Chitosan-Modified Stable Colloidal Gold Nanostars for thePhotothermolysis of Cancer CellsIvan 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 plasmonicgold nanostars (Au NSs) modified with a biopolymer chitosanare reported. The colloidal stability of Au NSs at thephysiological pH of 7.5 and their performance in thephotothermolysis of cancer cells in vitro were comparedwith those of gold nanorods (Au NRs). The opticalcharacteristics of chitosan-modified Au NSs dispersed in amedium with pH = 7.5 had higher stability than those ofchitosan-capped NRs because of the slower aggregation ofNSs. At pH = 7.5, the chitosan-modified Au NRs formedaggregates with highly nonuniform sizes. On the other hand,Au NSs formed small chain-like clusters, in which individualNSs were connected to one another, preferably via associationof branches with central cores. It is possible that the difference in areal charge density at these parts of NSs is responsible for theirpreferred association. Flow cytometry analysis showed the relatively nonequivalent distribution of the chitosan-capped Au NRsacross the cell line compared with that of Au NSs because of the fast and nonuniform aggregation of NRs. An in-vitrophotothermolysis experiment on J5 cancer cells showed that energy fluences of 23 and 33 mJ/cm2 are necessary to causecomplete death of J5 cells incubated with 4 μg/mL chitosan-capped Au NSs and NRs, respectively. When chitosan was used as asurface-capping agent, the Au NSs exhibited higher colloidal stability at the physiological pH than the NRs and lower energyfluence necessary for cell photothermolysis because of more uniform cellular uptake.

1. INTRODUCTION

Studies on noble metal nanoparticles (NPs) have attractedincreasing interest in the field of sensing, optoelectronics, andbiomedicine because of their enhanced optical propertiesincluding strong absorption and scattering of light in thevisible and near-infrared (NIR) wavelength regions related tothe localized surface plasmon resonance (LSPR).1−3 In thebiomedicine field, nanomaterials are selected with considerationof biocompatibility, colloidal and chemical stability, and abilityto functionalize depending on the intended application.4,5 Theunique combination of optical properties and chemical stabilitymakes gold nanoparticles (Au NPs) ideal nanostructures for awide range of biomedical applications.6−13 However, colloidalnanomaterials tend to aggregate rapidly. This underlines theneed for surface-protective agents, which would preventaggregation and enhance the properties of NPs for theintended application. A large number of surface-modifyingagents have been proposed.14,15 The most commonly used arepoly(ethylene glycol) derivatives, which provide high colloidal

stability as well as efficient masking of Au surface charge, whichminimizes unintentional adsorption.15 Polystyrene sulfonate(PSS) is also often used because of the simple modificationprocedure.16 However, negatively charged polycationic com-pounds such as PSS cover NPs over the existing cetyltrime-thylammonium bromide (CTAB) micelle capping used in NPsynthesis.17 This creates danger of unintended release of toxicCTAB. The natural choice for the safe surface-capping agents isbiomolecules that are innate in many types of biologicalorganisms. Chitosan is a derivative of chitin, an abundantnatural biopolymer, which underlines its advantage ofbiocompatibility and biodegradability. Hence, chitosan isextensively used in medicine, and chitosan NPs are studied innanobiology as nanocarriers for drugs and functionalgroups.18−21 The properties of chitosan in aqueous media

Received: November 14, 2012Revised: January 10, 2013Published: January 15, 2013

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strongly depend on the pH.22,23 The chitosan molecule ispositively charged in acidic medium because of the protonationof its amino groups, which results in increased solubility. In abasic environment, protonated amino groups lose H+ and thechitosan molecules become neutral. Thus, the solubility ofchitosan decreases. The positive charges in a weak acid providechitosan with an ability to bind to negatively charged Au NPsurfaces via an electrostatic interaction.24 In addition, thechitosan-based composites were found to have prolongedcirculation in the blood (up to 72 h) as well as goodaccumulation in tumors via the leaky vasculature and impairedlymphatic drainage system phenomenon (enhanced perme-ability and retention effect).25−27

In a basic environment owing to the absence of electrostaticrepulsion, chitosan-capping layers can form interpolymeric linkswith 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 AuNPs or production of chitosan films with embedded NPs viapH variations.28 However, in biomedicine, unintendedaggregation is dangerous because controlling the NP assemblyand predicting the properties of the formed aggregates areextremely difficult.29 This is true for chitosan since thephysiological pH is ∼7.4. Thus, optimal NP types with minimaltendency to aggregation that exhibit the necessary propertiesfor targeted applications must be identified when chitosancapping is intended to be used. One of the many fields whereLSPR-enabled Au NPs have gained major interest is non-invasive photothermal therapy (PTT) of cancer.30 The PPTmethod is based on conversion of electromagnetic wavesenergy into local heat with the following hyperthermia ordestruction of cancerous cells. NIR laser irradiation is generallyused in this therapeutic approach because of the deeppenetration ability in living tissues.31 Au NPs with highintrinsic absorption efficiencies can function as mediators ofnonradiative photothermal conversion, which provide localdestructive heating in tumors loaded with Au NPs withoutaffecting healthy tissues. However, the optimal NP type suitablefor PTT remains to be determined because the LSPRcharacteristics of NPs and their behavior in biological mediumstrongly depend on their size and shape. Au nanorods (NRs),nanoshells, and hollow NPs are recognized as the mostpromising plasmonic nanomaterials in the field of PTT becauseof their highly tunable LSPR properties.32,33 Au NRs possessthe highest absorption efficiency per mass of gold and a narrowextinction band, which is tunable from the visible to the NIRwavelength range via simple adjustments in the aspect ratio ofNR.33,34 In addition, highly uniform Au NRs can be synthesizedon a large scale. The LSPR arises from the interaction of lightwith the free-electron gas at the boundary between metal NPsand a dielectric medium.1,2 This results in amplification of theelectric field (E-field) near the NP surface and is evidenced bystrong absorption and scattering of light. E-field enhancementis particularly high at the sharp edges and acute tips of metalNPs such as nanocubes, triangular nanoplates, and nano-stars.35−40 This phenomenon is called the lightning rod effectand is particularly interesting for surface-enhanced Ramanscattering (SERS) analysis,41−43 which is based on enhance-ment of Raman scattering signal of analyte by the optical fieldcreated by the LSPR near the metal surface. Interest in SERSapplications has spurred studies on star-shaped gold NPs (AuNSs), 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 theirpotential use as mediators in the noninvasive PTT of cancercells.45−47

Although the synthesis of zero- and one-dimensional NPs(nanospheres, nanoshells, and NRs) with specific morphologieshas become routine, precise control of the shape of NSsremains a big challenge. At present, no method is available toprepare uniformly shaped Au NSs. Therefore, an increasingnumber of synthetic routes are being developed to improve thecontrollability of the morphology of Au NSs.43,48 Preparation ofbranched nanostructures as the products of a versatile seed-mediated synthesis was reported by our group.49 In this study,NSs with optimized morphologies were prepared. Surfacemodification of as-prepared Au NPs was performed usingchitosan to improve their biocompatibility and colloidalstability in dilute solutions. The present investigation aims toconduct a comparative study of the colloidal stabilities ofchitosan-capped Au NSs and NRs at pH −7.5 in near-physiological conditions as well as their performances inphotothermolysis of cancer cells in vitro using a pulse NIRlaser.

