Electron Compensation Effect Suppressed Silver Ion Release and

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Letter Cite This: Nano Lett. 2019, 19, 4478−4489

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Electron Compensation Effect Suppressed Silver Ion Release and Contributed Safety of Au@Ag Core−Shell Nanoparticles Yanlin Feng,†,‡ Guorui Wang,§ Yun Chang,† Yan Cheng,† Bingbing Sun,∥ Liming Wang,*,⊥ Chunying Chen,⊥ and Haiyuan Zhang*,†,‡

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Laboratory of Chemical Biology, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P.R. China ‡ University of Science and Technology of China, Hefei, Anhui 230026, P.R. China § Key Laboratory for UV Light-Emitting Materials and Technology of Ministry of Education, Northeast Normal University, Changchun 130024, P.R. China ∥ School of Chemical Engineering, Dalian University of Technology, Dalian 116024, P.R. China ⊥ CAS Key Lab for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, Institute of High Energy Physics, and National Center for Nanoscience and Technology of China, Chinese Academy of Sciences, Beijing 100049, P.R. China S Supporting Information *

ABSTRACT: Silver nanoparticles (Ag NPs) have promising plasmonic properties, however, they are rarely used in biomedical applications because of their potent toxicity. Herein, an electron compensation effect from Au to Ag was applied to design safe Au@Ag core−shell NPs. The Ag shell thickness was precisely regulated to enable the most efficient electron enrichment in Ag shell of [email protected] NPs, preventing Ag oxidation and subsequent Ag+ ion release. Xray photoelectron spectroscopy and X-ray absorption near-edge structure analysis revealed the electron transfer process from Au core to Ag shell, and inductively coupled plasma optical emission spectroscopy analysis confirmed the low Ag+ ion release from [email protected] NPs. Bare [email protected] NPs showed much lower toxicological responses than Ag NPs in BEAS-2B and Raw 264.7 cells and acute lung inflammation mouse models, and PEGylation of [email protected] NPs could further improve their safety to L02 and HEK293T cells as well as mice through intravenous injection. Further, diethylthiatri carbocyanine iodide attached [email protected] NPs exhibited intense surface-enhanced Raman scattering signals and were used for Raman imaging of MCF7 cells and Raman biosensing in MCF7 tumor-bearing mice. This electron compensation effect opens up new opportunity for broadening biomedical application of Ag-based NPs. KEYWORDS: Au@Ag, core−shell, electron compensation, safety, biomedical application

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Ag surface is still vulnerable to the etchants in biological media and the thick overlayer can also diminish the original plasmonic property.10,11 Thus, a “safe-by-design” strategy is necessary to be implemented to prevent Ag+ release from Ag NMs and retain their plasmonic property for biomedical application. Safer-by-design strategy by integration of design synthesis and safety assessment has been regarded as a feasible approach for promoting the wide applications of nanomaterials.12,13 Many safe-by-design approaches have been proposed to develop safer nanomaterials including coating,14−16 loading,17−19 and grafting,20,21 however, hazardous species (e.g., polymer degradation products and ions) release or individual nanostructures dissociated from the nanocomposites may potentially cause adverse effects. 22 As a result,

lasmonic nanomaterials made from gold (Au) and silver (Ag) are an important class of nanomaterials with unique optical and electronic properties, opening up multiple opportunities toward practical biomedical applications, such as therapeutics, diagnosis, and sensing applications.1 Generally, Ag nanomaterials (NMs) can exhibit more intense plasmonic property than Au NMs of similar size, shape, or structure.2,3 However, the biomedical applications of Ag NMs are not preferred as much as those of Au NMs because of their less chemical stability and worse biocompatibility, where the susceptible oxidation of Ag NMs can deteriorate the plasmonic performance and facilitate Ag ion (Ag+) release.4,5 Recently, Au−Ag core−shell (Au@Ag) NPs were found to exhibit more promising plasmonic properties than Au or Ag NPs alone and other Au/Ag nanostructures,6−9 which further raises concerns about how to improve the safety of the Ag shell. Although various inorganic or organic surface layers have been modified on the surface of Ag NMs to overcome these shortcomings, the © 2019 American Chemical Society

