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Functional Nanostructured Materials (including low-D carbon)

Au Nanobottles with Synthetically Tunable Overall and Opening Sizes for Chemo-Photothermal Combined Therapy Han ZHANG, Jianli Chen, Nannan Li, Ruibin Jiang, Xiao-Ming Zhu, and Jianfang Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b19163 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 19, 2019

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ACS Applied Materials & Interfaces

Au Nanobottles with Synthetically Tunable Overall and Opening Sizes for Chemo-Photothermal Combined Therapy Han Zhang,† Jianli Chen,‡ Nannan Li,† Ruibin Jiang,§ Xiao-Ming Zhu,*,‡ and Jianfang Wang*,† †Department ‡State

of Physics, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China

Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science

and Technology, Avenida Wai Long, Taipa, Macau SAR, China §Shaanxi

Engineering Lab for Advanced Energy Technology, School of Materials Science and

Engineering, Shaanxi Normal University, Xi’an 710119, China KEYWORDS: gold nanobottles, asymmetric metal nanostructures, plasmon resonance, drug delivery, photothermal therapy, magnetic plasmon resonance

ABSTRACT: Highly asymmetric Au nanostructures, such as split Au nanorings and Au nanocups, exhibit attractive plasmonic properties due to their asymmetric geometries. To facilitate their plasmonic applications, effective and facile synthetic methods for producing asymmetric Au nanostructures with controllable sizes and uniform shapes are highly desirable. Herein we report on an approach for the synthesis of largely asymmetric colloidal Au nanobottles with synthetically tunable overall and opening sizes. Au nanobottles with overall

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sizes in the range of ~100–230 nm are obtained through the sacrificial templating with differently sized PbS nanooctahedra. The opening sizes of the produced Au nanobottles can be tailored from ~10 to ~120 nm by either adjusting the Au/PbS molar ratio in the growth process or controlling the oxidation degree. The achieved size tunability allows the plasmon resonance wavelength of the Au nanobottles to be varied in the range of ~600–900 nm. Our uniform Au nanobottles, which possess controllable sizes, large cavity volumes and tunable plasmon resonance wavelengths in the visible to near-infrared range, have been further applied for anticancer drug delivery and photothermal therapy. The effects of the surface coating and opening size of the Au nanobottles on the drug encapsulation efficiency and initial burst drug release are systemically evaluated. A high doxorubicin encapsulation efficiency and low initial burst drug release are realized with the dense silica-coated Au nanobottles having the opening size of 44 nm. In addition, chemo-photothermal combined therapy has been demonstrated with the doxorubicin-loaded Au nanobottles. Our results will be helpful for the design of the Au nanobottles with different sizes and plasmonic properties as well as ample opportunities for exploring various plasmon-enabled applications out of the Au nanobottles.

1. INTRODUCTION Colloidal plasmonic metal nanocrystals have been extensively studied owing to their rich and attractive localized surface plasmon resonance (LSPR) properties and their broad applications in linear and nonlinear optics,1,2 photocatalysis,3,4 chemical and biological sensing.5 Moreover, because of their great chemical stability and synthetically controllable plasmonic properties, colloidal Au nanocrystals have been examined intensively in the fields of medical diagnostics


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and nanomedicine, including optical coherence tomography,6 controlled drug delivery7,8 and photothermal therapy (PTT).9,10 Over the last decade, a number of works have been carried out on the design of colloidal plasmonic metal nanocrystals,11,12 because the sizes, shapes and elemental compositions of plasmonic nanocrystals all play important roles on the plasmonic properties.13,14 For instance, the plasmon resonance wavelengths of plasmonic metal nanocrystals can be synthetically tailored from the ultraviolet to the near-infrared (NIR) region through the variation of the shape and/or size.15 Elongated metal nanostructures with high aspect ratios, such as Ag nanorods, possess multipolar plasmon modes, making them an ideal candidate for applications based on multipolar plasmons.16 Moreover, highly asymmetric plasmonic nanostructures can exhibit interesting plasmonic properties that are of great difference from those of their symmetric counterparts. For instance, split Au nanorings and Au nanocups can support interesting magnetic plasmon modes.17,18 Magnetic resonance is generally required for the design of metamaterials, which show broad applications in the fields of directional light scattering,19,20 electromagnetically induced transparency,21 perfect light reflection/absorption,22 negative refraction,23,24 cloaking and superlensing.25,26 Up to date, most colloidal plasmonic nanocrystals and nanostructures only possess electric plasmon resonance modes but lack magnetic plasmon resonance modes owing to their high structural symmetries. Breaking the symmetry of colloidal nanostructures at nanoscale by chemical methods has remained challenging because the growth of metal nanocrystals in solutions often proceeds in an isotropic manner under thermodynamical and/or kinetic control.12 So far, asymmetric nanostructures supporting strong magnetic resonance modes have mainly been fabricated by conventional physical methods, such as focused ion beam and electron beam lithography.18,20,27 However, in general, these methods are tedious, expensive, less flexible, and


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typically consist of multiple steps.28 Moreover, metal nanostructures fabricated by physical methods are polycrystalline and commonly attached to substrates through the use of an adhesive Cr or Ti layer. They therefore suffer from considerable plasmon damping.28,29 The inferior plasmonic properties can affect the performance of the fabricated plasmonic devices. Therefore, the wet-chemistry synthetic methods of plasmonic nanostructures with strong magnetic resonance are highly desired. Only a few types of Au nanostructures possessing magnetic resonance modes, such as Au nanocups, can be made by chemical methods. Such chemical methods rely mainly on partial surface blockage during growth or galvanic replacement combined with surface blockage.30,31 Recently, our group has developed a facile wet-chemistry synthetic method for colloidal Au nanocups.32 The synthesis starts with single vertex-initiated Au overgrowth on PbS nanooctahedra, which is followed by the etching of the PbS component with HCl. The obtained Au nanocups possess a magnetic (transverse) and an electric (axial) plasmon mode at spectrally separated positions. When the excitation light is polarized perpendicular to the symmetry axis (transverse), there are a great electric field enhancement at the cup edge and a great magnetic field enhancement in the cup opening region. In addition, another group has also successfully synthesized Au nanocups by thermal dewetting of Au shell encapsulated in between an inner SiO2 core and an outer SiO2 shell.6 Although these methods have been shown to be highly feasible, it has remained unexplored to synthesize Au nanocups in high quality and with good control over their important dimensional parameters, such as the overall size and opening size, both of which play important roles in their optical properties.32 In this work, we have demonstrated the systematic control of the overall and opening sizes of Au nanocups. The synthesized bottle-like Au nanostructures can have a much smaller opening size than their hollow cavity size and overall size, and therefore they are called Au nanobottles in