2. EXPERIMENTAL SECTIONChemicals. Hydrogen tetrachloroaurate(III) hydrate, triso-

dium citrate dehydrate (99.9%), silver nitrate (99%), ascorbicacid (AA) (99%), CTAB (99%), low molecular weight chitosan(99%; MW ≈ 17 000; deacetylation ratio approximately 70%),and fluorescein isothiocyanate (FITC) (99%) were obtainedfrom Acros Organics and used without further purification. Allwater used in the study was reagent grade and produced using aMilli-Q SP ultrapure water purification system (NihonMillipore 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, 0.3mL of ice-cold, freshly prepared 0.01 M aqueous NaBH4 wasadded, and the solution was stirred for 5 min. The seed solutionwas maintained at room temperature for ∼2 h prior to use.

Preparation of Au NSs in Solution. Au NSs weresynthesized using a procedure similar to that described by Chenet al.,49 with several modifications in the synthesis time andAgNO3 concentration. An aqueous solution consisting of 0.1 MCTAB, 0.25 mM HAuCl4, and 0.03, 0.04, 0.05, or 0.06 mMAgNO3 was used as the growth solution. This solution wasmaintained at 27 °C throughout the experiment. The Au seeds(0.1 mL) were placed in a beaker. Three volumes (1, 10, and100 mL) of the growth solution were then mixed with 0.06(first), 0.6 (second), and 6 mL (third) of freshly prepared AAsolution (10 mM), respectively. These three colorless solutionswere added stepwise to the quiescent Au seed solution atintervals of 30 s. The growth solution was maintained at 25 °Cfor 24−48 h and then centrifuged twice at 10 000 rpm for 10min to remove excess CTAB.

Preparation of Au NRs. AuNRs with aspect ratios of 3.5were prepared according to a previously reported procedure.50

First, 10 mL of a solution containing 0.1 M CTAB and 0.25mM HAuCl4 was mixed with 0.6 mL of ice-cold 0.01 M NaBH4to prepare the Au seeds. Au seed solution was then kept at 25°C for 2 h. Afterward, 10 mL of the growth solution containing0.1 M CTAB, 0.007 mM AgNO3, and 0.5 mM HAuCl4 wasmixed with 70 μL of 0.0788 M AA. About 12 μL of the seedsolution was then added to the growth solution with vigorousstirring, and the mixture was kept at 27 °C for 3 h. The Au NRs

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were then centrifuged twice at 10 000 rpm for 10 min toremove 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%) wasdissolved in 10 mL of 1% acetic acid. The resulting solution wassonicated for 1 h. The solution was then added dropwise into100 mL of the Au NPs solution. The mixture was vigorouslystirred for 4 h and then set aside for another 4 h. The Au NSand Au NR solutions were centrifuged twice at 7000 and 9000rpm for 7 min, respectively, to remove excess electrolytes.Surface Modification Using FITC-Conjuncted Chito-

san. FITC-labeled chitosan was prepared using a proceduresimilar 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 thedark, the FITC-labeled chitosan (FITC−CS) was precipitatedby increasing the pH to 10 via addition of 0.1 M NaOH. Theunreacted FITC was washed with distilled water and thenseparated by repetitive centrifugation with subsequent redis-persion and sonication until no fluorescence was detected inthe supernatant. Au NP surfaces were modified with the FITC-labeled chitosan following a procedure similar to that formodification of Au NPs using pure chitosan.Electrical Charge Density and Resistive Heating

Simulations. In the simulations, the commercial ComsolMultiphysics software was used to calculate the fielddistributions, which are based on the finite element method(FEM), to compare the resistive heating properties of Au NSsand NRs. The three-dimensional figures of the NPs werecarefully constructed in accordance to their transmissionelectron microscopy (TEM) images. In addition, thepermittivity of Au NPs was determined by the Drude−Lorentzmodel at a plasmon frequency ωp = 8.997 eV and a dampingconstant Γp = 0.14 eV in the optical frequency region. On theother hand, the refractive index of the surrounding solution is1.33 (water). The incident source is an x(y)-polarized planewave that propagates along the z axis. The wavelength wasselected using the value that corresponds with the calculatedspectral absorbance peaks from the calculations, namely, 700and 810 nm for NSs and NRs, respectively. Furthermore, thefield distributions were obtained using a lateral sectionperpendicular to the wave-propagating direction to facilitatethe observations.Characterization. TEM was performed to characterize the

overall morphology of the samples. TEM images were capturedusing a JEM-2010 (JEOL, Japan) electron microscope. High-resolution transmission electron microscope (HRTEM) imagesand electron diffraction patterns were obtained using a JEOLJEM-2100F electron microscope. The specimens were obtainedby placing several drops of the colloidal solution onto a carbon-covered copper grid and evaporating the solution in air at roomtemperature. Prior to specimen preparation, the colloidalsolution was sonicated for 1 min to promote dispersion ofparticles on the copper grid. Synchrotron radiation X-raydiffraction patterns were acquired on a beamline 01C2 at theTaiwan National Synchrotron Radiation Research Center(NSRRC) using a wavelength of 0.774907 Å. The Auconcentration in the colloidal solution was determined byinductively coupled plasma mass spectrometry (ICP MS) usinga Thermo X-Series II spectrometer (Thermo Fisher ScientificInc., USA). The transmittance Fourier transform infrared(FTIR) spectra of the Au NPs dispersed in KBr pellets were

acquired using a Varian FTIR-640 spectrometer (AgilentTechnologies, USA). UV−vis light absorption spectra of thecolloidal NP solutions were obtained using a Shimadzu UV-700spectrophotometer (Shimadzu Scientific Instruments, Japan)with a 1 cm quartz cell at room temperature. The hydro-dynamic size (zeta size) distribution and potential (zetapotential) of the NPs in solution were determined at 25 °Cby dynamic light scattering at 633 nm using Zetasizer 3000(Malvern Instruments Ltd., England).

Cytotoxicity Assay. Four cell lines were used. Oral mucosafibroblasts (OMF) were cultured in a minimum essentialmedium (MEM, Gibco, USA). Oral epithelial (S-G) andhuman liver cancer (J5) cell lines were cultured in Dulbecco’smodified Eagle’s medium (DMEM, Gibco, NY, USA). TheBEAS-2b cells were maintained in a bronchial epithelial cellbasal medium (BEBM, Lonza, Walkersville, MD, USA) thatcontained bronchial epithelial cell growth medium (BEGM)SingleQuots. All media were supplemented with 100 units/mLpenicillin (Gibco, USA) and 100 mg/mL streptomycin (Gibco,USA). About 2000 cells/100 μL were seeded onto 96-wellplates and incubated overnight at 37 °C in a 5% CO2atmosphere. Afterward, 10 μL of a solution containing 0,0.01, 0.1, 1, and 10 μL of CTAB (80 μg/mL) or chitosan (160μg/mL) Au nanomaterial was added to the wells. Cell cultureswere then incubated for 72 h. Cell viability was thendetermined using an AlamarBlue reagent in accordance withthe procedure from the manual. Cells were incubated for amaximum of 3 h at 37 °C. Absorbance was read on a platereader at a single wavelength of 590 nm.

In-Vitro Experiments. Cell cultures were grown as amonolayer in 35 mm dishes filled with 1 mL of DMEM at 37°C and in 5% CO2. In the confocal microscopy and cellphotothermolysis experiments, the cell monolayer was grownover a glass plate (Deckglaser, Germany) that was placed at thebottom of a dish. Approximately 0.5 million to 1 million cells inthe monolayer were prepared for flow cytometry andmicroscopic observations, whereas ∼2 million cells wereprepared for laser photothermolysis to yield a gap-freemonolayer.