Received: March 29, 2019 Revised: June 11, 2019 Published: June 19, 2019 4478

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Nano Letters manipulation of the intrinsic electronic property of Ag NMs probably is a fundamental and efficient approach without complex surface postmodification while retaining their desired functions. An electron compensation phenomena from Au to Ag has been found in Au−Ag nanostructures, such as Au−Ag core− shell structures and alloys,23−28 which potentially is beneficial for electron enrichment on Ag side and prevention from Ag oxidation. This electronic interaction between Au and Ag is counterintuitive: the higher electronegativity of Au relative to Ag can drive the electron transfer from Ag to Au; thereafter, the lopsided electron balance between Au and Ag is corrected for backdonation from Au to Ag, resulting in a depletion of d electron in Au and an increase of d electron in Ag. This electron compensation effect was significant at the interface of Au and Ag. Although the underlying reason for this electron compensation mechanism remains unclear, the electron enrichment on the Ag side potentially can be utilized to inhibit Ag oxidation and reduce Ag ion release, making Agbased NMs safer for in vivo applications. In the present study, a “safe-by-design” strategy based on Au−Ag electron compensation effect was applied to obtain safe Au@Ag core−shell NPs with excellent plasmonic property (Figure 1a). Twenty-six nanometer Au cores were coated with different thicknesses of Ag shell (2.4, 5.1, 7.9, and 10.1 nm) to form Au@Agx (x represents the thickness of the Ag shell) NPs, aiming at optimizing the electron enrichment condition of Ag shell. This electron enrichment can inhibit the surface Ag oxidation and reduce the Ag+ release of the Ag shell while keeping its plasmonic property (Figure 1b). Electron compensation effect from Au core to Ag shell was confirmed by X-ray photoelectron spectroscopy (XPS) and X-ray absorption near-edge structure (XANES) analysis, and the reduced Ag+ release behavior was determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis. Biosafety of bare Au@Ag NPs and PEGylated Au@Ag NPs (pAu@Ag NPs) was evaluated in different cells and mouse models dependent on their exposure or biomedical application approaches (Figure 1c). Bare [email protected] NPs were found to show the lowest Ag+ release among various Au@Agx NPs and could cause the mildest toxicity and inflammatory responses to human epithelial (BEAS-2B) and murine alveolar macrophages (RAW 264.7) cells and acute lung inflammation mouse models. PEGylation could ultimately improve Au@ Ag2.4 NPs showing negligibly toxic to human hepatocyte (L02) and kidney (HEK293T) cells as well as Balb/c mice after intravenous administration, confirming their excellent safety. Because the electromagnetic field-driven surface-enhanced Raman scattering (SERS) effect of plasmon NPs is greatly beneficial for biosensing and disease diagnosis, SERS enhancement of [email protected] NPs was investigated through coupling with a near-infrared Raman reporter molecule, diethylthiatricarbocyanine iodide (DTTC) (Figure 1d). The in vitro and in vivo SERS signals of DTTC attached [email protected] (pDAu@ Ag2.4 NPs) NPs were more intense than those of pure Au or Ag NPs, demonstrating the potential biomedical application of safe Au@Ag NPs. Fabrication and Characterization of Au@Ag NPs. Au and Ag NPs were synthesized by a citrate reduction method.29 Au@Agx NPs were obtained through Ag+ reduction on the Au NPs.9 The thickness of the Ag shell could be modulated by controlling the Ag+ dose. Transmission electron microscopy (TEM) images (Figure 2a) and the enlarged ones (Figure S1)

Figure 1. Electron compensation effect contributed safety and biomedical application of Au@Ag NPs. (a) Electron compensation effect occurring at the interface of Au and Ag atoms where the Au atom first accepts electrons from the non-d orbital of the Ag atom and then donates more electron to the d orbital of Ag atoms again. (b) Reduced Ag oxidation and Ag+ release of the Ag shell of Au@Ag NPs with the Ag shell getting thinner. (c) Safety assessment of Au@Ag NPs or pAu@Ag NPs in the mouse model through oropharyngeal aspiration or intravenous injection approaches. (d) SERS enhancement based biomedical application of safe Au@Ag NPs after fabrication with DTTC and PEG.

showed that core−shell nanostructure has been achieved. After we measured the sizes of Au and Au@Agx NPs by 4479

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Figure 2. Physicochemical characterization of the synthesized Au, Au@Agx, and Ag NPs. TEM (a), STEM images (b), and the corresponding element mapping (c), XRD pattern (d), and UV−vis spectra (e).