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this study. The edge length of PbS nanooctahedra and the Au/PbS ratio have been found to be the key factors during the Au overgrowth process for tailoring the sizes and hence the plasmon wavelengths of the Au nanobottles. Moreover, the sizes of the Au nanobottles and therefore their plasmon wavelengths can also be finely controlled after overgrowth through mild anisotropic oxidation at room temperature. To our knowledge, the anisotropic oxidation of Au nanobottles for finely controlling their morphology and plasmon wavelengths has not been demonstrated before. The overall size and opening size of the Au nanobottles can be respectively varied in the range of ~100–230 and ~10–120 nm. The magnetic plasmon resonance can be accordingly varied from ~600 to 900 nm. Furthermore, the large cavity volume, small opening size and tunable plasmon resonance wavelengths of the Au nanobottles enable them to act as an ideal candidate for anticancer drug encapsulation and combined chemo-photothermal therapy. The effects of the surface coating and opening size of the Au nanobottles on the drug encapsulation efficiency and initial burst drug release profile have been systematically examined, and the combined chemo-photothermal therapeutic effect of the doxorubicin (DOX)-loaded Au nanobottles has been investigated. Our study is valuable for guiding the design of highly asymmetric Au nanostructures with tunable dimensions. It also provides rich opportunities for the exploration of the Au nanobottles in various plasmon-enabled optical and biomedical applications, especially where magnetic plasmon resonance is desired.

2. EXPERIMENTAL SECTION 2.1. Synthesis of the PbS Nanooctahedra. For the growth of the PbS nanooctahedra, 2.04 mL of thioacetamide (TAA, 0.5 M), 2.57 mL of cetyltrimethylammonium bromide (CTAB, 0.1 M), 2.04 mL of lead acetate (Pb(Ac)2, 0.5 M) and 4.10 mL of acetic acid (HAc, 1 M) solutions


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were injected into 34.26 mL of water. The solution was heated at 60–90 °C for 2–8 h and then centrifuged at 3000–7000 rpm to give the PbS nanooctahedra with different edge lengths. Each obtained PbS nanooctahedron sample was redispersed into 45 mL of an aqueous CTAB (5 mM) solution for storage. Deionized water was used in all of the experiments. 2.2. Synthesis of the Au Nanobottles. The preparation procedure of the Au nanobottles is similar to that described in our previous work.32 0.5 mL of HAuCl4 (0.01 M) and 0.5 mL of ascorbic acid (0.1 M) solutions were sequentially added into 10 mL of a CTAB solution (0.075 M) to prepare the growth solution. After the resultant growth solution was swirled for 0.5 min, 0.01–0.2 mL of the differently-sized PbS nanocrystal solution was added to control the overall and opening sizes of the Au nanobottle products. The solution was stirred for 120 s and then kept undisturbed at room temperature for 2 h. After overgrowth, the solution was centrifuged at 4000– 6000 rpm for 10 min, followed by the removal of the supernatant. The product was redispersed in 10 mL of a CTAB solution (0.1 M). To remove the templating PbS component, 0.5 mL of HCl (5 M) was added into the solution and heated at 60 °C overnight. The Au nanobottles were finally obtained by centrifugation at 4500–6500 rpm for 10 min. They were redispersed in 20 mL of water for further characterization. 2.3. Oxidation of the Au Nanobottles. The Au nanobottles were anisotropically oxidized by adding 400–800 μL of HCl (1 M) and 400–800 μL of H2O2 (6 wt %) solutions into 10 mL of the as-grown Au nanobottle solution in the presence of CTAB (0.1 or 0.001 M). The oxidized Au nanobottles with different opening sizes and shapes were obtained by centrifugation (5000–8000 rpm, 10 min) and redispersion into water. 2.4. Preparation of the Dense Silica-Coated Au Nanobottles. Thiol-terminated methoxy poly(ethylene glycol) (mPEG-SH, RAPP Polymere, Germany, MW 5000) was first used to


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replace the CTAB molecules on the Au nanobottles. 20 mL of the Au nanobottle solution was centrifuged at 4500–6500 rpm for 10 min, followed by the collection and redispersion of the precipitate in 10 mL of water. 1 mL of an mPEG-SH (polymer chain concentration 1 mM) solution was subsequently added. The resultant solution was kept undisturbed at 25 °C for 6 h and then centrifuged twice at 4000 rpm for 10 min to remove the excess mPEG-SH molecules. The resultant mPEG-coated Au nanobottles were redispersed in a mixture solution containing 2.25 mL of water, 7.5 mL of absolute ethanol and 0.15 mL of NH3∙H2O (30 wt%). After that, 0.02 mL of the silica precursor solution (10 vol% tetraethylorthosilicate in ethanol) was injected into the Au nanobottle solution three times at an interval of 90 min under ice-cold and ultrasonication conditions, with the total volume of the added silica precursor solution being 0.06 mL. The obtained dense silica-coated Au nanobottles were centrifuged twice and redispersed in 10 mL of water. 2.5. Simulations. The finite-difference time-domain (FDTD) method was employed to simulate the plasmonic properties of the Au nanocrystals. All simulations were performed using FDTD Solutions v8.7 (Lumerical Solutions). An electromagnetic wave pulse ranging from 300 to 1200 nm in wavelength was applied on a specific nanoparticle that was contained in a threedimensional box. The box containing the nanoparticle was divided into a mesh of 0.5 nm in size. The refractive index of the surrounding medium was set to be equal to that of water (1.33). The dielectric function of Au was acquired through fitting the measured data of Christy and Johnson. The average size of the Au nanobottle was chosen according to the measurements from the scanning electron microscopy (SEM) images. The models of the Au nanorod and the Au nanosphere were designed to have the same particle volume as the Au nanobottle in the simulations. The Au nanorod was modeled as a cylinder with two hemispherical ends.