Cell Flow Cytometry. The J5 cells were incubated in 1 mLof DMEM with the FITC-labeled, chitosan-capped Au NSs andNRs each at 4 and 10 μg/mL Au concentrations. Incubationmedium was then removed, and cells were rinsed with PBSsolutions several times. Cells were detached using 0.5 mL of a0.05% trypsin−EDTA solution in PBS and then incubated for 2min. Approximately 0.5 mL of the DMEM was then added toeach well. The obtained specimens were then used for the flowcytometry experiment. The emission (FITC) at 525 nm wasacquired at an excitation wavelength of 488 nm using a BDFACSCanto (BD Bioscience, USA) flow cytometer. For the in-flow imaging on ImageStreamX Mark II (Amnis, USA), cellnuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI)and the cells were imaged using a laser at 488 and 405 nm forFITC and DAPI fluorescence, respectively. Images wereacquired 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 NSsand NRs each at concentrations of 4 and 10 μg/mL todetermine the average uptake per cell of Au NPs. Afterincubation, the medium was removed and the cells were rinsedtwice with PBS solutions. Cells were then dispersed in 0.5 mLof the 0.05% trypsin−EDTA solution in PBS. Two 10 μL

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probes were then collected from the dispersions and mixedwith 10 μL of trypan blue. The cells in 10 μL of the mediumwere then counted using an automatic cell counter (Invitrogen,USA) and visually observed using a microscope. The remainingcell dispersions were dissolved in 1 mL of aqua regia(HCl:HNO3 = 3:1) for 1 h, diluted 50 times, and thenanalyzed 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 AuNSs was determined using a Leica TCS-SP5-SMD confocalmicroscope (Leica Microsystems GmbH, Germany). The J5cells were treated in fresh DMEM with Au NSs (16 μg/mL)capped with FITC-modifyed chitosan for 12 h incubation. Onthe next day, cells were stained with 5 μg/mL LysotTacker RedDND-99 and 50 μg/mL Hoschst 33342 (Invitrogen, USA) for30 min as given in the user instruction. Stained cells were thenobserved by confocal microscopy. In the cell photothermolysisexperiment, cell monolayers were incubated for 12 h in 1 mL ofDMEM with the chitosan-capped Au NP solutions at 10 and 4μg/mL of Au mass concentrations. After incubation, the culturemedium was replaced to remove excess Au NPs. Target cellswere irradiated with a 3.11 W NIR femtosecond (fs) pulse laserat a wavelength of 765 nm and an estimated maximum powerin the pulse of approximately 18, 26, 41, 60, 90, 105, 140, and170 mW (measured using a Newport Instrumentsoptical powermeter model 842 PE). The full-width at half-maximum (fwhm)of the laser pulse was determined as 88 fs, and the periodbetween pulses was 20.5 ns. The scanned area was a squarewith a length of 500 μm in the sides that contained around 518± 31 cells. The time per scan was 1.314 s. After irradiation, thecells were incubated for an additional 2 h. The DMEM wasthen removed, and 0.5 mL of trypan blue was added to stain thecells with membrane permeability. Dead cells (necrotic) withleaky membranes appeared blue in the microscope images. Todetermine the viability of J5 cells upon laser treatment theamount of cells with consistent membranes was countedmanually relative to the total number of cells within thescanned area using an optical microscope. The experiment onlaser scanning was conducted at 25 °C. An oil-immersionobjective lens with a numerical aperture (NA) of 0.7 was used.Energy fluences were calculated in accordance with a previouslyreported method.52,53 A 512 × 512 pixel area was scanned at arate of 1.314 s. The exposure time of each pixel per scan was5.01 μs. The focal spot area was calculated as πd2/4, where d isthe fwhm of the beam waist and was calculated from theformula d = 0.61λ/NA = 0.67. In this condition, the totalexposure time for Au NPs was estimated as (focal spot area/pixel area) ×5.01 μs = 0.832 μs per scan. In this study, thescanning of different excited power densities at maximum inpulse, namely, 0.2, 0.31, 0.46, 0.67, 1.01, 1.18, 1.57, and 1.92kW/cm2, at a focal spot area was applied to the specimens. Thenumber of scans was either 30 or 60. Energy fluences werecalculated from the product of the power density, number ofscans, and total exposure time per scan.

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

synthetic process. Au NPs with spherical morphologies wereinitially used as seeds in the Au growth solution. As the Auseeds grew, the silver cation complexes were adsorbed on theAu surface and functioned as growth inhibitors.49 Hence, after agrowth time of 24 h (Figure 1a), irregular plate-like Au NPswith few short branches were observed because the cappingability of the silver ions results in the blocking of most of theAu NP surface.49 Thus, the remaining unblocked parts on thesurface of irregular Au NPs expanded in a specific direction,which eventually formed branches. The diameter of the Au NPcores increased only slightly between a growth time of 24 and48 h. Continuous growth predominantly occurred on thecorners of the irregular plate-like Au NPs and in a particulardirection, which results in branching (Figure 1b and 1c). Silvercapping is effective on specific facets of initial Au crystallitesand results in growth of branches rather than expansion of thecore. HRTEM was performed to characterize branched Au NPsin detail and identify their crystalline boundaries (Figure 1c).Visible boundaries reveal that a branched Au NP consists of

Figure 1. TEM images of CTAB-encapsulated branched Au NPs thatwere obtained from a growth solution that contained 0.04 mM AgNO3within a growth period of (a) 24 and (b) 48 h; (c) HRTEM images ofbranched Au NPs grown for 42 h; (d) lattice fringes on a single branchtip; (e) synchrotron XRD pattern of Au NP (λ = 0.774907 Å).

Scheme 1. Stages of Au NP Growth and Modification withChitosan

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numerous single Au crystallites. The boundaries are in themiddle of the branches, which indicates the occurrence ofpreferential growth along the boundary of two crystallites. Thisresult suggests that Au NPs initially grew in the form ofirregular hexagonal or pentagonal multicrystalline structures(Figure 1a), which function as initial intermediate products inthe growth of branched Au NPs. If the AgNO3 concentration inthe growth solution is 0.04 mM and the synthesis period doesnot exceed ∼42 h, then most of the formed Au NPs have two-dimensional (2D) star-like structures with 3−5 branches(Figure 1c). When the growth time is increased to 48 h orwhen the AgNO3concentration is 0.05 mM, more irregularthree-dimensional (3D) Au NSs with more than 6 branches areformed (Figure 1b). The HRTEM image of a single branch(Figure 1d) shows lattice fringes with an average interfacetdistance of ∼0.23 nm, which corresponds to the space betweenthe ⟨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 inthe presence of Ag+ cations.49,54 The crystalline structure of AuNPs was analyzed using synchrotron XRD (Figure 1e). Thediffraction peaks of an as-prepared sample coincide with thoseof a standard Au (ICSD 58393). On the basis of the Scherrerequation, the primary crystallite size was estimated at 16.2 nmfrom the fwhm of the ⟨111⟩ reflex,55 which is considerablysmaller than the particle sizes observed by TEM. These resultsconfirm that each of the Au NSs is multicrystalline, i.e.,constructed from several single crystals. Following formation ofbranched Au NPs, chitosan was used to replace CTAB as astabilizer on the Au surface for subsequent application.The CTAB-protected Au NPs form a highly stable