TEM, Gaussian fit gave an average size. The size distribution of Au and Au@Agx NPs (Figure S2) showed that Au cores had a primary size of 26.4 ± 1.0 nm, whereas the final Au@Agx NPs’ sizes were 31.2 ± 1.3, 36.6 ± 1.4, 42.2 ± 1.6, and 46.6 ± 1.8 nm. As a result, the thicknesses of the Ag shell were determined by using the average size of Au@Agx NPs to subtract the average size of Au NPs and then to divide by 2, resulting in the thickness of Au@Agx NPs of 2.4, 5.1, 7.9, and 10.1 nm, respectively. For comparison, Ag NPs with a primary size about 47 ± 2.1 nm (close to the size of [email protected] NPs) were also prepared. Scanning transmission electron microscope (STEM) images (Figure 2b) of Au@Ag NPs showed the very bright center region surrounded by a less bright peripheral area, suggesting the Au core with various thickness of Ag shell. Energy dispersive X-ray (EDS) elemental mapping images (Figure 2c) of Au@Ag NPs clearly indicated that both the Au M line (green color) and the Ag L line (red color) existed in Au@Ag NPs where Au is confined to the core region of the particle whereas Ag surrounds the Au core with different thickness. Moreover, a high-resolution transmission electron microscopy (HR-TEM) image of [email protected] NPs was supplied to show the different crystalline planes of Ag (200) and Au (111) that are ascribed to Ag shell and Au core (in Figure S3), respectively. The X-ray diffraction (XRD) patterns further verified the successful synthesis of Au, Au@Agx, and Ag NPs (Figure 2d). Au@Agx NPs had the cubic phase of Au (JCPDS, 4-0784) and Ag (JCPDS, 4-0783). All the Au@Agx NPs had the same crystal structures. All these Au, Au@Agx, and Ag NPs

could be well dispersed in distilled water, showing hydrodynamic sizes of 40 ± 1.2, 59 ± 6.5, 72 ± 2.7, 75 ± 6.0, 81 ± 5.7, and 80 ± 7.9 nm, respectively, as determined by dynamic light scattering (DLS) (Figure S4a). Zeta potential measurement revealed the negative surface charges of −30.2 ± 2.4, −32.4 ± 1.9, −31.9 ± 2.2, −31.8 ± 1.9, −31.1 ± 1.2, and −32.3 ± 3.0 mV for these NPs, respectively (Figure S4b). The optical properties of Au@Agx NPs were analyzed by UV−vis absorption spectra (Figure 2e). Pure Au and Ag NPs exhibited strong absorption peaks at 526 and 417 nm, respectively, and Au@Agx NPs involved two different absorption peaks relative to Au and Ag. With the increase of the Ag shell thickness, the absorption peak of the Au core was gradually blue-shifted and attenuated, and simultaneously, the peak of Ag shell formed and became evident. Electronic Compensation Effect from Au Core to Ag Shell in Au@Ag NPs. The electron transfer behavior between Au core and Ag shell was first investigated by XPS analysis. Figure 3 shows the XPS spectra of Ag 3d (Figure 3a) and Au 4f (Figure 3b). With Ag shell getting thinner, Ag 3d3/2 and 3d5/2 peaks shifted toward the lower binding energy whereas the Au 4f5/2 and 4f7/2 peaks shifted toward the higher binding energy, meaning the Ag shell accepts more electrons whereas the Au core donates more electrons during this process. This result suggests an electron compensation occurring from the Au core to the Ag shell, and the most pronounced effect was found in [email protected] NPs that have the thinnest Ag shell. The electron compensation potentially can affect the Ag shell oxidation 4480