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2.6. Characterization. SEM imaging and energy-dispersive X-ray (EDX) analysis were carried out on an FEI Quanta 400 FEG microscope under an electron acceleration voltage of 20 kV. Transmission electron microscopy (TEM) images were recorded on an FEI Tecnai Spirit microscope under an electron acceleration voltage of 120 kV. The overall and opening sizes of the Au nanobottles were measured from their SEM images. The extinction spectra were acquired on a Lambda 950 ultraviolet/visible/NIR spectrophotometer (Perkin Elmer). Inductively coupled plasma atomic emission spectrometry (ICP-AES) results were obtained on an Agilent 7500a ICP-AES system. 2.7. Drug Loading and In Vitro Drug Release. DOX, camptothecin (CPT) and actinomycin D (ACTD) were loaded into the Au nanobottles as follows. DOX was first dissolved in water, while CPT and ATCD were dissolved in dimethyl sulfoxide (DMSO). 1 mL of each nanobottle solution (opening size 13, 44 or 68 nm, 400 μg Au mL–1) was centrifuged at 4500 rpm for 10 min, and the obtained nanobottle precipitate was redispersed in 100 μL of each drug solution (0.5 mg mL–1) by gentle ultrasonication. After being shaken under 600 rpm at 25 ℃ overnight, the mixture was centrifuged at 6000 rpm for 10 min. The supernatant solution was subsequently subjected to the absorption measurement. The concentration of the solution was calculated from the standard curve for each drug, which was established in advance. The drug encapsulation efficiency (EE) was calculated as the percentage of the loaded drug amount relative to the total drug amount, where the loaded drug amount was taken as the difference between the total drug amount and the amount of the drug in the supernatant. The initial drug burst release profile of the DOX-loaded nanobottles was studied after the drug-loaded nanobottles were dispersed in water at 80 μg Au mL–1 and allowed for standing for 6 h. The mixture was then centrifuged at 6000 rpm for 10 min, and the DOX concentration of the


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supernatant was measured. The drug release profile of the DOX-loaded nanobottles was also studied in 1 mL of a citrate buffer (20 mM, pH 4.5, 80 μg Au mL–1) and 1 mL of a phosphatebuffered saline (PBS, pH 7.4, 80 μg Au mL–1). After being shaken for 6, 12, 24, 36 and 48 h, the mixture was centrifuged, and a portion of the supernatant solution at 20 μL was taken out, followed by the addition of 20 μL of the corresponding buffer solution and the redispersion of the precipitate by shaking. The DOX concentration in the removed supernatant was measured, and the release percentage was calculated. 2.8. Cell Culture and Cell Viability. Human glioblastoma U-87 MG cells were from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in alpha-modified Minimum Essential Medium (α-MEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific) at 37 ℃ in a humidified incubator with 5% CO2. The cell viability was determined by a Cell Counting Kit-8 (CCK-8) assay. Briefly, 4000 U-87 MG cells were seeded in each well of a 96-well plate. After 24-h incubation, the medium was replaced with 100 μL of a fresh medium containing the nanobottles (opening size 44 nm) at different concentrations (25–200 μg Au mL–1). After further 48-h incubation, the medium in each well was changed with 100 μL of a fresh medium containing 10% of the CCK-8 solution, and the plate was incubated for another 1 h. All determinations were carried out in triplicate, and the cell viability of each sample was calculated by comparison with the control. 2.9. Photothermal Therapy. 4000 U-87 MG cells were seeded into each well of a 96-well plate. After 24-h incubation, the medium was changed with a phenol red-free α-MEM containing the Au nanobottles (100 μg Au mL–1, opening size 44 nm). After further 24-h incubation, the cells were irradiated with an 808-nm laser at different optical power densities (0, 10.9, 12.5 and


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14.1 W cm–2) for 3 min. The laser spot size was adjusted to equal to that of the bottom of each well. The cell viability was determined separately using CCK-8 assay and calcein-AM staining after 24-h incubation. The cells were washed by a fresh culture medium after 30-min incubation with calcein-AM (2 μM). An Olympus IX71 microscope was used to detect the green fluorescence emitted from the live cells. 2.10. Chemo-Photothermal Combined Therapy. 4000 U-87 MG cells were seeded into each well. After 24-h incubation, the medium of the U-87 MG cells was changed with 100 μL of a fresh phenol red-free medium containing the DOX-loaded nanobottles (100 μg Au mL–1, 100 ng DOX mL–1). After further 24-h incubation, the cells were exposed to the irradiation of the 808-nm laser for 3min, with the optical power adjusted at 12.5 W cm–2. After further 24-h incubation, the CCK-8 assay and calcein-AM staining were performed to measure the cell viability.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Size Control of the Au Nanobottles. The Janus Au/PbS nanostructures were produced through a combination of the synthesis of differently sized colloidal PbS nanooctahedra and asymmetric Au overgrowth. The core PbS component was then dissolved by HCl to produce the Au nanobottles, as schematically illustrated in Figure 1a. Thermal treatment of Pb(Ac)2 and TAA in aqueous CTAB solutions gave the PbS nanooctahedra. The prepared PbS nanooctahedra are seen under SEM imaging to have uniform edge lengths (l) with the vertices slightly truncated (Figure 1b). The PbS nanooctahedra act as the sacrificial template, and therefore determine the size parameters of the Au nanobottles. As the sulfur atoms come from the thermal decomposition of TAA, the temperature is a key factor for the decomposition rate of


