 100 nm were prepared via the solution route in the presence of silver cations. TEM results showed that Au NSs have a two-dimensional morphology when the silver concentration in the growth solution is 0.04 mM and the synthesis time is 36−42 h. An increase in the silver content or growth time leads to formation of three-dimensional Au NSs. The surfaces of as-prepared Au NSs were modified with biopolymer chitosan to improve biocompatibility and colloidal stability in diluted solutions of as2408



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MY3 and NSC 101-3113-P-002-021) for financially supporting this research.

prepared Au NSs. The electrical charge distribution simulation based on FEM showed the free electron density and electric field enhancement of the surface because the LSPR reached the maximum value at the vertices on NS branches. The LSPR extinction spectra of the as-prepared NSs consist of two bands in the visible region at ∼610 and 627 nm, and the NIR band centered within a range of 853 to 995 nm is tunable via the synthesis time and silver content in the growth solution. The optical characteristics of colloidal chitosan-modified Au NSs dispersed in a PBS buffer and high serum medium with pH = 7.5 appeared to be substantially more stable than those of Au NRs because of the slower aggregation of NSs. Chitosanmodified Au NRs at pH = 7.5 formed aggregates with highly nonuniform sizes. The Au NSs form small chain-like clusters in which individual NSs connect to one another preferably via association of acute branch tips and central cores. The substantial charge distribution gradients on the Au NSs surface where acute branch tips surrounded by a high aerial electron density and the surface of central cores of NSs possess a negligible free charge density are presumably responsible for the preferred affinity of those parts of NSs during the aggregation process. This restriction on the mutual orientation of Au NSs needed for association also slows down the aggregation of chitosan-capped Au NSs. Flow cytometry analysis results showed a relatively nonequivalent distribution of chitosan-capped Au NRs across the cells in specimen compared with Au NSs because of highly nonuniform aggregation and cellular uptake of NRs. An experiment on the photothermolysis of J5 liver cancer cells in vitro demonstrated that an energy fluence of 23 mJ/cm2 was enough to cause the death of J5 cells that were incubated with chitosancapped Au NSs. For complete photothermolysis of cell samples treated with Au NRs an energy fluence of 33 mJ/cm2 was necessary. When chitosan was used as a surface capping agent, the Au NSs had a higher colloidal stability at a physiological pH of 7.5 than Au NRs and are more suitable mediators in cell photothermolysis because of the slower aggregation and more uniform cellular uptake.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed experimental information of FTIR, size distribution, and TEM images for characterization of chitosan-modified gold nanostars; 3D analysis of localization of chitosan-modified gold nanostars in J5 cells; transmittance photos of photothermolysis of J5 cancer cells; flow cytometry detection of FITC-chitosancapped gold nanostars; microscope images after treating with various concentrations of gold nanostars. 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]; dptsai@ phys.ntu.edu.tw. Author Contributions

The first two authors contributed equally to this work.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the National Science Council of the Republic of China and Taiwan (Contract Nos. NSC101-2113-M-002-0142409



Article

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Chitosan-Modified Stable Colloidal Gold Nanostars for the

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