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Figure 3. Electron compensation characteristic of Au, Au@Agx, and Ag NPs. XPS spectra in the Ag 3d (a) and Au 4f (b) area; XANES spectra of Au L2-edge of Au foil, Au NPs, and Au@Agx NPs (c) and d-orbital hole number (Δh3/2 + Δh5/2) variation in Au@Ag NPs from Au foil (d); XANES of Ag K-edge (e) and Ag+ release of NPs (equivalent to 25 μg mL−1 Ag) in BEGM culture medium after 24 h of incubation (f).

by Au L2- and L3-edges.27 XANES spectrum of Au@Ag NPs was collected to offer a more rigorous understanding of the electronic compensation effect based on the d-electron donation behavior and d-orbital hole density in Au core. Au L2-edge and L3-edge XANES spectra of Au foil, Au NPs, and Au@Agx NPs were collected as shown in Figure 3c and Figure S6, respectively. The absorption band at the white line region was paid much attention because the band corresponds to the electron transition from 2p3/2 core level state to the vacant 5d3/2 and 5d5/2 states at L3-edge (close to 11920 eV) or from 2p1/2 to 5d3/2 at the L2-edge (close to 13742 eV), which is directly related to the density of unoccupied d-states.30 The

behavior in Au@Agx NPs. The oxidation state of Ag was further investigated by decomposing the Ag 3d3/2 (or 3d5/2) XPS spectra into Ag0 and Ag oxide spectra (Figure S5). It could be found that pure Ag NPs had evident Ag0 and Ag oxide peaks in both Ag 3d3/2 and 3d5/2 XPS spectra, however, Au@Agx NPs displayed a prominent Ag0 peak and gradually attenuated Ag oxide peaks with the Ag shell getting thinner. This result provides solid evidence that the thin Ag shell of Au@Agx NPs is not readily oxidized, which is significantly different from the oxidation behavior of pure Ag NPs. This compensated electron potentially arises from a depletion of d electrons at the Au site, which can be probed 4481

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Figure 4. In vitro and in vivo biosafety evaluation of bare Au@Ag NPs in BEAS-2B cells and acute lung inflammation mouse models. (a) Viability of BEAS-2B cells treated with various doses of bare Au@Agx and Ag NPs (dosed as Ag) for 24 h assessed by MTS assays. (b) Live/dead cell staining assay of BEAS-2B cells treated with bare Au@Agx and Ag NPs (25 μg mL−1, dosed as Ag) for 24 h. Scale bar, 100 μm. Neutrophil counts (c), KC levels in the BALF (d), and H&E staining of lung sections (e) of Balb/c mice after oropharyngeal aspiration with 20 μg of each of the NPs. Scale bar, 100 μm.

Electron donation of Au core can facilitate electron enrichment in Ag shell, inhibiting the surface oxidation of Ag shell. Ag K-edge XANES spectra (Figure 3e) was collected to characterize the speciation of oxidation states of Ag element in Au@Agx NPs and Ag NPs.32,33 Two reference samples (Ag foil and Ag2O) were used to fit XANES results of test samples. On the basis of the least-squares fitting (LSF) analysis, Ag NPs were found containing 89.46% of elemental Ag and 10.54% of Ag2O (Table S2), however, [email protected], [email protected], [email protected], and [email protected] NPs contained 99.98, 99.06, 97.27 and 92.19% of elemental Ag, respectively. Obviously, the thin Ag shell can be well protected from oxidation. All of the above XPS and XANES analyses demonstrate that the thinner Ag shell exhibits the more pronounced Au core electron donation. Electron compensation takes place only in the vicinity of the interface

inset in Figure 3c displays the profile of the white line at the L2-edge, where the Au foil showed almost zero “white-line” because of the almost completely filled 5d state whereas Au@ Agx NPs showed gradually increased intensity with the decrease in Ag shell thickness. The similar phenomenon was also found in L3-edge (Figure S6). These results clearly reveal the more potent d-electron donation behavior of Au core with the Ag shell getting thinner. The d-electron donation of Au core can generate the d-orbital hole, the number of which can be calculated based on the parameters derived from Au L2- and L3-edge spectra (Table S1).27,31 Figure 3d shows the d-orbital hole number (Δh3/2 + Δh5/2) variation in Au@Agx NPs, where the [email protected] NPs with the thinnest Ag shell exhibit the most pronounced d-orbital hole numbers, followed by [email protected], [email protected], and [email protected] NPs. 4482