suspension. However, relatively weak electrostatic bondsbetween CTA+ and the Au surface result in a CTAB cappinglayer 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 Aumass concentration of 80 μg/mL to target 10 and 4 μg/mLresults in immediate precipitation of Au NPs because theCTAB content in solution is reduced below its critical micellarconcentration and cannot retain stable and continuous cappingmicelles around NPs.56 Thus, the CTAB on the surface of AuNPs was replaced by the biopolymer chitosan to increase thebiocompatibility and stability of NPs in solution. FTIR spectra(Figure S1, Supporting Information) show replacement ofCTAB by chitosan on the surface of Au. The characteristicpeaks of chitosan are at 3449 cm−1 for ν(OH) (stretchingdeformation), 1084 cm−1 for ν(C−O−C), 1597 cm−1 for−NH2 bending, and 1650 cm−1 for ν(CO) or ν(C−N). Thesharp band at 1386 cm−1 corresponds to formation of a −CH3group. Comparison with the FTIR spectra of chitosan in FigureS1, Supporting Information, shows that all peaks in the FTIRspectra of the chitosan-modified Au NPs can be indexed to thefunctional groups of chitosan. The intensity of the peak at 1525cm−1, which corresponds to an −NH2 group, is considerablyreduced, and the peak shifts to higher energy because theinteraction between NH2 and the heavy Au atom changes thevibration mode of NH2 and affects its conformation. FiguresS1c and S1d (Supporting Information) show the FTIR spectraof the CTAB-modified Au NPs and pure CTAB. These spectrashow a strong and sharp characteristic band at 2918 cm−1,which corresponds to the stretching vibration of −CH2 groupsand can be compared with those of the chitosan-modified AuNPs.57 FTIR spectra of the chitosan-modified Au NPs show no

absorption band for CTAB, which indicates that CTAB was notpresent after chitosan treatment.Figure 2 shows UV−vis spectra of the as-prepared branched

AuNPs in various synthesis conditions. Shorter and longer

wavelength LSPR extinction bands appear because of the highanisotropy of the NS LSPR properties. By analogy with the AuNR spectra, shorter wavelengths contributions are assigned totransverse LSPR whereas longer wavelengths are assigned tolongitudinal LSPR.33,34,41 The changes in the extinction bandintensities and peak positions (Figure 2a and 2b) correspond tothe morphological evolution of the branched Au NPs withgrowth time and variation in the silver content, respectively.The contribution of the longitudinal LSPR to the extinctionspectra increases in intensity and shifts to a considerably longerwavelength (red shift) following the increase in the amount andlength of the branches, respectively. Extinction spectra of theCTAB-coated Au NPs can be deconvoluted in at least threedifferent bands, namely, T, T′, and L, as shown in Figure 2c and2d for growth times of 24 and 48 h, respectively. The simulateddistributions of the electric energy density of both NSs andNRs are shown in Figure 3a−d. The NS figure is constructedfrom the HRTEM image from Figure 1c. For simplification, themultifacet branches and central core were approximated withconic and spherical shapes, respectively. For each structure, thex- and y-polarized incidence cases were calculated to determinethe various modes of LSPR. Figure 3e−h shows thedistributions of the time-average resistive heating of theparticles excited by these LSPR modes during polarizedillumination. For the star-shaped NPs, the strongest fieldenhancement is achieved around the branch vertices when thelongitudinal axis of the branches is in maximum alignment withthe polarization direction (in the XY plane). In addition, thegreater enhancement of the E-field is found at more acutevertices (Figure 3a and 3b), which is consistent with previousreports on experimental and theoretical E-field enhancement ofAu NSs.35−40 A significantly greater areal density of free

Figure 2. UV−vis SPR spectra of branched Au NPs obtained (a) froma 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 24h; (d) deconvolution of absorbance spectraof Au NSs sample grownfor 48 h.

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electrons was observed for sharper vertices, which indicatesexcitation of a considerably stronger electrostatic field. Thisphenomenon is called the lightning rod effect, in which anincrease in the vertex angle results in aggregation of freeelectrons 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 atsharper corners of metal nanostructures such as cubes,triangular nanoplates, and branched structures with high tipcurvatures. Moreover, sharper vertices should induce a red shiftof the LSPR wavelength.35 The sharp tips on the NS branchesthat are not aligned with the E-field polarization direction (inthe XY plane) also cause substantial field enhancements (Figure3b) because of plasmonic coupling with other strongly excitedparts of NS.35,41,42 Areas of high free electron density are alsolocated at sharp vertices in intersections between the branchesand the central core or between closely situated branches.Meanwhile, two different particle-aligned cases are shown forthe Au NRs: one with the main axis parallel to the excited E-field (longitudinal plasmon) and the other perpendicular to theE-field (transverse plasmon). A comparison of Figure 3c and 3dshows that the particles aligned with the E-field polarizationcontribute a greater field enhancement. This result is alsoconsistent with the previously mentioned lightning rod effectand coincides with previous reports.58 In a similar manner,when an E-field polarization is perpendicular to all branch axesof the NS (Z direction in Figure 3i), the field enhancement issignificantly smaller than when the E-field is aligned with theXY plane. By using the calculated contributed E-field intensitiesas well as the imaginary part of Au permittivity, thedistributions of the time-average resistive heating can be

simulated based on the equation that describes the dissipativeelectric energy qe

37

ε ω ε ω= | |q E12

Im ( )e 02

(1)

where ε0 is the permittivity of vacuum, Im ε (ω) is theimaginary part of the permittivity of Au, ω is the frequency ofthe 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 NPsindicate that dissipative heating can be mostly generated in tworegions. One region is close to the branch vertices, whereas theother is corners at the intersection between the branches andthe Au core structures (Figure 3e and 3f). For the verticesaligned with the E-field, a strong field enhancement can beinduced based on the lightning rod effect. The enhanced near-field accelerates the free electrons in the NPs, which theninteract with phonons, which results in heat generation. Theother mechanism is due to the surface roughness in theintersection between the branch and the Au core structure. Theoscillated electrons are scattered by surface defects andgenerate heat. The simulated results for the NRs show thatthe strongest resistive heating was obtained at the middle of thestructure (Figure 3g). These results indicate that the conductivecharges are mostly accumulated at the edges and the highestenhanced field distribute outside the charge-accumulatedregions. Thus, the strongest contributed electrical field is atthe middle of the NRs, at which most of the heat is generated.In addition, a comparison of Figure 3g and 3h indicates that theresistive heating is significantly stronger when the particles arealigned with the E-field. These results are consistent with thediscussed results obtained from calculation of the electricenergy density distribution. The simulation shows the strongresistive heating ability of Au NSs under NIR irradiationbecause of the multiple sharp morphological features likebranch tips and branch−core intersections. However, thenonuniform shapes of the NSs result in a broad LSPRabsorption efficiency distribution across a relatively widewavelength range. The broadening of the extinction spectraof Au NSs is disadvantageous in comparison with Au NRs,which possess a highly narrow longitudinal absorptiondistribution 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 Figure2c and 2d can be attributed to the different calculated LSPRbands of Au NSs shown in Figure 3j. The presented simulationshows that when the electric field polarization is parallel to oneof the longitudinal branch axes (Figure 3b), a longerwavelength extinction band Y with a calculated resonantwavelength of 700 nm is obtained. Hence, L denotes thelongitudinal LSPR extinction on the Au NP branches. If the E-field is not perfectly aligned with any of the long branch axes(Figure 3a), a shorter wavelength band X centered at 670 nmappears. Therefore, T should be attributed to the transverseLSPR bands of the Au NP branches, which correspond to theE-field polarization that is perpendicular to the branch axes inthe XY plane (Figure 3i). However, the simulated spectradeviate 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 goldnanostar when the E-field is applied in the (a) X direction and (b) Ydirection, a gold nanorod when the E-field is applied in the (c) Xdirection and (d) Y direction; distribution of time-average resistiveheating produced by interaction of light with surface plasmons of agold 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) Xdirection and (h) Y direction; (i) simulation conditions for a two-dimensional nanostar; (j) simulated extinction spectra of a goldnanostar for an E-field polarization in the X, Y, and Z directions.

