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Near-IR–Absorbing Gold Nanoframes with Enhanced Physiological Stability and Improved Biocompatibility for In Vivo Biomedical Applications Liying Wang, Yunching Chen, Hsin Yao Lin, Yung-Te Hou, Ling-Chu Yang, Aileen Y. Sun, Jia-Yu Liu, Chien-Wen Chang, and Dehui Wan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12591 • Publication Date (Web): 10 Jan 2017 Downloaded from http://pubs.acs.org on January 11, 2017

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ACS Applied Materials & Interfaces

Near-IR–Absorbing Gold Nanoframes with Enhanced Physiological Stability and Improved Biocompatibility for In Vivo Biomedical Applications

Liying Wang1, Yunching Chen1, Hsin Yao Lin1,4, Yung-Te Hou2, Ling-Chu Yang1, Aileen Y. Sun1, Jia-Yu Liu1, Chien-Wen Chang3, and Dehui Wan1,*

1

Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu, Taiwan

2

Department of Bio-Industrial Mechatronics Engineering, National Taiwan University,

Taipei, Taiwan 3

Department of Biomedical Engineering & Environmental Sciences, National Tsing Hua

University, Hsinchu, Taiwan 4

Department of Neurosurgery, MacKay Memorial Hospital, Taipei, Taiwan

*

To whom correspondence should be addressed. E-mail: [email protected]

ACS Paragon Plus Environment


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Abstract This paper describes the synthesis of near-infrared (NIR)–absorbing gold nanoframes (GNFs) and a systematic study comparing their physiological stability and biocompatibility with those of hollow Au-Ag nanoshells (GNSs), which have been used widely as photothermal agents in biomedical applications because of their localized surface plasmon resonance (LSPR) in the NIR region. The GNFs were synthesized in three steps: galvanic replacement, Au deposition, and Ag dealloying, using silver nanospheres (SNP) as the starting material. The morphology and optical properties of the GNFs were dependent on the thickness of the Au coating layer and the degree of Ag dealloying. The optimal GNF exhibited a robust spherical skeleton composed of a few thick rims, but preserved the distinctive LSPR absorbance in the NIR region—even when the Ag content within the skeleton was only 10 wt%, fourfold lower than that of the GNSs. These GNFs displayed an attractive photothermal conversion ability, great photothermal stability, and could efficiently kill 4T1 cancer cells through light-induced heating. Moreover, the GNFs preserved their morphology and optical properties after incubation in biological media (e.g., saline, serum), whereas the GNSs were unstable under the same conditions because of rapid dissolution of the considerable silver content with the shell. Furthermore, the GNFs had good biocompatibility with normal cells (e.g., NIH-3T3 and hepatocytes; cell viability for both cells: >90%), whereas the GNSs exhibited significant dose-dependent cytotoxicity (e.g., cell viability for hepatocytes at 1.14 nM: ca. 11%), accompanied by the induction of reactive oxygen species. Finally, the GNFs displayed good biocompatibility and biosafety in an in vivo mouse model; in contrast, the accumulation of GNSs caused liver injury and inflammation. Our results suggest that GNFs have great potential to serve as stable, biocompatible NIR-light absorbers for in vivo applications, including cancer detection and combination therapy.

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Keywords: gold nanoframes, hollow Au-Ag nanoshells, localized surface plasmon resonance, photothermal agents, physiological stability, biocompatibility

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Introduction Light-active nanomaterials, which convert photon energy efficiently into chemical, electronic, or thermal energy, have been used widely with many practical applications in fields ranging from energy to health.1-4 Among common light sources, near-infrared (NIR) light is particularly suitable for excitation of light-responsive nanomaterials in the human body because it can penetrate into deep biological tissues, the result of low absorption by water and blood as well as weak scattering from soft tissue in the NIR region.5 Therefore, NIR light–absorbing nanomaterials are attracting attention for use in biomedical applications—including in vivo imaging, controlled drug release systems, and photothermal and photodynamic cancer therapy—in which incident NIR light is converted into fluorescence, heat, or reactive oxygen species (ROS).6-8 Recently, there has been an explosion of research in the development of various NIR light absorbers, including organic dyes9 and carbon-based,10 polymer-based11, semiconductor,13,

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and noble-metal nanomaterials.15,

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Among these platforms, gold

nanostructures are the most attractive because of their high absorbance efficiency, great photostability, and low toxicity, arising from the inert nature of gold17-19 and its unique local surface plasmon resonance (LSPR) properties.6,

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In particular, the LSPR peaks of gold

nanostructures can be readily tuned into the NIR region through variations in their shapes, structures, and inter-particle distances.7 Gold-based nanostructures having several non-spherical shapes or complex structures have been developed, including nanorods,20 nanostars,21 and nanovesicles.22 Although these gold nanostructures have highly tunable LSPR peaks in the NIR region, their fabrication generally require specific shape- or structure-directing agents, which may induce potential toxicity or instability. For instance, several groups have reported the cellular toxicity of cetyltrimethylammonium bromide (CTAB),19 a capping agent commonly used for the anisotropic growth of gold nanostructures (e.g., nanorods). In addition, the preparation of the copolymers used to form gold 4


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nanovesicles

can

be

complicated

and

time-consuming.