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Figure 5. In vitro and in vivo biosafety evaluation of pAu@Agx NPs in L02 and HEK293T cells as well as mice through intravenous administration. (a) Viability of L02 cells treated with various doses of pAu@Agx and pAg NPs (dosed as Ag) for 24 h and assessed by MTS assays. (b) Cellular ROS level analyzed by flow cytometry based on DCF assays. (c) Cellular GSH level assessed by DTNB assays. (d) HO-1 and MT expression analyzed by Western blot. (e) IL-8 production assessed by Elisa. (f) Confocal fluorescence images of cells stained by JC-1 (green) and MitoSox Red (red). Scale bar, 50 μm. For (b−f), L02 cells were treated with 6.25 μg mL−1 NPs (equivalent to Ag content) for 24 h. (g) H&E staining of major organ tissues harvested from Balb/c mice that were intravenously injected with [email protected] and pAg NPs (equivalent to 10 mg Ag/kg mice) at 21 days postinjection. Scale bar, 100 μm.

between the Au core and the Ag shell. Two reasons are potentially responsible for thinner Ag shell leading to the higher electron compensation: (1) the thinner Ag shell compared with the thicker one is more active to prompt electron transferring at the interface of Au and Ag; (2) the transferred electrons are more sufficient to compensate the

thinner Ag shell that has less Ag species compared with the thicker one with more Ag species. The same trend is observed for the chemical shift of the Ag 3d level for the same series of Au@Ag NPs.9,28 The significantly suppressed Ag shell oxidation in Au@Agx NPs can probably result in low level of Ag+ release.34 Ag+ 4483

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process is often used to reduce protein adsorption in vivo to avoid the reticuloendothelial system (RES) and improve the biocompatibility of NPs in biomedical application processes,46−48 the biosafety of pAu@Agx NPs was further examined. Figure S10 reveals that even lower Ag+ release level of these PEGylated NPs compared with bare NPs, meaning PEGylation process can further contribute to the inhibition of Ag+ release to some extent. The cytotoxicity of pAu@Agx NPs was assessed in L02 and HEK293T cells by MTS assay. All of these NPs could be well dispersed in RPMI (for L02 cells) or DMEM (for HEK293T cells) culture medium, showing hydrodynamic sizes at the range of 77.3− 106.3 nm (Table S5) and negative zeta potentials (Table S6). MTS assay was still employed to assess the cytotoxic effects of pAu@Agx and pAg NPs at a dose range of 0−25 μg mL−1 (according to Ag content) for 24 h. Figure 5a and Figure S11 revealed that PEGylation did improve the safety of all these pAu@Agx or pAg NPs in L02 and HEK293T cells, where [email protected] NPs were even nontoxic at this dose range. Meanwhile, live/dead cell staining assay of L02 and HEK293T cells (Figures S12 and S13) further confirmed that [email protected] NPs could keep a high level of live cells (green color), consistent with MTS results. Obviously, pAu@Agx NPs with the thinner Ag shell exhibit the weaker toxicity, which is ascribed to the weaker Ag oxidation and lower Ag+ release endowed by the fact of electron compensation. Cellular uptake and hierarchical oxidative stress toxicological responses of pAu@Agx NPs were investigated to correlate with their electron compensation-attenuated toxicity. The ICP-OES analysis indicated that cells treated with pAu@Agx and pAg NPs showed similar cellular Ag contents in L02 and HEK293T cells (Figure S14). It seems reasonable because the smaller particles that have less Ag content could be more efficiently taken into cells than the larger particles with the high Ag content, which results in similar cellular Ag content in these NP-treated cells. Intracellular ROS production and GSH depletion usually are typically indicative of oxidative stress injury.49 Flow cytometer analysis was carried out to detect the intracellular ROS level using H2DCFDA,50 while the classic 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) method was employed to determine the intracellular GSH level51 after 24 h of exposure to pAu@ Agx, and pAg NPs (6.25 μg mL−1). Figure 5b and Figure S15a show pAg NPs induced the strongest fluorescence intensity in L02 and HEK293T cells, followed by [email protected], [email protected], [email protected], and [email protected]. Simultaneously, cellular GSH level (Figure 5c and Figure S15b) was notably reduced by these NPs, and the GSH reduction trend was consistent with the ROS enhancement trend. Both ROS production and GSH depletion studies corroborate that pAu@Agx NPs can elicit oxidative stress cell injury in an Ag shell-dependent manner, which closely correlates with their Ag+ release performance. Oxidative stress injury is usually associated with hierarchical biological responses, including heme oxygenase-1 (HO-1)45 and metallothionein (MT) expression,52,53 chemokine or cytokine release,49,54 and mitochondrial dysfunction.55 HO-1 and MT expression levels in L02 and HEK293T cells were analyzed by Western blot analysis. Figure 5d and Figure S15c show that pAu@Agx, and pAg NPs could increase both HO-1 and MT expression in cells, that pAg NPs could induce the highest expression levels, and that [email protected], [email protected], [email protected], and [email protected] NPs induced gradually reduced levels. HO-1 expression, considered as the lowest level of