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observed ones. The main reasons for this disagreement are theapproximations performed for the simulated electric energydensity distributions and, more importantly, the nonuniformshapes 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 853to 995 nm, which corresponds to the increase in the length ofthe branches as shown in the corresponding TEM images(Figure 1a and 1b) because the SPR effect is strongly sizedependent. The fitted T peaks at 604 and 627 nm in Figure 2cand 2d represent the shifts in the positions of the transverseLSPR bands of the branches. Relatively broad shapes of T andL bands suggest various lengths of transverse and longitudinalaxes because the branch sizes are nonuniform. The T band isonly slightly red shifted by ∼23 nm, whereas the red shift of theL band is almost ∼150 nm when the synthesis time wasincreased from 24 to 48 h. This result suggests that theanisotropic growth of branches occurs preferably along thelongitudinal axes. According to the lightning rod effect, moreacute vertices yield a longer wavelength LSPR extinction.35 Themuch slower growth of the transverse axes compared withlongitudinal ones results in the decrease of branch verticesangles, which is the primary reason for a substantial red shift ofthe longitudinal LSPR band with increasing synthesis time.Moreover, formation of branches on the Au NPs in theproposed conditions is slow and time-dependent because thegold concentration in the solution gradually diminishes. Thisfact can be exploited to tune the optical properties of Au NSs.The narrow band T′ of the transverse region that is centered ataround ∼610 nm is assigned to the extinction that correspondsto the E-field polarization perpendicular to the NSs plane (Zdirection in Figure 3i). The relative intensity of T′ decreaseswith increasing growth time and AgNO3 concentration becauseof the increasing share of three-dimensional NRs according toTEM investigation. Growth of branches outside the XY plane(as depicted in Figure 3i) results in the appearance of anadditional longitudinal axis in the Z direction as well as agradual red shift of the corresponding LSPR contribution fromT′ to L. The morphology of Au NSs and their absorbancespectra are also highly sensitive to the silver cation content inthe growth solution. At a growth time of 42 h, the relativeintensity of the longitudinal part of the extinction spectrainitially increases until the Ag+ concentration reaches 0.05 mM,as displayed in Figure 2b. These results are attributed to theincrease in the number of branches and in the appearance ofthree-dimensional NSs. The relative intensity of the longi-tudinal band declines when the growth solution contains 0.06mM 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 Lband, which is obviously due to the increase in the averageamount of elongated branches per NPs, the longitudinal LSPRwavelength remains within the relatively narrow range from∼932 to 943 nm and does not suffer a noticeable shift when thesilver content in the growth solution increases from 0.03 to0.06 mM (Figure 2b). In accordance with the lightning rodeffect,35,37 the vertex angle on the tips and the average length ofbranches do not significantly change within the investigatedvariation of the silver concentration for the same growth timeperiod. However, the amount of branches per particle increaseswhen the Ag+ concentration is increased from 0.03 to 0.05 mM.The desired morphological (2D or 3D NSs) and opticalproperties can be obtained by setting both the growth time and

the Ag+ content in solution. Au NSs that were obtained with agrowth time of 42 h and an Ag+ concentration of 0.04−0.05mM have a longitudinal absorption band that completely coversthe NIR light transparency window of living tissues within630−950 nm.59,60 Increasing the synthesis time to 48 h shiftsthe NIR absorption maximum above the NIR transparencylimit of 950 nm, where the light absorption of water issubstantially higher.Figure 4 presents the normalized absorbance spectra of as-

prepared branched Au NSs capped with CTAB and after being

modified with chitosan. The normalized spectra of chitosan-capped Au NSs demonstrate a slight blue shift in the transverseLSPR extinction band by ∼5 nm from that of the CTAB-capped NPs. The LSPR absorption properties of Au NPs areknown to be strongly affected by the local dielectric function ofthe surrounding medium.17,61 Another reason for thisphenomenon could be the slight plasmon coupling that resultsfrom association of chitosan-capped Au NPs. The interaction oftwo or more surface plasmons of closely situated metal NP canlead to the appearance of a completely new plasmon field thatalso results in a change in the extinction band intensity andwavelength.36 Thus, the variations in the intensity and shape ofthe absorbance spectra of Au NPs provide a simple way toevaluate the stability of Au NPs dispersion in solution. Thepolymer chitosan and its derivatives were previously used forboth the stabilization of Au NPs in solution and the controlledassembly via chitosan linkers.22,62 Hydrodynamic diametermeasurements show that the average sizes (zeta average) ofCTAB- and chitosan-modified Au NSs in DI water are 26.1 and36.7 nm, respectively. The increased hydrodynamic diameterfor chitosan-capped NSs shows a higher degree of agglomer-ation, which is likely due to hydrogen bonding and van derWaals interaction between chitosan molecules that envelop AuNPs. In a DI-water solution, this interaction is compensated bythe electrostatic repulsion between charged capping layers ofAu NSs. The chitosan molecules are able to bind hydrogencations in an acidic medium and detach them in a basic solutionbecause of the −NH2 groups. Therefore, the properties of thechitosan capping layer such as surface potential strongly dependon the composition and pH of the medium. Water-solublechitosan-capped NPs are stabilized better in weak acids.22 Thesolution of as-prepared chitosan-capped Au NPs in DI waterhas a slightly acidic reaction with a pH of 5.9. Hence, Au NPsremain dispersed because protonated chitosan layers carry apositive 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

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

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when dispersed in DI water. After a long period of time, NPsprecipitate but can be easily redispersed with simple shaking orsonication.Figure 5 displays the time-dependent evolution of the

extinction bands of chitosan-capped Au NRs and NSsdispersions (10 μg/mL) at 37 °C in a phosphate buffer saline(PBS) and DMEM medium with pH = 7.5. Figure 5a and 5bshows spectra for Au NRs and NSs in PBS, respectively. AuNRs with an aspect ratio of ∼3.5 were prepared according tothe method described by El-Sayed et al.50 and used forcomparison purposes. The water dispersion of Au NRs has anextremely narrow and strong longitudinal LSPR absorptionband at 765 nm because of the size and shape uniformity of theNRs. An initial intensity at the peak of the longitudinal band forthe chitosan-capped NRs solution in water is about 2.5 timeshigher 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 animmediate and drastic change when PBS solution was usedinstead of DI water because of aggregation. However, the

reaction proceeds at substantially different rates for rod- andstar-shaped nanoparticles (Figure 5a and 5b). Chitosanmolecules contain NH2 groups that function as a weak base,which bind H+ cations in acidic media and detaches them inbasic solution. The average zeta potential of chitosan-cappedAu NSs in DI water is +36.4 mV at a pH of 5.9. However, thisvalue sharply decreases to about +3 mV in PBS because of thedeprotonation of amino groups in chitosan molecules. Theincrease in pH to 7.5 (PBS buffer) results in degradation of thepositive charge of the capping layer, and as a consequence, theelectrostatic repulsion forces between nanoparticles decrease toa point where they cannot sustain a stable dispersion. As aresult, chitosan-coated NPs become aggregated, which isconfirmed by a remarkable shift and broadening of extinctionbands. In the Au NRs solution, the extinction spectra shiftsfrom ∼765 (in a water solution) to ∼870 nm immediately upondissolution in PBS with a sharp drop in peak intensity (Figure5a). The longitudinal LSPR band of chitosan-capped Au NRs inPBS is significantly broadened. The hydrodynamic diametermeasurement results for Au NRs upon dissolution in PBS at

Figure 5. Time-dependent evolution of extinction spectra of dispersion of chitosan-capped (a) Au NRs and (b) Au NSs with gold massconcentrations 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-NSaggregate); time-dependent evolution of extinction spectra of a dispersion of (e) chitosan-capped Au NRs and (f) Au NSs with gold massconcentrations of 10 μg/mL treated at 37 °C in DMEM medium.