Furthermore,

vesicle-like

morphologies can be crushed into smaller particles after a single shot of laser radiation,22 making nanovesicles unsuitable for repeated photothermal treatment.11 Another concern is that nanorods and nanostars may also be reshaped after absorption of an excessive amount of energy and, thus, lose their NIR absorbance.23 Besides, core-shell gold nanoparticles produced by coating dielectric particles with shells of Au (e.g., SiO2@Au) have been reported.24 However, the core-shell gold nanoparticles have a typical diameter in the range of 100–400 nm, making them less attractive for most in vivo applications,7 and also display a relatively poor photothermal conversion ability due to the strong scattering.13 Therefore, the challenge remains to develop stable and biocompatible NIR-absorbers for future clinical use. Alternatively, an increasing number of studies have been conducted on hollow Au-Ag nanoshells (GNSs) comprising a thin alloyed shell and a hollow interior.25-28 The attractive features of GNSs include high photothermal transduction efficiency resulting from the strong excited electronic field;29 high catalytic ability resulting from the large surface area;30 and high drug payloads resulting from the hollow vacancy.31 In addition, cytotoxic surfactants (e.g., CTAB) are not used during the formation of GNSs. Notably, GNSs can transfer NIR photon energy efficiently to generate local heat causing hyperthermia and triggering drug release27 or to produce cytotoxic ROS to kill cancer cells in a form of photodynamic therapy.25, 26 GNSs have been also used for in vivo imaging in, for example, photoacoustic tomography (PAT)28, 32 and surface-enhanced Raman scattering (SERS).33 Therefore, GNSs have great potential to serve as multifunctional theranostic agents for cancer detection and combination therapy.25-28, 32 GNSs are commonly synthesized through galvanic replacement reactions between gold ions and silver nanocrystals as sacrificial templates.34 For in vivo applications, GNSs must typically be small (sub-100 nm) to allow passive uptake in tumors,35 with strong absorbance near 800 nm, matching the NIR laser wavelength. They also typically contain a considerable 5



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Ag content (ca. 40–60 at%) within the Au-Ag alloyed shell.36 Notably, the residual silver may result in the GNSs displaying instability and toxicity, especially in biological media. For example, Halas and co-workers reported that GNSs are unstable in vivo because the residual Ag atoms can transform into Ag ions because of the high ionic strength, low pH, and dissolved O2.37 Such GNSs would break into small fragments and lose their NIR absorbance capability in vivo. More importantly, multiple researches have reported that Ag nanoparticles (NPs) have high cytotoxic risk,17, 38 originating from the released Ag ions and the induced ROS arising from the chemical transformation of the Ag atoms.39, 40 In particular, the strong induction of ROS can result in DNA damage, mitochondrial membrane destabilization, activation of transcription factors, and initiation of cell death through ROS-dependent apoptotic pathways and IKK/NF-κB pathways.41-43 Moreover, several in vivo toxicity studies of Ag NPs have been reported.44-46 Xue et al. observed that Ag NPs administrated in mice resulted in an inflammatory response in the liver,44 with the NPs accumulating predominantly in the liver and spleen upon intravenous injection of Ag NPs.46 Furthermore, De Jong et al. found, through a clinical chemistry investigation of intravenous Ag NP toxicity in rats, that the Ag NPs caused liver damage.45 In addition, they also observed significant suppression of natural killer cell activity in the spleen, indicating that the Ag NPs might also have adverse effects on the immune system.45 To the best of our knowledge, no in vitro and in vivo toxicity studies of GNSs have been reported; previous studies have focused predominantly on evaluating the toxicity of solid Au and Ag NPs.17, 19 Therefore, further investigations of the potential toxicity of GNSs remains necessary if they are to find future nanomedical applications. Several strategies have been employed to enhance the chemical stability of GNSs by modifying their composition and morphology. The most common method is dealloying of the unstable residual silver, including adding an etchant [e.g., Fe(NO3)3 and NH4OH]47 or using a gold salt solution at ultralow concentration in the galvanic replacement reaction.48 In these 6


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cases, gold nanoframes (GNFs) composed of several thin rims, with extremely low Ag contents, have been formed. These nanostructures are, however, very sensitive to the amount of etchant, making it difficult to precisely control the final morphology and the LSPR peak position. Alternatively, coating solid materials as protective layers onto the surfaces of GNSs has been used to enhance their chemical stability. For example, Xu et al. employed gold as a protective layer, observing a significant enhancement in the chemical stability of the Au-coated GNSs.49 Nevertheless, the as-synthesized nanostructures contained greater than 35 at% of silver. In addition, a few reports of pure GNFs have been published.50-52 For example, Ling et al. and Gao et al. employed a site-selective deposition method to synthesize GNFs using AgCl50 or AgI52 nanocrystals, respectively, as sacrificial templates, followed by sequential etching of the nanocrystals. The silver could not, however, be removed completely from these nanostructures. Generally, the reported GNFs have had strong, tunable LSPR peaks in the NIR region, as well as a limited Ag contents, but the research has focused only on the synthetic strategies, the morphological evolution, the optical properties, and the SERS applications of GNFs.50-53 Therefore, the stability of GNFs in biological media and their toxicity remain poorly understood. In addition, no studies of the nanomedical applications of GNFs have been reported. In this study, we prepared NIR-absorbing GNFs having a low Ag content by modifying a reported three-step method;53 we then systematically investigated their physiological stability and biocompatibility, especially compared with those of GNSs. We used polycrystalline Ag nanospheres (SNPs) as templates to form GNSs, then transformed the GNSs into GNFs by coating with a Au layer, and finally dissolved the residual Ag with HAuCl4. The morphology and optical properties of the GNFs could be varied by adjusting the thickness of the Au coating layer and the degree of Ag dealloying. Notably, the resultant GNFs exhibited robust skeletons and preserved their distinctive LSPR peak near 840 nm. The Ag content of these GNFs decreased to approximately 10 wt%—much lower than that of the GNSs (ca. 40 wt%). 7



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In addition, the GNFs displayed great photothermal stability under repeated exposure to the NIR laser and could kill cancer cells efficiently as a result of light-induced heat. Moreover, we conducted a series of in vitro and in vivo evaluations to compare the physiological stability and biocompatibility of GNSs and GNFs. Our results indicated that the as-synthesized GNFs exhibited significantly enhanced stability in biological media, as well as improved in vitro/in vivo biocompatibility, relative to those of the GNSs, suggesting that GNFs have great potential to serve as stable, biocompatible, NIR-light absorbers for in vivo applications (e.g., cancer detection, combination therapy).