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TAA. By simply controlling the reaction temperature and duration, the average edge length l of the PbS nanooctahedra can be roughly varied from 20 to 200 nm (Figure S1a and b, Supporting Information). The obtained PbS nanooctahedra were then employed as the seeds for the growth of the Janus Au/PbS nanostructures. Au was preferentially deposited at one of the six equivalent vertices of each PbS nanooctahedron. The further Au deposition occurred selectively on the formed nucleus along the four facets that were adjacent to the Au-deposited vertex (Figure 1c). This selective Au deposition and overgrowth has been ascribed to the electron transfer from the PbS nanooctahedron to the Au nuclear once the latter is formed at one vertex.33 Because the number of electrons on each PbS nanooctahedron is finite, the electron transfer makes the PbS nanooctahedron electron-deficient. The electron deficiency causes Au atoms to be preferentially attached to the existent nuclear and prevents Au nucleation on the other vertices.32 The Au nanobottles were finally produced by removing the PbS components from the Au/PbS Janus nanostructures through HCl etching, with the Au components unaffected. The obtained Au nanobottles show a uniform bottle-like shape (Figure 1d), and the overall and opening sizes are 160 ± 6 and 40 ± 7 nm, respectively. In addition, the inner surface of the Au nanobottles is faceted, which can be verified by SEM imaging on the nanobottles with a widely opened mouth (Figure S2, Supporting Information).


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Figure 1. Synthesis of the Au nanobottles. (a) Schematic illustrating the synthetic process. (b‒d) SEM images of a PbS nanooctahedron sample, the correspondingly produced Janus Au/PbS nanostructures and Au nanobottles, respectively. (e) Normalized extinction spectra of the PbS nanooctahedron, Janus Au/PbS nanostructure and Au nanobottle samples. (f) Low-magnification SEM image of another Au nanobottle sample.

The normalized extinction spectra of the shown PbS nanooctahedron, Janus Au/PbS nanostructure and Au nanobottle samples are displayed in Figure 1e. No extinction peak is present in the visible or NIR region for the pure PbS nanooctahedron sample (Figure 1e and Figure S1c, Supporting Information). The Janus Au/PbS nanostructure sample shows a clear extinction peak at 550 nm, while the etching of PbS induces a redshift of the peak. The resultant


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Au nanobottles exhibit a strong extinction peak at 740 nm and a weak peak at 570 nm. By our method, Au nanobottles with high size uniformity and high yield (~100%) can be produced. As one example, Figure 1f and Figure S3 (Supporting Information) show the low- and highmagnification SEM images of another Au nanobottle sample with a smaller opening size (138 ± 5 nm) and a smaller overall size (30 ± 4 nm). We measured the opening sizes (d) and overall sizes (D) of the Au nanobottles, where d represents the mouth width in the opening region of the bottle, and D refers to the diameter of the Au nanobottle in the direction perpendicular to its symmetry axis (Figure 2a). These two size parameters affect the plasmonic properties of the Au nanobottles.32 Au nanobottles with different d and D values were synthesized by varying the amount of the PbS nanooctahedra (l = 47 ± 5 nm) during Au overgrowth. The effect of the Au/PbS molar ratio on the morphology of the Janus Au/PbS nanostructures (Figure S4, Supporting Information) allows for the control of the d and D parameters of the Au nanobottles. As the Au/PbS ratio is increased, the d value of the obtained Au nanobottles first increases, reaches a maximum and then decreases, while the D value and the cavity volume inside the bottle keep increasing (Figure 2a‒e and Figure S5, Supporting Information). The produced Au nanobottles are relatively uniform in size and morphology. We performed EDX analysis to examine the composition of the Au nanobottles. Two examples are provided in Figure S6 (Supporting Information). One is for a nanobottle sample with a widely opened mouth, and the other is for a nanobottle sample with a nearly closed mouth. The EDX results show only the presence of Au atoms in the nanobottle samples. PbS is dissolved away by HCl, with its amount below the detection limit of our EDX system.


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Figure 2. Au nanobottles with different opening and overall sizes. (a‒e) Schematics and corresponding SEM images of the Au nanobottles. D and d denote the overall size and the opening size of the bottle. As more Au precursor is supplied, the D value and the cavity volume of the produced Au nanobottles increase, while the d value first increases and then decreases. (f) Normalized extinction spectra of the five Au nanobottle samples. The Au nanobottles were synthesized using the PbS nanooctahedra with an edge length of l = 47 ± 5 nm.

The extinction spectra of the five differently-sized Au nanobottle samples are shown in Figure 2f. All of these Au nanobottle samples have a major plasmon peak and a weak one at the shorter-wavelength side. As the overall size is increased, the major plasmon peak gradually redshifts and the weak plasmon peak becomes stronger. As have been reported,32 the major


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plasmon peak originates from the transversely polarized excitation, with the polarization direction being perpendicular to the symmetry axis of the nanobottle. The weak plasmon peak at the shorter-wavelength side arises from the axially-polarized excitation. The transverse plasmon mode possesses the character of magnetic plasmon resonance. The role of the edge length l of the PbS nanooctahedra on the morphology of the Au nanobottles was examined next. As shown in Figure 3a, if the opening size is kept fixed, the cavity volume and the overall size of the Au nanobottles both increase as the edge length l of the PbS nanooctahedra is increased. This is verified by the SEM images of six Au nanobottle samples whose opening sizes are all adjusted to be 40–50 nm (Figure 3b) and four other samples whose opening sizes are adjusted to be 30–35 nm (Figure S7, Supporting Information). The overall size D of the Au nanobottles can be synthetically varied in the range of ~100‒230 nm using the PbS nanooctahedra with increasing edge lengths. Their opening sizes can be adjusted by controlling the Au/PbS ratio. In addition, the cavity volume of the Au nanobottles also increases with the overall size due to the increasing size of the templating PbS nanooctahedra. These results demonstrate that a facile and robust wet-chemistry method has been developed for the synthesis of Au nanobottles with variable overall and opening sizes. Except the two small nanobottle samples, the other Au nanobottle samples show two distinct plasmon peaks (Figure 3c), which can be ascribed to their small opening sizes compared to their large overall sizes and cavity volumes. The major plasmon peak of the Au nanobottles redshifts from ~600 to ~900 nm with increasing overall sizes. The synthetically tunable magnetic plasmon resonance wavelength endows our Au nanobottles with advantages for many plasmonic applications, especially where magnetic plasmon resonance is desired.