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first show an average size of 147 nm with a relatively narrowdistribution (Figure S2a, Supporting Information). This value isequal to about three times the size of NRs along thelongitudinal axis. The instrument is unable to provide correctmeasurements of hydrodynamic size after prolonged treatmentsin PBS at 37 °C because of the high polydispersity of particlesin the Au NR solution sample. This originates from a verybroad size distribution of formed aggregates and from thedecrease in the effective particle concentration in thesuspension. The longitudinal LSPR band of the Au NRsolution becomes asymmetric during PBS treatment (Figure5a), which also indicates that the obtained NRs aggregates hada wide size and shape distribution.58 In Figure 5b, a substantialdrop in the extinction intensity for the Au NS solution in PBSwas confirmed only after ∼1 h (at 37 °C), which implies aslower aggregation of the as-prepared gold NSs compared withNRs. Immediately upon dissolution the average hydrodynamicsize of Au NSs in PBS is 106 and 174 nm after 1 h of treatmentat 37 °C. The histograms of hydrodynamic size distributionsare 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 AuNRs form clusters that comprise tens or even hundreds ofrandomly associated particles. This clustering leads to adecrease in the effective concentration of particles in thesolution as well as the complex interaction of surface plasmonsof NRs that results in a substantial change in its absorbancecharacteristics (Figure 5a). The star-shaped nanoparticlespredominantly form chain-like aggregates comprising arelatively small amount of NPs compared with Au NRs. Ahigh-resolution TEM image (inset in Figure 5d) illustrates themain aggregation route of chitosan-capped Au NSs. Branch tipswith a high curvature preferably associate with the low-curvature surfaces of NS, which are ordinarily the central coresor flat parts of the branches. This result can be explained by theelectrostatic interaction between different parts of star-shapedNPs. As noted before, the zeta potential of chitosan-capped AuNPs drastically decreases to around +3 mV at a pH of 7.5 inPBS from +36.4 mV in DI water. The measured averagepotential in PBS refers to the aggregates of several NSs, whichsuggests that the chitosan capping layer of individual Au NSscould be even smaller. A diminished positive charge of thechitosan layer decreases its ability to mask a negative chargelocated at the surface of Au NSs. The areas of a stronger LSPRE-field enhancement upon interaction with light of resonantwavelength (polarization in the XY plane as displayed in Figures3a and 3b) reflect parts of NSs with a higher aerial electrondensity. On the basis of the FEM simulation results, the highestcharge density is concentrated around the acute branch tips(lightning rod effect). The similar electric field distributionsacross the Au NSs occur because of an electrostatic interactionof the charged surface of Au NSs with electrolyte medium.Moreover, the aerial free electron density is directly propor-tional to the surface area of a specific part of the Au NP and isinversely proportional to the corresponding volume.35 Themuch larger specific area to volume ratio at the sharp verticesresults in a greater electric field at the branch tips. Thus, ahigher surface charge density at the vertices should cause astronger electrostatic repulsion between the tips of thebranches. Hence, acute tips preferably associate with low-curvature cores or flat parts of the NPs that possess a muchlower charge density at the surface. In an ideal scenario, one

chitosan-capped NSs can associate with two other NSs via theconnection to their branches by different sides of the centralcore (opposite sides of the XY plane of a NSs in Figure 3i),which results in chain-like structures. This restriction on themutual orientation of NSs also makes the agglomerationprocess slower compared with NRs. Au NRs have a muchsmaller surface charge density gradient along the longitudinalaxis (Figures 3c). Hence, the aggregation of chitosan-capped AuNR in PBS predominantly occurs in a random way (Figure 5c).Chain-like aggregation was also observed for Au NPs preparedvia direct reduction of NAuCl4 by chitosan.63 The tail-to-middle assembly into stair-like chains as a result of the mutualorientation of Au NRs that were modified by N-methyl-2-pyrrolidone was also reported recently.58 The observed chain-like aggregation of NSs could be also the consequence of thelow probability of contact between branch tips of different NSsbecause the as-prepared Au NSs have a relatively big size(∼70−90 nm in diameter). Therefore, ultrasmall (∼30−40nm) branched gold NPs were prepared via HEPES reduction ofHAuCl4 according to Xie et al.64 to check if chain-likeaggregation is induced by a branched structure or is aconsequence of the large size of the as-prepared NSs. Aftermodification with chitosan, the concentrated NP solution wasdissolved in PBS to produce a gold mass concentration of 10μg/mL. This solution was kept for 4 h at 37 °C. TEM results(Figure S3, Supporting Information) also reveal a chain-likeaggregation similar to Au NSs that were investigated in thepresent study but comprised a larger amount of nanoparticles.Moreover, the smaller Au NSs produce highly branched chainsthat can associate further in ring-like structures, probablybecause of the high ratio of three-dimensional NSs produced bythe HEPES reduction method as opposed to two-dimensionalAu NSs that were investigated in the present study. Chitosan-capped Au NSs with sizes within 30−40 nm also experienced amuch slower intensity degradation of the extinction band in thePBS 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 DMEMsolution (10% serum), which was used as a cell culture mediumfor the in-vitro study. In DMEM, the change in the spectralcharacteristics of chitosan-capped Au NPs is not as sharp as thatin PBS despite the same pH value. In the case of Au NSs, aninsignificant red shift was observed in the band maximumposition and almost no degradation was observed in theintensity of the extinction spectra even after 24 h at 37 °C. Thisresult suggests a stable dispersion. On the other hand,hydrodynamic diameter measurement results for the chitosan-capped Au NS dispersion in DMEM clearly show a slight shiftof the zeta size distribution toward larger sizes as timeprogressed (Figures S2d and S2e, Supporting Information).Moreover, the zeta size histogram shows an additional peakwithin a range of 500−800 nm after 4 h, which is a clear sign ofaggregation. However, in DMEM, this process appears to bemuch slower than in the PBS solution. The amino groups inchitosan molecules can create hydrogen bonds with theproteins in DMEM.20,21 A strong electrostatic interaction isunlikely due to a substantially decreased charge of the chitosancapping layer at pH = 7.5. Relatively large protein moleculescan form linkers between chitosan-capped gold NPs. At thesame time, these protein molecules can function as separatinglayers that prevent the coalescence of chitosan layers that

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envelop NPs and the strong interaction between the surfaceplasmons of neighboring particles. Therefore, the shifts of theextinction bands of Au NPs dispersions in DMEM shown inFigure 5e and 5f are insufficient compared with those of PBSsolutions (Figure 5a and 5b) despite agglomeration. The factthat the intensity of the extinction band of Au NSs in DMEMdoes not noticeably decrease within the investigated period (24

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

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 withmaximum concentration of Au for 72 h, image of (c) a blank J5 cell culture (incubated without Au NSs), (d) J5 cell culture treated with chitosan-coated 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 LysotrackerDND-99 fluorescence, (c) overlap of bright field with FITC, Lysotracker, and blue Hoschst-33342 fluorescences, (d−f) respective images offluorescence only for the J5 cell culture incubated for 12 h with 16 μg/mL of FITC-labeled chitosan-capped Au NSs; (g−i) confocal microscopicimages of untreated J5 cells (incubated without Au NSs). Scale bar is 20 μm.