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Results and Discussion Figure 1 illustrates the morphological and structural changes involved in the formation of the GNFs from SNPs, and the hypothetical relationships among the content of residual silver, physiological stability, and biocompatibility of the nanostructures. Our synthetic approach had three steps: galvanic replacement, Au deposition, and Ag dealloying (Figure 1a). First, we prepared GNSs through a galvanic replacement reaction using nanospheres, rather than nanocubes, as sacrificial templates54 (Step 1). The synthesis of single-crystalline cubes typically requires strictly controlled reaction conditions,55 potentially decreasing their practicality for mass-scale production of nanomedicines for clinical applications. During the galvanic replacement reaction, the gold ions were reduced, because of their higher reduction potential (0.99 V for Au; 0.80 V for Ag34), and then deposited onto the surfaces of templates, while the silver atoms in the nanocrystals were simultaneously oxidized. Controlling the amount of aqueous HAuCl4 solution allowed us to tune the LSPR wavelength into the NIR region. Notably, the residual silver within the GNSs could be further dissolved by adding more HAuCl4 into the system. When too much HAuCl4 was added, however, the hollow structure cracked into smaller pieces, accompanied by a significant blue-shift of the LSPR peak.54 To solve this problem, in Step 2, a pure Au layer was deposited directly onto the surface of the GNSs by reducing HAuCl4 with ascorbic acid (AA) to avoid structural collapse in the following dealloying process. In this step, GNS@Au particles of various shell thicknesses were formed by adjusting the volume of the HAuCl4 solution added. Notably, the shell thickness played a key role affecting the formation of the final nanostructures. Step 3 involved dissolving the Ag atoms from the Au-Ag alloyed shells by adding HAuCl4 solution to induce a dealloying process. In the initial stages of the dealloying process, tiny pores appeared on the surfaces; they gradually enlarged to form a frame-like skeleton comprising several thick rims. Finally, we obtained GNFs having significantly low silver contents. Notably, the hollow structures could be maintained during the dealloying process because of 9



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the thickened shells formed in Step 2, suggesting that the GNFs would retain their strong LSPR peak in the NIR region.

Figure 1 (a) Schematic representation of morphological and structural changes involved in the formation of a GNF. The major steps include (1) formation of a GNS through galvanic replacement; (2) deposition of a Au layer onto the GNS by directly reducing HAuCl4 with AA; (3) a dealloying process with HAuCl4, enlarging the pores on the surface, and forming a GNF made of pure Au. (b) Schematic representation of the hypothetical relationships among the content of residual silver, the physiological stability, and the biocompatibility of GNSs and GNFs. As displayed in Figure 1b, we suspected that the residual silver within the GNSs would transform to Ag ions in biological media,37 potentially changing the structure and LSPR wavelength of the GNSs, with the released Ag ions inducing intracellular ROS generation as well as cytotoxicity. In this case, the GNSs would cause further damage or inflammation to the organs, especially the liver; that is, they would have poor in vivo biocompatibility. In contrast, we suspected that the GNFs would have enhanced physiological stability because of 10


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their much lower Ag contents and the strengthened Au skeletons. Accordingly, the induction of ROS and the cytotoxicity of the GNFs would be suppressed dramatically because of the limited release of Ag ions. Moreover, we also expected the GNFs to exhibit greater biocompatibility in vivo and less significant injury to the organs, especially the liver. To examine the physiological stability of the GNFs and GNSs, we compared the changes to the morphologies and extinction spectra of the nanostructures before and after incubation in various environments: (i) phosphate-buffered saline (PBS)—conditions of high ionic strength; (ii) tumor microenvironment (TME)—conditions of high ionic strength, weak acidity, and an abundance of ROS; and (iii) fetal bovine serum (FBS)—conditions presenting real, complicated physiological environment with abundant biomolecules (e.g., sugars, lipids, proteins). Furthermore, we evaluated the dose-dependent cytotoxicities of the nanostructures in two types of normal cells—mouse embryonic fibroblasts (NIH-3T3) and primary rat hepatocytes—by using LIVE/DEAD, WST-1, and MTT assays. We also assessed the cellular ROS generation of the nanostructures through a DCFH-DA assay. Finally, to examine the in vivo toxicities of the GNFs and GNSs, we injected the nanostructures intravenously into C57BL/6 mice and evaluated the liver injury through clinical chemistry and liver histology. First, we used transmission electron microscopy (TEM) to monitor the morphological evolution of the SNPs to GNFs. Figure S1 reveals that the SNPs exhibited a sphere-like, polycrystalline morphology with an average diameter of 56 nm; the as-synthesized GNS featured a thin shell having an average outer diameter of 66 nm. Figures 2a–c display TEM images obtained from aqueous dispersions of GNS@Au after reducing various amounts of the HAuCl4 solution with AA. Prior to deposition, the GNSs had a uniform shell thickness of approximately 6.4 nm with a few tiny pores on the surfaces (Figure S1b). When the HAuCl4 solution was added under the reductive conditions, a gradual increase in the shell thickness of the GNS@Au species occurred. The average shell thicknesses after the addition of 2.5, 3.5, and 7.5 mL of the HAuCl4 solution were 7.4, 8.0, and 10.4 nm, respectively. In addition, we 11