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Figure 3. Au nanobottles with different overall sizes and cavity volumes. (a) Schematic illustrating the synthesis of the Au nanobottles using the PbS nanooctahedra of increasing sizes. The Au nanobottles have increasing overall sizes and cavity volumes but similar opening sizes. (b) SEM images of the representative Au nanobottle samples. Their average D and d values together with their standard deviations are given above the SEM images. (c) Normalized extinction spectra of the ten Au nanobottle samples. The major plasmon peak redshifts as the overall size is increased. The curves 2, 3, 5, 7, 8 and 10 are for the samples shown in (b), and the curves 1, 4, 6 and 9 are for the samples shown in Figure S7 (Supporting Information), with both being in the order of increasing overall sizes.


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3.2. Anisotropic Oxidation of the Au Nanobottles. The morphology of the Au nanobottles and therefore their plasmon wavelength can be further finely controlled through anisotropic mild oxidation. Oxidation has proven to be a facile approach for reshaping metal nanocrystals and reducing their sizes.34 Anisotropic oxidation of Au nanobottles for adjusting their morphology and plasmon wavelength has not been demonstrated before. In our study, we chose H2O2 as the mild oxidizing agent to oxidize Au(0) into Au(III) and used HCl to control the oxidation rate, because the oxidation ability of H2O2 is affected by the solution pH.35 H2O2 and HCl solutions were sequentially added into the Au nanobottle (D = 195 ± 12 nm, d = 38 ± 6 nm) solution in the presence of 0.1 M CTAB, which was also used during the growth of the Au nanobottles. The opening size of the Au nanobottles was found to increase with the oxidation time, as schematically illustrated in Figure 4a. The SEM images of the eight representative products are shown in Figure 4b and Figure S8 (Supporting Information). They were obtained at 0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 and 5.0 h after the oxidation was started. Upon the addition of H2O2, oxidation occurs preferentially at the position with high curvature on the Au nanobottles. The small mouth of the Au nanobottles is first oxidized and becomes larger, with the largest opening size being 120 ± 11 nm. At the same time, the rim of the opening is rounded while the entire wall of the Au nanobottles becomes thinner. As oxidation goes on, it becomes less anisotropic, with the overall size decreasing gradually. The Au nanoparticles obtained at the final stage of oxidation are still asymmetric in morphology and have the shape of red blood cells. To obtain Au nanobottles with specific morphologies and sizes from oxidation, aliquots of the reaction solution were pipetted out at different time points and the oxidation reaction was terminated rapidly through centrifugation. The extinction spectra (Figure 4c) taken at an interval of 30 min during the oxidation process show that the original Au nanobottles have two clear


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extinction peaks. The two peaks come from the axial (short wavelength, electric) and transverse (long wavelength, magnetic) plasmon modes, respectively.32 With prolonging oxidation time, the axial plasmon peak slightly blueshifts, and its intensity decreases. In comparison, as the oxidation goes on, the transverse plasmon peak initially blueshifts and then redshifts, with its intensity decreasing (Figure 4d). At the final stage, the two plasmon peaks are located at ~565 and ~830 nm, respectively.

Figure 4. Anisotropic oxidation of the Au nanobottles with 0.1 M CTAB. (a) Schematic illustrating the morphological evolution during oxidation. (b) SEM images of the six


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representative nanobottle samples. The samples were obtained at 0, 1.5, 2.5, 3.5, 4.0 and 5.0 h after oxidation. (c) Extinction spectra recorded at an interval of 30 min after the oxidizing agents were added. (d) Time-dependent variations of the plasmon peak wavelengths and intensities.

With the increase of the oxidation time, the rough outer surface of the Au nanobottles becomes smooth and the faceted inner surface of the Au nanobottles becomes rounded. In the presence of 0.1 M CTAB, the morphology of the Au nanobottles during oxidation still remains asymmetric. The asymmetric morphology is also implied by the spectral evolution. This anisotropic oxidation process can be attributed to three factors. First, the packing density of the stabilizing CTAB molecules at the rim of the mouth is smaller than that on the round surface because of the curvature effect.13,36 The oxidizing species can more easily access the metal surface at the rim due to the lower density of the surfactant molecules. Second, the Au atoms at the sharp rim have more dangling bonds and are more undersaturated than those at the other positions. The more undersaturated Au atoms at the rim of the opening are easier to react with the oxidizing species diffusing in from the solution.37 Third, a previous study has shown that CTAB at high concentrations can slow down the oxidative transformation of Au nanorods into Au nanospheres due to the existence of CTAB micelles.38 CTAB micelles are also believed to play a similar role in our study, where the asymmetric structure of the Au nanobottles is protected by CTAB at the high concentration in the reaction solution. We also carried out anisotropic oxidation on the same Au nanobottle sample in the presence of 0.001 M CTAB to examine the role of the surfactant concentration on the oxidation process. The oxidation-induced morphological evolution is schematically illustrated in Figure 5a. The SEM images of the products obtained after different periods of the oxidation time are shown


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in Figure 5b. At the beginning stage, oxidation occurs preferentially at the opening rim where less CTAB molecules are present due to the high curvature. The oxidation behaviors at the beginning stage are similar at the two CTAB concentrations. As oxidation goes on, the Au nanobottles quickly transform into the spherical shape. The shape transformation can be ascribed to the lack of the stabilization by the surfactant molecules at the low CTAB concentration, where the Au nanoparticles tend to assume the thermodynamically stable state, i.e., spherical nanoparticles. Figure 5c shows the evolution of the extinction spectra of the Au nanobottles during the oxidation process. The axial plasmon peak stays nearly unchanged at the spectral position throughout the oxidation process, but its intensity linearly decreases and finally disappears. (Figure 5d). The transverse plasmon peak wavelength stays nearly unchanged in the beginning stage and then blueshifts gradually due to the transformation from the bottle shape to the sphere shape. The transverse plasmon peak intensity decreases throughout the oxidation process due to the continuous reduction in the overall size. At the end stage of oxidation, there exists only one plasmon peak located at ~580 nm.