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spectra of chitosan-capped gold NRs in DMEM appears to beonly partially reversible even upon sonication (Figure 5e).Thus, chitosan-capped Au NPs can retain at least a partialdispersion in a high-serum DMEM medium for a prolongedperiod of time (up to 24 h) despite a basic medium with a pHof 7.5.An in-vitro cytotoxicity analysis was further performed by

incubating several cell lines (BEAS-2b, OMF, S-G, J5) withchitosan- and CTAB-modified branched Au NSs for 72 h, asdisplayed in Figure 6. CTAB-modified Au NPs show anobvious cytotoxicity toward all cell types when theirconcentration exceeds 0.08 μg/mL. This result is consistentwith previous findings on the bioeffect of CTAB-cappedNPs.16,17 The viability of BEAS cells declines to less than 60%and then to ∼0% as the concentration of CTAB-capped NPsincreases to 0.8 and 8 μg/mL, respectively. The cell lines usedin the study have different tolerances to the CTAB-modifiedNPs. The viability of the four cell lines that were incubated with8 μg/mL of CTAB-modified Au NPs were all ∼0%, whichindicates a high cytotoxicity that is disadvantageous tobioapplications such as thermal therapy and drug delivery.Concentrations of CTAB-modified Au NP that are less than 0.8μg/mL have no effect on viability except for BEAS-2b cells.

This effect may be the result of the negligible uptake of CTAB-capped Au NSs at very low concentrations in solution.53

Another potential reason for this phenomenon is thesubstitution of weakly bonded CTAB on the surface of NPsby various species in the culture medium. By contrast, thechitosan-modified Au NSs exhibit no significant cytotoxicitytoward any cell line. Figure 6c−e presents photographs of theuntreated J5 cells and J5 cells that were incubated with 16 μg/mL of chitosan-modified Au NPs for 72 h and 8 μg/mL ofCTAB-capped Au NPs. Cells that were incubated with CTAB-capped Au NSs all have a spherical shape, which indicates theirdeath. The state of the cells that were treated with chitosan-capped NPs remains mostly unchanged after 72 h, whichconfirms the biocompatibility of chitosan-modified branchedAu NPs. Cytotoxicity assay results shows that the cytotoxicityof 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 itshighest value (16 μg/mL). This result suggests a concentrationlimit in the safe uptake of chitosan-capped Au NSs, which alsodepends on the cell type. Internalization of Au NP in largeamounts may have an effect other than toxicity on the viabilityof cells or may disrupt important intracellular processes.65 The

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 anincubation DMEM medium; flow cytometry FITC fluorescence histograms of (b) blank J5 cells incubated for 12 h with (c) a 10 μg/mL solution ofFITC-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 AuNRs, (e) a 4 μg/mL solution of FITC-chitosan-capped Au NSs.

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uptake and localization of chitosan-capped Au NSs by J5 cancercells was verified by a three-dimensional analysis of thefluorescence distribution via confocal microscopy. Cells weretreated with a modified green-fluorescent FITC dye-labeledchitosan Au NSs with a concentration of 16 μg/mL. Bluefluorescent Hoschst 33342 was added to mark positions ofnuclei in J5 cells, and LysoTracker Red DND-99 was used todetermine whether internalized Au NSs are localized inlysosomes. Figure 7 shows the bright-field images, FITC,LysoTracker Red DND-99, and Hoschst 33342 fluorescenceimages and their overlaps with Hoschst 33342-fluorescence forthe J5 cells incubated with FITC-labeled Au NSs in comparisonwith untreated J5 cells. The intracellular staining result showedthat the green-fluorescent spots of Au NSs were generalymerged with lysosomal markers (Figure 7c and 7f). The yellowspots indicate merging of FITC fluorescence with lysosomalprobe in cytosol. The positions of Au NSs are remarkablycolocalized with red-fluorescent LysoTracker. A three-dimen-sional analysis (Figure S5, Supporting Information) showedover a 58% colocalization ratio. It suggests that Au NSs in J5cell 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 ofchitosan-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/mLof gold concentrations in incubation medium are presented.However, the mechanism and kinetics of chitosan-modified AuNPs internalization by cells were not a subject of detailedinvestigation in this study. The flow cytometry results of the J5cells that were incubated with fluorescent FITC-chitosan-capped Au NPs are presented in Figure 8c−f. Figure 8b displays

the fluorescence hystogram of the control J5 cells. Figure 8c−fshows the fluorescence distributions of the J5 cells that wereincubated for 12 h with different contents of FITC-chitosan-modified Au NSs and NRs. A shift in the overall distributiontoward a higher fluorescence in reference to the control sampleindicates the internalization of fluorescent nanoparticles. Thehistograms for the J5 cells that were incubated with Au NRs(Figure 8c and 8e) appear to be noticeably broader comparedwith 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 cancercells using a femtosecond laser source with a wavelength of 765nm, which corresponds to the maximum absorption efficiencyof as-prepared Au NRs in a water solution. After lasertreatment, cell samples were incubated for two more hoursand stained with trypan blue to indicate dead cells. In thisstudy, an energy fluence of 86 mJ/cm2 was found to be enoughto cause a substantial effect on the control J5 cells (without AuNPs), as shown Figure S6 in the Supporting Information. In theliterature, the energy fluence that causes Hela cell mortality isgiven as 113 mJ/cm2.52 Figure 9a−e and 9f−j shows the effectsof pulse laser scanning (area of 500 μm × 500 μm) on the cellsthat were incubated with 10 μg/mL of chitosan-capped goldNSs and NRs, respectively. The average amount of cells in thearea is 518 ± 31. The photothermal impact on cells is relativelystrong for both investigated types of NPs because of the highconcentration of Au NPs. In cell samples treated with chitosan-capped Au NSs solution starting from an energy fluence of ∼12mJ/cm2 (0.46 kW/cm2 power density and 30 scans), almost∼100% percent of cells are destroyed as seen in Figure 9e. Incell samples incubated with 10 μg/mL of chitosan-capped AuNRs (Figure 9g−j), only an energy fluence of ∼17 mJ/cm2

Figure 9. J5 cell monolayers stained with a trypan blue dye pulsed laser scanning with energy flunces: cells incubated with 10 μg/mL of chitosan-capped 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; cellsincubated with 4 μg/mL of chitosan-capped Au NRs (p) 33, (r) 17, (s) 23, (t) 12, and (u) 15 mJ/cm2.