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observed some changes in the shell features during the deposition of pure Au. In the initial stages, the shell thickness increased slightly, but some tiny pores remained on the shell surface (Figure 2b). When the amount of Au precursor increased gradually, the deposited Au atoms filled the pores on the surface and, consequently, thickened the shell, accompanied by shrinkage of the pores (Figure 2a). Notably, however, unexpected self-nucleation occurred when the amount of added HAuCl4 was too large, as displayed in Figure 2c (red arrows). Thus, self-nucleation of Au could still occur spontaneously in solution, even though AA is a relatively weak reducing agent. Presumably, self-nucleation occurred when the concentration of Au0 monomers was higher than the nucleation threshold, when an excessive amount of the gold precursor was added.56

Figure 2 (a–c) TEM images of GNS@Au species having shell thicknesses of (a) 8.0, (b) 7.4, and (c) 10.4 nm, obtained by varying the amount of aqueous HAuCl4 solution (0.25 mM): (a) 3.5, (b) 2.5, and (c) 7.5 mL. (d–i) TEM images of the corresponding nanostructures obtained 12


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after the GNS@Au species in (a–c) had been titrated with an aqueous HAuCl4 solution (0.25 mM): (d–f) 0.5 and (g–i) 3 mL. Red arrows indicate the unwanted self-nucleation of gold; blue arrows indicate broken nanostructures. Insets: TEM images of individual nanostructures at higher magnification; scale bar: 20 nm.

The GNS@Au species having various shell thicknesses were then reacted with Au ions to form the GNFs. Figures 2d and 2g display TEM images obtained from aqueous dispersions of GNS@Au species having a shell thickness of 8.0 nm after they had been reacted with 0.5 and 3 mL of HAuCl4 solution (0.25 mM), respectively. In the initial dealloying stage, several pores appeared on the surfaces of the GNS@Au species; they grew larger as more Ag atoms were dissolved upon the addition of HAuCl4 (inset to Figure 2d). As more Au ions were added, adjacent pores gradually enlarged and merged into larger pores. Eventually, spherical GNFs having a robust skeleton and several rims were obtained after adding 3 mL of HAuCl4 solution (Figure 2g). The average diameter of the spherical GNFs was 80 ± 11 nm. Notably, the hollow and porous structure of the GNFs was successfully protected from collapse of the skeleton, indicating that the additional Au coating strengthened the shells. Interestingly, previously reported GNFs have typically been synthesized from single-crystalline templates (e.g., cube50, 51, 53 or octahedron51), combined with selective deposition of Au atoms onto the ridges and vertices of the single-crystals, due to their higher surface energies. After the selective deposition of Au atoms, the templates were removed to form the frame-like Au nanostructures. In our case, however, the polycrystalline, sphere-like Ag templates had no obvious ridges or vertices on their surfaces (see Figure S1a). Thus, we suspect that the Au atoms still preferentially deposited onto some surface sites exhibiting higher energy (e.g., defects, grain boundaries). Meanwhile, the deposited Au atoms might also migrate to fill the vacancies generated by the dissolved Ag atoms during the dealloying process. Thus, robust GNFs could also be obtained from polycrystalline, spherical silver templates when using this

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current approach. More importantly, use of polycrystalline templates suggests that the synthesis of robust GNFs might potentially be developed for massive-scale production. Figures 2e and 2h present TEM images obtained from aqueous dispersions of GNS@Au species, having a shell thickness of 7.4 nm, after their reactions with 0.5 and 3 mL, respectively, of the HAuCl4 solution. As expected, we observed morphological changes similar to those of the samples having 8.0-nm shells . The tiny pores on the surface developed to formed porous nanostructures (Figure 2e). The thinner shell was not, however, strong enough to maintain the hollow structure, which eventually cracked, due to the dissolution of silver, after adding 3 mL of the HAuCl4 solution (blue arrows in Figure 2h). In contrast, for the samples having a shell thickness of 10.4 nm, only a few tiny pores emerged on the surface of the GNS@Au species after the addition of 0.5 mL of the HAuCl4 solution (Figure 2f). Thus, the Au precursors had more difficulty dissolving the residual Ag atoms embedded in the thick shell through the galvanic replacement reaction. After the addition of 3 mL of the HAuCl4 solution, GNFs with thick rims remained (Figure 2i). We also observed, however, that some irregular, solid nanostructures formed and grew when increasing the amount of HAuCl4 (red arrows in Figures 2f and 2i). Thus, the Au atoms not only deposited onto the surface of the GNS@Au species to form GNFs during the dealloying process but also deposited onto the small nuclei originating from the self-nucleation of Au atoms in the step of pure Au deposition (red arrows in Figure 2c). In this case, the uniformity of GNFs decreased significantly, because the irregular byproducts were difficult to remove. Accordingly, the shell thickness of the GNS@Au species played a key role affecting the formation of the GNFs: uniform GNFs having spherical skeletons were formed only when the shells of the GNS@Au species were sufficiently thick for support during the dealloying process. If the shells were too thin, the hollow structure of the GNS would eventually collapse during the dealloying process. If the amount of HAuCl4 was too high in the Au deposition step, however, self-nucleation of Au would occur and, thereby, generate undesired irregular Au 14