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Figure 5. Anisotropic oxidation of the Au nanobottles with 0.001 M CTAB. (a) Schematic illustrating the morphological evolution during oxidation. (b) SEM images of the six representative nanobottle samples. The samples were obtained at 0, 1.0, 1.5, 2.0, 3.0 and 5.0 h after oxidation. (c) Extinction spectra taken at an interval of 30 min after the oxidizing agents were supplied. (d) Time-dependent variations of the plasmon peak wavelengths and intensities.

3.3. Drug Encapsulation. Nanomaterials have shown great potential for loading drugs and delivering them to target positions. Drug molecules can either be entrapped inside nanomaterials


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or adsorbed to their surfaces. Compared with free drugs, nanoscale formulations have the advantages of either passive or active tumor targeting.39 Porous Au nanoparticles have been considered as attractive drug carriers.40 Due to their large inner cavity space and synthetically tunable plasmon resonance wavelength, the Au nanobottles have the potential for anticancer drug encapsulation and for cancer PTT. In order to investigate the effect of the opening size of the Au nanobottles on the drug encapsulation efficiency, three Au nanobottle samples with different opening sizes of 68 ± 8, 44 ± 7, and 13 ± 4 nm were synthesized (Figure 6a, top row and Figure S9, Supporting Information). The corresponding overall sizes are 153 ± 9, 175 ± 12 and 211 ± 13 nm, respectively. We find that our Au nanobottle samples are relatively stable when kept in aqueous CTAB solutions. As one example, the shape, size and plasmon wavelengths of the sample with the opening size of 44 nm were found to remain nearly unchanged after storage in an aqueous CTAB solution for 5 months from SEM imaging and extinction measurements (Figure S10, Supporting Information). The slight reduction in the axial plasmon peak and the background rise in the long-wavelength region might be caused by the slight enlargement of the opening of the nanobottles and particle aggregation. The transverse plasmon resonance peaks of the three samples were adjusted to be around 808 nm for the PTT tests. The Au concentrations of the samples were measured by ICP-AES (Figure S11, Supporting Information). The Au concentrations of the Au nanobottle solutions with 68-, 44- and 13-nm opening sizes are 84.1, 82.8 and 89.9 µg mL–1, respectively. They are nearly equal. In addition, the ICP-AES measurements indicate that there is no residual Pb in the Au nanobottle samples.


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Figure 6. Three Au nanobottle samples with different opening sizes for drug encapsulation. (a) SEM images (top row) of the CTAB-stabilized Au nanobottles without silica coating and TEM images (bottom row) of the corresponding Au nanobottle samples coated with dense silica. The thicknesses of the coated silica layers are 8, 5 and 3 nm for the Au nanobottle samples with opening sizes of 68, 44 and 13 nm, respectively. (b) Normalized extinction spectra of the three Au nanobottle samples before and after dense silica coating.

Besides the removal of the toxic PbS component, biocompatible surface modification of the Au nanobottles is also necessary. The prepared Au nanobottles are capped with CTAB molecules, which have been known to be cytotoxic.41 The exchange of the CTAB molecules with thiolated PEG and the coating of silica has been reported to be effective for improving the biocompatibility of CTAB-capped Au nanoparticles.42 We therefore attached mPEG-SH molecules on the Au nanobottles to eliminate the CTAB molecules and coated them with dense silica (Figure 6a, bottom row and Figure S12, Supporting Information). The thickness of the coated dense silica layer can be synthetically varied from ~3 to 35 nm (Figure 6a, Figures S13 and S14, Supporting Information). We further characterized the surface morphology of the three


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samples deposited on indium tin oxide-coated glass slides by SEM at a low acceleration voltage of 5 kV to verify that the mouths of the Au nanobottles are not sealed by dense silica (Figure S15, Supporting Information). In addition, the extinction spectra show that the coating of dense silica causes a slight redshift on the transverse plasmon peak of the Au nanobottles (Figure 6b). The transverse plasmon peaks of the three nanobottle samples after silica coating are located at 769, 797 and 828 nm, respectively. Colloidal Au nanocups have recently been modified with a photosensitizer, chlorin e6, on the surface for phototherapy.43 However, the effect of the morphology of the Au nanocups or Au nanobottles on the drug encapsulation efficiency and release profile has not been studied. We therefore examined the effects of the opening size and surface coating of the Au nanobottles on the drug encapsulation efficiency. Three common anticancer drugs, DOX, CPT and ACTD (Figure S16, Supporting Information), were used for encapsulation. DOX is highly watersoluble, while CPT and ACTD have low solubilities in water. The three Au nanobottle samples described above with different opening sizes (68, 44 and 13 nm) coated with PEG or dense silica were employed for drug loading. The PEG-coated Au nanobottles show poor and unstable DOX encapsulation (Figure S17, Supporting Information). In contrast, remarkably higher DOX encapsulation efficiencies were obtained with the dense silica-coated Au nanobottles (Figure 7a and b). This result can be ascribed to the fact that DOX has a high binding affinity to silica.44 Among the three Au nanobottle samples, the one with the opening size of 44 nm gives the highest DOX encapsulation efficiency of (23.6 ± 1.1)%. Therefore, the drug encapsulation efficiency of the Au nanobottles is highly dependent on their opening size. The Au nanobottles of larger or smaller opening sizes lead to lower drug encapsulation efficiencies. We also found that the Au nanobottles show poor drug encapsulation efficiencies for both CPT and ACTD


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(Figure S18, Supporting Information). This result suggests that the drug itself also affects its encapsulation efficiency. Based on these results, we focus on DOX below to study its release behavior and therapy performance.