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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 massconcentration of gold is ∼10 μg/mL, the photothermal impactmediated by Au NPs from laser power density over 0.46 kW/cm2 is strong enough to cause a very intense cavitation andbubble 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 aroundbig clusters of Au NPs because of intense evaporation. Theproblem in this case is that vapor bubbles are also efficientscattering 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 toeliminate the evaporation effect. The results are presented inFigure 9k−o and 9p−u for cells treated with chitosan-cappedAu NSs and Au NRs, respectively. Laser irradiation starts tocause photothermolysis of the cells that were incubated with 4μg/mL of Au NPs at a power density of 0.31 kW/cm2 after 60scans, which corresponds to an energy fluence of 15 mJ/cm2 forboth Au NSs and NRs in Figure 9o and 9u, respectively.However, at this level of laser power density only a smallfraction of cells is killed (less than 50% for the sample treatedwith Au NRs). An increase in the power density leads to asmaller amount of scans needed to achieve similar result. At0.46 kW/cm2, up to 80% of the cells are killed after 30 scans,which corresponds to an energy fluence of 12 mJ/cm2. Anenergy fluence of 23 mJ/cm2 (power density 0.46 kW/cm2 and60 scans) results in an almost complete photothermolysis ofcells that were incubated with 4 μg/mL of chitosan-capped AuNSs as shown in Figure 9m. A similar result for cells that wereincubated with 4 μg/mL of Au NRs can be achieved when theenergy fluence reaches 33 mJ/cm2 (at a power density of 0.67kW/cm2 and 60 scans). For Au NSs-treated samples, 6%, 5%,9%, 8%, and 27% of cells retain a consistent membrane afterlaser scanning with energy fluences of 33, 23, 17, 12, and 15mJ/cm2, respectively. On the other hand, for samples incubatedwith Au NRs, 5%, 12%, 16%, 19%, and 52% of the cells retain aconsistent membrane after irradiation with energy fluences of33, 23, 17, 12, and 15 mJ/cm2, respectively. In samples treatedwith Au NRs, a higher amount of cells generally remainsundamaged in comparison with those incubated with Au NSs.This result is the opposite of what was expected from theextinction measurement data for Au NSs and NRs at awavelength of 765 nm. Au NRs have about a 150% higherextinction 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 765nm measured in the water solution because of the aggregationof chitosan-capped NRs. However, as shown before (Figure 5e)in a protein-rich medium, the shape and position of theextinction spectra do not significantly change. Second, Figure5e and 5f shows that the agglomeration of chitosan-capped AuNRs (unlike that of Au NSs) in a DMEM medium still causes anoticeable degradation in the absorbance intensity in the NIRregion probably because the extinction in the cross-section ofthe sample decreases. In addition, the absorption efficiency isoptimal when the longitudinal axis of the rod-shaped particle isaligned with the E-field polarization during laser irradiation,which is impossible to achieve for all NRs in the samplebecause aggregation restricts an independent orientation ofNRs 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 inthe as-performed experiment, no substantial difference wasobserved in the photothermolysis efficiency between two kindsof investigated NPs to claim a significant advantage of any typefrom the obtained data. However, every specimen after laserscanning contains a specific amount of cells with consistentmembranes whose amount decreases with increasing appliedlaser energy fluence. The most obvious reason for incompletephotothermolysis is that the survived cells had internalized asmaller amount of Au NPs or, in other words, as a result of thenonuniform distribution of gold across cells in the specimen.Figure 8c and 8e reveals a substantial broadening of histogramsof the FITC fluorescence distribution versus cell count forspecimens that were incubated with Au NRs modified byFITC-labeled chitosan compared with the histograms ofcontrol cell samples and samples treated with Au NSs (Figure8d and 8f). The overlays of normalized histograms for goldconcentrations of 10 and 4 μg/mL are presented in Figure S8,Supporting Information. Broader histograms obtained fromsamples treated with Au NRs mean that FITC fluorescence hasa more nonequivalent distribution across the cell line. Thisresult suggests that the distribution of FITC-chitosan-cappedAu NRs among cells is more nonuniform compared with that ofAu NSs. The examples of in-flow images of the J5 cells thatwere incubated with Au NPs and modified by FITC-labeledchitosan are presented in Figure S9, Supporting Information.Very large FITC fluorescence areas that mark positions of AuNRs clusters can be spotted in some cells treated with FITC-chitosan Au NRs, whereas other cells show very little or nofluorescence at all. The highly nonuniform aggregation of thechitosan-capped Au NRs and as a consequence of non-equivalent mass distribution of gold NRs across cell lines arethe most likely reasons for a greater amount of surviving J5 cellsin the specimens that were incubated with Au NRs comparedwith those treated with Au NSs after laser irradiation. Adifference in the aggregation of chitosan-capped Au NSs andNRs in incubation wells can also be observed in the opticalmicroscope as displayed in Figures S10, Supporting Informa-tion. Rare but relatively large agglomerates (black spots) can beseen in images taken using an optical microscope in cellsamples after 4 h of incubation in a DMEM culture mediumcontaining chitosan-capped Au NRs. In this case, largeaggregate formation proceeds mostly in the vicinity of cellmembranes. In the samples treated with both 4 and 10 μg/mLof chitosan-capped Au NSs for 4 h a high amount of muchsmaller aggregates with a relatively even distribution over thedish bottom and cell layer can be observed. A nonuniformdistribution of Au NRs across the cell line results in a strongphotothermal effect on cells that internalized larger aggregates,which leads to membrane destruction that can be detected viatrypan blue staining. Cells with a smaller amount of internalizedAu NRs can remain unaffected at specific energy fluences.

4. CONCLUSIONIn summary, Au NSs with sizes between 70 and 100 nm wereprepared via the solution route in the presence of silver cations.TEM results showed that Au NSs have a two-dimensionalmorphology when the silver concentration in the growthsolution is 0.04 mM and the synthesis time is 36−42 h. Anincrease in the silver content or growth time leads to formationof three-dimensional Au NSs. The surfaces of as-prepared AuNSs were modified with biopolymer chitosan to improvebiocompatibility and colloidal stability in diluted solutions of as-

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prepared Au NSs. The electrical charge distribution simulationbased on FEM showed the free electron density and electricfield enhancement of the surface because the LSPR reached themaximum value at the vertices on NS branches. The LSPRextinction spectra of the as-prepared NSs consist of two bandsin the visible region at ∼610 and 627 nm, and the NIR bandcentered within a range of 853 to 995 nm is tunable via thesynthesis time and silver content in the growth solution. Theoptical characteristics of colloidal chitosan-modified Au NSsdispersed in a PBS buffer and high serum medium with pH =7.5 appeared to be substantially more stable than those of AuNRs because of the slower aggregation of NSs. Chitosan-modified Au NRs at pH = 7.5 formed aggregates with highlynonuniform sizes. The Au NSs form small chain-like clusters inwhich individual NSs connect to one another preferably viaassociation of acute branch tips and central cores. Thesubstantial charge distribution gradients on the Au NSs surfacewhere acute branch tips surrounded by a high aerial electrondensity and the surface of central cores of NSs possess anegligible free charge density are presumably responsible forthe preferred affinity of those parts of NSs during theaggregation process. This restriction on the mutual orientationof Au NSs needed for association also slows down theaggregation of chitosan-capped Au NSs. Flow cytometryanalysis results showed a relatively nonequivalent distributionof chitosan-capped Au NRs across the cells in specimencompared with Au NSs because of highly nonuniformaggregation and cellular uptake of NRs. An experiment onthe photothermolysis of J5 liver cancer cells in vitrodemonstrated that an energy fluence of 23 mJ/cm2 was enoughto cause the death of J5 cells that were incubated with chitosan-capped Au NSs. For complete photothermolysis of cell samplestreated with Au NRs an energy fluence of 33 mJ/cm2 wasnecessary. When chitosan was used as a surface capping agent,the Au NSs had a higher colloidal stability at a physiological pHof 7.5 than Au NRs and are more suitable mediators in cellphotothermolysis because of the slower aggregation and moreuniform cellular uptake.

■ ASSOCIATED CONTENT*S Supporting InformationDetailed experimental information of FTIR, size distribution,and TEM images for characterization of chitosan-modified goldnanostars; 3D analysis of localization of chitosan-modified goldnanostars in J5 cells; transmittance photos of photothermolysisof J5 cancer cells; flow cytometry detection of FITC-chitosan-capped gold nanostars; microscope images after treating withvarious concentrations of gold nanostars. This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: rsliu@ntu.edu.tw; mhsiao@gate.sinica.edu.tw; dptsai@phys.ntu.edu.tw.Author Contributions&The first two authors contributed equally to this work.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank the National Science Council of the Republic ofChina and Taiwan (Contract Nos. NSC101-2113-M-002-014-

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

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