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nanostructures in the final products. Based on these observations, we chose GNS@Au species having 8.0-nm shells to prepare the optimal GNFs for the following studies. We used ICP-MS to analyze the elemental compositions within various nanostructures, from the SNPs to GNFs, to examine the changes in their Ag contents. Figure 3a plots the weight ratios of Ag and Au in the samples, including the SNPs, the GNSs, the GNS@Au species (sample in Figure 2a), and the GNFs (sample in Figure 2g). The as-synthesized GNS contained approximately 40 wt% of Ag. After the deposition of Au atoms, the Ag ratio of the GNS@Au species decreased to 20 wt%. As more Ag was dissolved by HAuCl4 in the dealloying process, a porous structure gradually formed and the Ag content decreased further. Finally, the Ag ratio in the resultant GNFs decreased significantly, reaching just 10 wt%. We also used scanning TEM (STEM) with energy dispersive X-ray spectroscopy (EDX) to verify the composition and spatial distribution of elements in the GNFs. The typical bright-field (BF) and high-angle annular dark-field (HAADF) STEM images of an individual GNF confirmed the frame-like morphology, comprising a hollow interior and several thick rims. More importantly, EDX mapping images indicated that the principal component of the GNFs was Au atoms (green spots in Figure 3d), with Ag atoms rarely appearing in this nanostructure (red spots in Figure 3e). Thus, the optimal GNFs maintained a hollow, porous metallic skeleton, with almost all of the Ag atoms completely removed.

Figure 3 (a) Weight ratios of Ag (red) and Au (green) in a set of samples: SNPs, GNSs, GNS@Au species having 8.0-nm-thick shells, and GNFs formed from the Au-coated GNSs 15



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titrated with 3 mL of the HAuCl4 solution; insets: representative TEM images of individual nanostructures at higher magnification. (b) BF and (c) HAADF STEM images of an individual GNF; (d, e) EDX mapping of (d) Au and (e) Ag.

Next, we examined the spectral evolution during formation of the GNFs. Figure 4a displays the extinction spectra of aqueous suspensions containing GNSs before and after their reaction with increasing volumes of the HAuCl4 solution containing AA. During the deposition of pure Au, a gradual blue-shift of the extinction peaks of the samples occurred, with the peak shifting from 830 to 726 to 707 to 678 nm after the addition of 0, 2.5, 3.5, and 7.5 mL, respectively, of the HAuCl4 solution. This shift can be attributed to the shrinkage of the pinholes in the shells and the gradual increase in shell thickness.57 Figure 4b presents extinction spectra of aqueous suspensions containing GNS@Au species (shell thickness: 8.0 nm; λ: 707 nm) after their reactions with various volumes of the HAuCl4 solution to form GNFs. During the dealloying process, the LSPR position gradually red-shifted (from 745 to 845 nm) after more Au ions had been added into the reaction system (from 0.5 to 3 mL). The peak shift may have resulted from the dissolution of Ag atoms in the shells and the formation of frame-like skeletons of increasing porosity.37 These spectroscopic changes agree well with the observed morphological and compositional evolutions of the corresponding samples in Figures 2 and 3. Notably, the optimal GNFs displayed a significant absorbance in the NIR region, suggesting that they might have great potential as efficient light absorbers for in vivo biomedical applications (e.g., photothermal therapy, medical imaging).6, 7

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Figure 4 Optical and photothermal properties of GNFs. (a) UV–Vis spectra of aqueous suspensions of GNS and the nanostructures in Figures 2a–c. (b) UV–Vis spectra of the samples obtained by titrating GNS@Au species having a shell thickness of 8.0 nm with different volumes of an HAuCl4 aqueous solution. (c) Typical thermal images and temperature-rise profiles of H2O, GNS, and GNF solutions under irradiation with an 808-nm CW laser for 5 min; the images and temperatures were recorded using a thermal camera. (d) Temperature-rise profile of a GNF solution during 10 cycles of laser irradiation; one cycle: light on for 2 min, light off for 8 min (2 W/cm2); insets: TEM images of an individual GNF before and after 10 cycles of laser treatment. (e) LIVE/DEAD double staining of 4T1 cells treated with or without GNFs (0.57 nM) and irradiated with or without laser exposure (1 W/cm2, 5 min); scale bar: 200 µm.

To examine the photothermal behavior of the GNFs, we used a thermal camera to record the temperature of the samples in situ under irradiation with an NIR laser. Figure 4c displays the temperature profiles and thermal images obtained from the samples during a 17



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5-min period of irradiation from an 808-nm continuous-wave (CW) laser of tunable power density. The temperatures of the GNF solutions increased rapidly and steadily within 2 min, reaching equilibrium values of 48.5, 55.1, and 66.5 °C at light densities of 1, 1.5, and 2 W/cm2, respectively; as expected, DI water displayed negligible enhancement. In addition, at the same concentration (1.14 nM), the final temperature of the GNF solution was slightly higher than that of the GNS solution (65.2 °C) at 2 W/cm2. Thus, the GNFs could convert NIR light to heat efficiently while displaying photothermal conversion ability comparable with that of GNSs, which have been used widely for biomedical applications.25-28 Next, we examined the photothermal stability of the GNFs by performing repeated laser irradiations with 10 cycles of light exposure at 2 W/cm2 (one cycle: 2 min light on, 8 min light off). In Figure 4d, we observe that the temperature of the GNF solution rapidly reached a high value of approximately 58 °C as the light was turned on in each illumination cycle, indicating that the GNFs retained great photothermal conversion ability after repeated cycles of irradiation. TEM images of the GNFs revealed no significant morphological changes before and after treatment through 10 cycles of light exposure (see insets to Figures 4d and S7), consistent with the great photothermal stability of the GNFs. We also found that the GNFs displayed better photothermal stability, as compared with gold nanorods (see Figure S8). Moreover, the GNF displayed efficient photothermal killing of cancer cells. For example, we incubated mouse 4T1 breast cancer cells with GNFs (0.57 nM) for 4 h and then subjected them to NIR laser irradiation (1 W/cm2, 5 min) to induce hyperthermia. After treatment, we stained the cells in a LIVE/DEAD assay, using calcein acetoxymethyl (calcein AM, green for live cells) and ethidium homodimer-1 (EthD-1, red for dead cells). Figure 4e reveals that all of the cells were killed, while the cells incubated with the GNFs or treated with laser irradiation alone remained alive. These observations suggest that GNFs have potential as efficient photothermal agents in cancer therapy. Notably, the GNFs would be especially suitable for developing photothermal therapies requiring repeated laser irradiation, which has been 18