Figure 7. Au nanobottles for DOX loading. (a) Chemical structure of DOX. (b) DOX encapsulation efficiencies in the three Au nanobottle samples with different opening sizes. (c) Initial burst drug release values for the three nanobottle samples with different opening sizes. (d) Time-dependent DOX release profiles for the DOX-loaded Au nanobottles at two different pH


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values. (e) Bright-field image of U-87 MG cells after incubation with the Au nanobottles at 60 μg Au mL‒1. Dark granules arising from the uptaken Au nanobottles are evident in the cytoplasm around the nuclei of the cells. (f) Cell viabilities of U-87 MG cells after incubation with the Au nanobottles at different Au concentrations. Shown are the mean values together with half standard deviations. The nanobottles have an opening size of 44 ± 7 nm and are coated with dense silica.

The initial burst release refers to the drug release in the early beginning after drug-loaded nanoparticles get in contact with a medium. Since a high initial burst release rate might lead to tissue irritation or toxicity in human bodies, the burst release is regarded as an undesirable phenomenon in many cases. As an ideal drug carrier, the initial burst drug release should be prevented. The surface modification and the adjustment of the pore size of nanocarriers have been previously employed to reduce the initial burst release.45,46 As shown in Figure 7c, much lower initial DOX release was observed in the Au nanobottles with opening sizes of 44 and 13 nm, in comparison with those with the opening size of 68 nm, after the DOX-loaded nanobottles were kept in water for 12 h. This result is consistent with a previous study, which shows that drug carriers of small pore sizes dramatically reduce the initial burst drug release.47 A combination of our results on the drug encapsulation and the initial burst drug release indicates clearly that an appropriate opening size possessed by the Au nanobottles is pivotal for the encapsulation efficiency and initial drug release, and that the Au nanobottles with the opening size of 44 nm are superior for DOX encapsulation. We speculate that both of the inner surface area and the opening size affect the drug encapsulation efficiency. The nanobottles with the smallest opening size (13 nm) also have the smallest total inner surface area. Their drug


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encapsulation efficiency is therefore lower. Although the Au nanobottles with the opening size of 66 nm support a larger total inner surface area, which is favorable for drug encapsulation, the drug can easily diffuse out from the nanobottles (Figure 7c). As a result, the Au nanobottles with the opening size of 44 nm shows the highest DOX encapsulation efficiency. The DOX release profiles were next studied in a citrate buffer (pH 4.5) and PBS (pH 7.4), which simulate the intracellular lysosomal and cytoplasmic environment, respectively.48 Figure 7d shows the time-dependent DOX release profiles at the two pH values. A higher DOX release rate in the citrate buffer (pH 4.5) was found in comparison with that in PBS (pH 7.4). This result is attributed to that an acidic environment can promote the DOX release from the carrier. The biocompatibility property of the dense silica-coated Au nanobottles was examined by determining the cell viability by the CCK-8 assay after U-87 MG cells were incubated together with the Au nanobottles for 48 h. After incubation, the silica-coated Au nanobottles are readily internalized by the cells (Figure 7e). The intracellular Au nanobottles are mainly located at the cytoplasm around the nuclei. The silica-coated Au nanobottles show no cytotoxicity to U-87 MG cells, even when the concentration of Au reaches 200 μg mL‒1 (Figure 7f).

3.4. Chemo-Photothermal Combined Therapy. Hyperthermia induces cell death at temperatures above 43 °C.49 Plasmonic metal nanoparticle-mediated PTT has recently drawn enormous attention for cancer hyperthermia therapy because it is selective and noninvasive.50 As photothermal agents, plasmonic metal nanoparticles can absorb visible and NIR photons and convert them into heat.51–53 We coated dense silica on the Au nanobottle sample mentioned above (Figure 6) with the opening size of 44 nm, and first studied their photothermal conversion properties under 808 nm-laser irradiation. The transverse plasmon peak of the sample after silica


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coating is at 806 nm (Figure 8a). The temperature of the Au nanobottle solution (2 mL, 80 µg mL‒1) was monitored when the solution was irradiated at 1.5 W for 35 min and cooled to the ambient temperature (Figure 8b). Following our previously reported method,54 the efficiency of the photothermal conversion of the Au nanobottles was determined to be 52.2% from the recorded temperature rise and decay curves.

Figure 8. Chemo-photothermal combined therapy with the Au nanobottles. (a) Extinction spectra of the Au nanobottles. (b) Variation of the temperature in the Au nanobottle solution as a


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function of the laser irradiation time. The Au nanobottle solution (2 mL) was irradiated by the 808-nm laser at the optical power of 1.5 W for 35 min, and then left for natural cooling with the laser switched off. (c) Cell viabilities under the laser irradiation at different optical power densities. (d) Fluorescence images of the cells stained with calcein-AM upon the PTT at different optical power densities. (e) Cell viabilities for the chemotherapy with DOX, the PTT with the Au nanobottles, and the chemo-photothermal combined therapy. (f) Fluorescence images of the cells stained with calcein-AM subjected to the different therapies. Chemotherapy: DOX-loaded Au nanobottles, 100 μg Au mL‒1, 100 ng DOX mL‒1. PTT: Au nanobottles, 100 μg Au mL‒1, 808nm laser irradiation, 12.5 W cm‒2, 3 min. Combined therapy: DOX-loaded Au nanobottles, 100 μg Au mL‒1, 100 ng DOX mL‒1, 808-nm laser irradiation, 12.5 W cm‒2, 3 min. The cell viabilities were determined by the CCK-8 assay or calcein-AM staining. The data points in (c and e) represent the mean values and standard deviations. *P < 0.05 and **P < 0.01 are considered to be statistically significant. The nanobottles have an opening size of 44 ± 7 nm and are coated with dense silica.