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proven to enhance the therapeutic efficacy of cancer treatment;11 in this case, the photothermal agents should maintain their photothermal conversion ability even after repeated irradiation. Moreover, we evaluated the physiological stability of the GNSs and GNFs. First, we used a dialysis method58, 59 with PBS (pH 7.4) as the dialysate. The extinction spectra of the colloidal suspensions were measured before and after dialysis for 1 and 7 days (Figures 5a and 5b, respectively), with the dialysates at different time points further analyzed (ICP-MS) to monitor the amount of released Ag ions (Figure 5c). Figure 5a reveals that the extinction peak intensity of the GNSs decreased, accompanied by a significant red-shift, after 1 day, almost vanishing completely after 7 days, indicative of the poor stability of the GNSs under conditions of high ionic strength. The finding is consistent with the rapid dissolution of the Ag atoms within the alloyed shell (solid triangles in Figure 5c) and agrees well with the results of a previous study.37 In contrast, the extinction spectrum of the GNFs featured only a slight decrease in peak intensity after 7 days (Figure 5b), with the concentration of released Ag ions increasing slowly and reaching a value of 0.7 µg/L after 7 days (hollow triangles in Figure 5c), approximately fivefold lower than that (3.5 µg/L) from the GNSs. Thus, the GNFs could maintain their NIR absorbance under conditions of high ionic strength, presumably because the unstable Ag atoms within their skeletons had been removed almost completely while their principal component, Au atoms, was stable under the conditions. In addition to PBS, we also inspected the stabilities of the GNSs and GNFs in two more biological environments (TME, FBS). The TME contained PBS at a lower pH (ca. 6.2) and H2O2 (500 µM) to mimic an acidic tumor microenvironment accompanied by an abundance of ROS,60 which may accelerate the dissolution of Ag atoms.61 On the other hand, we used FBS to mimic real, complicated physiological conditions. Figure S9 presents the extinction spectra obtained from suspensions of GNSs and GNFs before and after incubation in TME or FBS for 1 and 7 days. For convenience, the extinction peak intensities of the nanostructures 19



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after incubation with PBS, TME, and FBS have been normalized to that of the corresponding nanostructures prior to treatment; the normalized maximum extinctions are summarized in Figures 5d and 5e. As expected, the extinction of the GNSs decreased after incubation for 1 day under all conditions (Figure 5d), while the extinction of the GNFs decreased only slightly after 7 days (Figure 5e). In addition, TEM images revealed that the GNSs became porous and that their hollow structures collapsed in these biological media after 7 days (insets to Figure 5d). Notably, the GNFs retained their frame-like skeletons, without any significant morphological changes, after treatment in PBS, TME, or FBS for 7 days (insets to Figure 5e). These spectroscopic and morphological results confirmed that the GNSs were unstable in biological media and would, consequently, lose their NIR absorbances; in contrast, the optimal GNFs displayed significantly enhanced physiological stability and would likely preserve their morphology and NIR absorbance during circulation in the blood and in the tumor extracellular matrix used for in vivo applications.

Figure 5 Physiological stability of GNSs and GNFs. UV–Vis spectra of (a) GNS and (b) GNF solutions obtained after dialysis in PBS at 0, 1, and 7 days. (c) Released silver ion profiles of GNSs and GNFs in PBS. (d, e) Normalized maximum extinction of (d) GNSs and (e) GNFs in various biological media (PBS, TME, FBS) at 0, 1, and 7 days. Insets: TEM images of an individual (d) GNS and (e) GNF before and after incubation in the biological media for 7 days. 20


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Furthermore, we verified the cytotoxicity of the nanostructures qualitatively and quantitatively on a normal cell line (NIH-3T3), using LIVE/DEAD and WST-1 assays, respectively. NIH-3T3 cells, originally established from primary mouse embryonic fibroblast cells, have been used widely to determine the cytotoxicity of nanomaterials.62 Figure 6a presents fluorescent images of NIH-3T3 cells incubated in the absence and presence of various nanostructures (SNPs, GNSs, GNFs) at 1.71 nM for 6 h, followed by staining with calcein AM and EthD-1. The GNFs had negligible toxicity toward the NIH-3T3 cells, while the SNPs and GNSs were comparatively toxic at the same concentration. To further study the dose-dependency of the cytotoxicity, Figure 6b reveals the viability of NIH-3T3 cells, determined using a WST-1 assay, incubated for 24 h in the presence of various nanostructures (SNPs, GNSs, GNFs) at different particle concentrations. At low concentration (0.14 nM), the SNPs were significantly toxic (viability: ca. 60%), while the GNS and GNF groups both displayed high cell viability (ca. 89 and 91%, respectively). Upon increasing the concentration, the cell viability of the GNS group decreased gradually, reaching 61% at the highest concentration (1.43 nM), indicating the dose-dependency of the cytotoxicity of the GNSs. In contrast, the cell viability of the GNF group remained greater than 90% over a broad range of concentrations (0–1.43 nM); thus, the GNFs exhibited negligible cytotoxicity, comparable with that of Au NPs reported in previous studies.63

21



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Figure 6 (a) LIVE/DEAD double staining of NIH-3T3 cells treated with PBS (control), SNPs, GNSs and GNFs (1.71 nM). (b) Cell viability of NIH-3T3 cells incubated with SNPs, GNSs, and GNFs for 24 h, analyzed using a WST-1 assay. (c, d) Cell viability of hepatocytes incubated with GNSs or GNFs for (c) 24 and (d) 72 h, analyzed using an MTT assay. Data are represented as mean ± standard deviation. Analysis of mechanical differences were calculated by using student’s t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001 (n = 3). Scale bar: 200 µm.

We used an MTT assay to compare the cytotoxicity of GNSs and GNFs toward normal rat hepatocytes from a primary cell culture. Figure 6c displays the cytotoxicity effects of those nanostructures toward hepatocytes at different exposure doses after incubation for 24 h. As expected, the GNSs were toxic toward hepatocytes, even at low concentration (0.14 nM); the cell viability decreased gradually upon increasing the concentration, reaching only 32% at 1.14 nM. The GNSs not only presented a significant, dose-dependent cytotoxicity toward hepatocytes but also were more toxic to hepatocytes than to NIH-3T3 cells. We also 22


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investigated the cytotoxicity of the GNSs and GNFs toward hepatocytes after a longer incubation time of 72 h (Figure 6d), because the clearance of nanomaterials by the liver typically requires such a period of time.64 Interestingly, the cell viability of the GNS group decreased dramatically to only 11–17% at each concentration, clearly indicating that the GNSs have a more significant cytotoxicity effect on hepatocytes after incubation for 72 h. More importantly, the results suggest that the in vivo accumulation of GNSs may potentially induce liver injury. For example, the usual dose of GNSs for photothermal therapy is 50–100 µg/mL26,

65

(i.e., ca. 0.17–0.34 nM for our GNS), possibly sufficiently high to induce

potential cytotoxicity on hepatocytes and, consequently, cause liver injury. Notably, in contrast to the GNSs, the GNFs maintained a surprisingly high cell viability of approximately 90% within the concentration range 0–1.14 nM after incubation for 24 and 72 h (Figures 6c and 6d); that is, the GNFs displayed negligible cytotoxicity toward the hepatocytes. According to these toxicity evaluations, based on two types of normal cells and three different assays, the GNFs should have great in vitro biocompatibility, even at high particle concentrations (up to 1 nM), while GNSs have poor in vitro biocompatibility, due to their significant dose-dependent cytotoxicity. We attribute the enhanced in vitro biocompatibility of the GNFs to their inert Au skeletons, considerably low Ag contents, and their suppressed release of Ag ions (see Figures 3 and 5c). These results are consistent with those of a previous study suggesting that the cytotoxicity of Au-Ag alloyed NPs is directly related to the Ag content.66 In addition, we used a DCFH-DA assay to examine the induction of cellular ROS from the GNSs and GNFs.67 Figure 7 presents fluorescence images of NIH-3T3 cells incubated with GNSs or GNFs at various concentrations from 0 to 1.71 nM for 6 h, followed by staining with DCFH-DA. At a low concentration of 0.57 nM, no ROS production was visible in either group. Upon increasing the concentration, however, the GNSs induced dose-dependent ROS production at concentrations in the range 0.89–1.71 nM (green spots in 23



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GNS group in Figure 7), potentially resulting in cell death and restricting the further applications of GNSs in biomedical fields. In contrast, no observable generation of ROS occurred in the GNF group at any concentration. These results are consistent with those of the cytotoxicity evaluations of the GNSs and GNFs (Figure 6), and strongly support the notion that the GNFs have in vitro biocompatibility greater than that of the GNSs.

Figure 7 Intracellular ROS production (green) of NIH-3T3 cells treated with GNSs and GNFs at 0, 0.57, 0.89, 1.14, 1.43, or 1.71 nM, analyzed using a DCFH-DA assay. Scale bar: 200 µm. Finally, we examined the biocompatibility of the GNSs and GNFs in vivo. The nanostructures were intravenously injected into C57BL/6 female mice and then the liver injury was evaluated using clinical chemistry and liver histology. First, we used ICP-MS to examine the biodistribution of the GNFs. These nanostructures accumulated predominantly in the liver and spleen (Figure S11). Notably, the accumulation of nanomaterials in the liver may induce inflammation, necrosis, and other pathological changes.68, 69 To examine the hepatic toxicity of the nanostructures, we injected the mice intravenously with PBS (control), SNPs, GNSs, and GNFs, each at the same dose (200 µL, 34 nM). After exposure for 24 h, the blood serum was collected and the hepatotoxicity markers—aspartate aminotransferase (AST) and alanine aminotransferase (ALT)—were examined to evaluate the liver injury (Table 1).45 After treatment with the SNPs, the levels of AST and ALT reached 2218 and 519 U/L, respectively, approximately 20-fold higher than those of the control group. In addition, we 24


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observed hemolysis after treatment with the SNPs (data not shown), consistent with the nanosilver causing liver damage and impairing liver function.70 Similarly, the GNS group also displayed statistically significant increases in the contents of AST and ALT, relative to the control group, suggesting that the accumulated GNSs also induced some liver damage. Notably, however, the AST and ALT levels of the GNF group were only 109.9 and 31.8 U/L, respectively; that is, no significant difference existed between the GNF group and the control group, suggesting that the GNPs are relatively safe and suitable for clinical applications.

Table 1 Serum levels of aspartate transaminase (AST) and alanine transaminase (ALT) at 24 h after intravenous injection of PBS (control), SNPs, GNSs, and GNFs. Control AST (U/L)

108.5±26.5

ALT (U/L)

30.5±4.1

SNP 2202.0±305.5

GNS ***

713.8±283.9**

179.8±22.1

GNF **

49.3±15.6*

109.9±20.0 31.8±3.8

Data are represented as mean ± standard deviation. Analysis of mechanical differences between control and NS were calculated by using student’s t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001 (n = 5).