FDTD simulations were carried out to compare the theoretical photothermal conversion efficiency of a single, averagely-sized Au nanobottle with those of a single Au nanosphere and a single Au nanorod (Figure S19, Supporting Information). The percentage of the absorption in the total extinction at the laser wavelength determines the photothermal conversion efficiency of plasmonic metal nanoparticles. The model of the Au nanobottle was designed according to the experimental sample used in Figure 8a. The particle volume of the Au nanorod and the Au nanosphere were adjusted to be the same as that of the Au nanobottle. The major plasmon wavelength of the Au nanobottle and the longitudinal plasmon wavelength of the Au nanorod


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were adjusted around 800 nm. The plasmon wavelength of the Au nanosphere is 668 nm. The plasmon peaks of the Au nanobottle sample in the experimental extinction spectrum are broader because of the inhomogeneous size distribution. The simulated photothermal conversion efficiencies are 8.1% for the Au nanobottle, 7.4% for the Au nanosphere and 5.5% for the Au nanorod. The simulated photothermal conversion efficiency of the Au nanobottle is higher than those of the Au nanosphere and nanorod with the same particle volume, which shows the potential of the Au nanobottles for PTT applications. We found that the simulated photothermal conversion efficiency of the Au nanobottle is much smaller than the experimental value. The discrepancy can be attributed to the re-absorption of the scattered light by the nanobottles in the ensemble solution.54 As the size of plasmonic metal nanoparticles is increased, their scattering capability increases and a fraction of the scattered light will be re-absorbed, which will contribute more to the overall photothermal conversion efficiency. In addition, it is also meaningful to measure the photothermal conversion efficiencies of the Au nanorod and the Au nanosphere for comparison. However, this is currently limited by the available approaches for the synthesis of Au nanorods of desired sizes over a broad size range. The PTT performance of the dense silica-coated Au nanobottles was evaluated by the CCK-8 assay as well as calcein-AM staining. As shown in Figure 8c, in the presence of the dense silica-coated Au nanobottles, the cell viability was reduced to (84.1 ± 4.8)%, (61.0 ± 6.3)% and (17.3 ± 2.9)% under the 808-nm laser irradiation at the optical power density of 10.9, 12.5 and 14.1 W cm–2, respectively. The reduced cell viabilities caused by the laser irradiation were confirmed by calcein-AM staining (Figure 8d). As the laser power density was increased, the cell viability decreased, and the cells became rounded. Most of the cells died after the laser irradiation at 14.1 W cm‒2.


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Photothermal heating to a high temperature (>50 °C) is commonly used in order to fully ablate cancer cells. However, such high hyperthermia temperatures can inevitably damage the normal tissue nearby the tumor owing to heat diffusion. On the other hand, due to the limited tissue penetration of NIR light, PTT is often insufficient for killing cancer cells situated in deep tumor tissues. Owing to these reasons, a combination of mild PTT and chemotherapy has recently attracted attention.55,56 It has proven to synergistically improve the performance of cancer therapy. Photothermal agents carrying anticancer drugs can simultaneously deliver heat and drugs to tumors. In addition, hyperthermia induced by PTT can also synergistically enhance the anticancer effects of chemotherapeutic agents.55,56 We evaluated the chemo-photothermal combined therapy performance using the dense silica-coated Au nanobottles loaded with DOX and compared it with those of the separate therapies (Figure 8e and f). The cell viabilities were reduced to (87.3 ± 2.0)% and (57.1 ± 6.3)% after the chemotherapeutic treatment with the DOXloaded Au nanobottles and the PTT treatment with the dense silica-coated Au nanobottles, respectively. The cell viability was synergistically reduced to (19.9 ± 4.8)% by the combination of the two treatments. We also found that the photothermal conversion promoted an additional ~20% DOX release from the Au nanobottles, and therefore believe that this is the possible mechanism for the enhanced anticancer effect of this combined treatment.

4. CONCLUSIONS In this study, a facile and robust method for producing highly asymmetric Au nanobottles with controllable sizes and in high uniformity has been demonstrated. The method relies on the use of differently sized PbS nanooctahedra (~20–200 nm) as the scaffolds for asymmetric Au overgrowth, followed by the scaffold dissolution to yield Au nanobottles. The overall size of the


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Au nanobottles can be synthetically varied from ~100 to ~230 nm through control of the edge length of the PbS nanooctahedra, while the opening size can be tailored by changing the Au/PbS ratio. In addition, the magnetic plasmonic wavelength of the Au nanobottles can be synthetically varied over a wide spectral range from ~600 to ~900 nm. The size and morphology of the asprepared Au nanobottles can be further tailored through mild anisotropic oxidation of the Au nanobottles in the presence of CTAB molecules. The Au nanobottles are further coated with dense silica for drug encapsulation. The opening size and the inner surface area of the Au nanobottles play vital roles in the drug encapsulation efficiency and initial burst drug release. The Au nanobottles with the opening size of 44 nm exhibit the highest DOX encapsulation efficiency of (23.6 ± 1.1)% and low initial DOX release. We have also found that the plasmonic photothermal conversion promotes an additional ~20% DOX release from the Au nanobottles, and that the drug-loaded Au nanobottles show great potential for chemo-phothermal combined cancer therapy. We believe that our results will be useful for exploring highly asymmetric Au nanobottles in different biomedical applications and employing the magnetic plasmon resonance of Au nanobottles to enhance different optical signals.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxx. Extinction spectra, SEM images and size control of the PbS nanooctahedra, schematics and SEM images of the Janus Au/PbS nanostructures, EDX spectra, SEM images and size histograms of the Au nanobottles, ICP-AES calibration curves, SEM images, TEM images and extinction


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spectra of the PEG-coated and silica-coated Au nanobottles, molecular structures, absorption spectra and calibration curves of the drugs, encapsulation efficiencies of the drugs, and simulated absorption, scattering, extinction cross-section spectra of the Au nanobottle, Au nanorod and Au nanosphere (PDF) AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (J.F.W.). *E-mail: [email protected] (X.M.Z.). ORCID Ruibin Jiang: 0000-0001-6977-3421 Xiao-Ming Zhu: 0000-0001-8545-1211 Jianfang Wang: 0000-0002-2467-8751 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the Research Grants Council (RGC) of Hong Kong (GRF, 14306817). H.Z. acknowledges the support from the RGC of Hong Kong through Hong Kong Postgraduate Fellowship Scheme (HKPFS). REFERENCES (1) Novotny, L.; van Hulst, N. Antennas for Light. Nat. Photonics 2011, 5, 83–90. (2) Kauranen, M.; Zayats, A. V. Nonlinear Plasmonics. Nat. Photonics 2012, 6, 737–748.


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