Next, we examined the histology of mouse liver sections through haematoxylin and eosin staining at 24 h post-injection of PBS (control), SNPs, GNSs, and GNFs. In Figure 8, we observe a brown pigment in the Kupffer cells of the liver samples that had been treated with SNPs, GNSs, and GNFs (black arrows); no such pigment can be found for the control group. We ascribe this pigment to the presence of metal nanostructures taken up by the Kupffer cells.45 For the SNP group, abnormal pathological changes, including necrosis and diffuse hemorrhage (yellow and green dashed lines), are clearly evident, suggesting that the SNPs induced serious hepatic injury accompanied by a high level of hepatic function enzyme released in the blood. The section of liver treated with GNSs displays an abundance of lymphocyte infiltration in the sinusoid accompanied by necrotic hepatocytes (purple dashed line), indicating the occurrence of inflammation in the liver. Oxidative stress is widely 25



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considered a toxic source capable of causing inflammatory responses. In this case, the liver inflammation can be attributed to the accumulation of GNSs, which would increase cellular ROS production and, accordingly, cause oxidative stress (see Figure 6). In comparison, the GNFs did not induce any abnormal pathological changes in the liver sections at the same concentrations, confirming the negligible hepatic toxicity of GNFs. In addition, we examined the histology of lung, spleen, and kidney samples at 24 h post-injection of the GNFs; we found no abnormal pathological changes (Figure S12).

Figure 8 Histological H&E staining of liver samples at 24 h after the intravenous injection of PBS (control), SNPs, GNSs, and GNFs. He, N, Ly, and NS represent hemorrhage, necrosis, lymphocyte, and nanostructure, respectively. Scale bar: 100 µm.

These toxicity evaluations of the nanostructures clearly support our assumption that GNFs have great biocompatibility in vitro and in vivo, whereas GNSs are relatively toxic to normal cells and to mice at the same particle concentration. Interestingly, previous studies have noted that key factors related to the toxicity of metal nanostructures include their surface charge, size, surface chemistry, and elemental composition.18, 19 In our case, the GNFs and 26


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GNSs had the same surface coating material (i.e., PVP), comparable sizes, and similar hollow structures; the major difference between them was their elemental composition. The Ag content within the GNFs was very low—only 10 wt%, fourfold lower than that (40 wt%) within the GNSs. Therefore, we believe that the enhanced biocompatibility of the GNFs can be attributed to the substantial lowering of the residual Ag content in the nanostructures. Although Halas and coworkers reported the in vivo instability of GNSs,37 the in vitro and in vivo toxicity of GNSs has not been investigated. To the best of our knowledge, this present study is the first to confirm that the instability of GNSs can induce dose-dependent cytotoxicity and can, accordingly, cause liver damage and inflammation. More importantly, relative to GNSs, our GNFs not only exhibited strong NIR absorbance and great photothermal stability but also significantly enhanced physiological stability and improved biocompatibility. Therefore, we suggest that GNFs have great potential to serve as stable, biocompatible NIR-light absorbers for future in vivo applications, including cancer detection and combination therapy.

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Conclusions We have prepared NIR-absorbing GNFs having limited Ag content and systematically investigated their physiological stability and biocompatibility, especially in comparison with those of GNSs. We employed spherical, polycrystalline SNPs as sacrificial templates to form the GNSs, which we then transformed into GNFs by coating with a pure Au layer and sequentially dissolving the residual Ag with HAuCl4. The morphologies and optical properties of the GNFs varied upon adjusting the thickness of the Au coating layer and the degree of Ag dealloying. Notably, the optimal GNFs had a robust spherical skeleton composed of a few thick rims, and preserved the distinctive LSPR peak near 840 nm, even though their Ag content had decreased to 10 wt%, much lower than that (40 wt%) of the GNSs. In addition, under NIR laser irradiation, the GNFs displayed efficient photothermal conversion ability, great photothermal stability, and had the ability to efficiently kill 4T1 cancer cells through light-induced heat. Moreover, we conducted a series of in vitro and in vivo tests to systematically compare the physiological stability and biocompatibility of the GNSs and GNFs. The residual Ag atoms within the GNSs rapidly transformed into Ag+ ions in saline and in serum, leading to collapse of the hollow nanostructures and disappearance of the NIR absorbance, thereby inducing the production of cellular ROS and dose-dependent cytotoxicity toward NIH-3T3 and hepatocytes and, accordingly, resulting in liver damage and inflammation in mice after intravenous injection. In contrast, the as-synthesized GNFs exhibited significantly enhanced physiological stability and improved biocompatibility as a result of the extensive reduction in their Ag content; thus, GNFs have great potential to serve as stable, biocompatible NIR-light absorbers for in vivo applications, including cancer detection and combination therapy.

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Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Experimental sections, additional experimental data on TEM images, EDX mapping images and UV–Vis spectra of nanostructures, calculated cross section spectra of a GNF, photothermal conversion efficiencies of GNFs and GNSs, photothermal stabilities of GNFs and Au nanorods, cytotoxicity evaluation on nanostructures with a CCK-8 assay, biodistribution of GNFs, histological H&E staining of spleen, kidney, and lung.

Acknowledgment This study was supported by the Ministry of Science and Technology, Taiwan (MOST-103-2113-M-007-007-MY2) and Chang Gung Memorial Hospital-National Tsing Hua University Joint Research Grant (104N2744E1). The authors thank Rih-Yang Huang for help with fluorescence microscopy.

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Near-IR-Absorbing Gold Nanoframes with ... - ACS Publications

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