release from Au@Agx and pure Ag NPs was examined in cell culture medium by ICP-OES analysis. It was shown that pure Ag NPs had high levels of Ag+ release in BEGM medium (bronchial epithelial basal medium (BEBM), supplemented with growth factors from the SingleQuot kit to reconstitute BEGM) (Figure 3f), Roswell Park Memorial Institute medium (RPMI) (Figure S7a), and Dulbecco’s modified Eagle’s medium (DMEM) (Figure S7b), whereas Au@Agx NPs showed gradually reduced Ag+ release levels with the Ag shell getting thinner, which was further supported by the Ag+ release profile in buffered phagolysosomal-simulant fluid (PSF, pH = 4.5) (Figure S8). In Vitro and in Vivo Biosafety Evaluation of Bare Au@ Ag NPs in BEAS-2B, Raw 264.7 Cells and Acute Lung Inflammation Mouse Models. Because the toxicity potential of Ag NMs is intensely dependent on Ag+ release tendency, the reduced Ag+ release in Au@Ag NPs probably can result in the lower toxicity compared to Ag NPs.35,36 Given the potential inhalation exposure route during manufacturing, transportation, consumption, and storage processes,37−41 the biosafety of bare Au@Ag NPs was evaluated in normal human bronchial epithelial (BEAS-2B) and murine alveolar macrophages (RAW 264.7) cells, which are representatives of a lung target for nanomaterials.42−45 Hydrodynamic sizes of these NPs in BEGM and DMEM medium were at the range of 61.48− 104.44 nm (Table S3) whereas their zeta potentials were kept at negative (Table S4). After 24 h of treatment with bare Au@ Agx or Ag NPs at the doses ranging from 0 to 25 μg mL−1 (according to Ag), and the viability of BEAS-2B and Raw 264.7 cells was assessed by MTS assay. Figure 4a shows that Ag NPs exhibited the most severe toxicity to BEAS-2B cells, and Au@ Agx NPs showed gradually reduced toxicity with the Ag shell getting thinner and thinner, whereas [email protected] NPs presented the lowest toxicity. Similar cell death potential of Ag and Au@ Agx NPs was also found in RAW 264.7 cells (Figure S9a). Moreover, live/dead cell staining assay further confirmed Ag NPs could cause the largest population of dead cells (red color) while [email protected] NPs still caused the lowest level of cell death (Figure 4b and Figure S9b). Both these assays reveal a consistent toxicity profile of Au@Agx NPs with the thinner Ag shell exhibiting the weaker toxicity, which is ascribed to the attenuated Ag oxidation and subsequent Ag+ release after electronic compensation occurring. Oropharyngeal aspiration in Balb/c mice was carried out to investigate the in vivo acute lung inflammatory response of Au@Agx NPs. Mice were instilled with Au@Agx or Ag NPs and sacrificed after 40 h of treatment. After collecting bronchoalveolar lavage (BAL) fluid, the characteristic markers for acute lung inflammation such as neutrophil cell counts and Keratinocyte-derived (KC) cytokine level were assessed. Figure 4c,d shows Ag NPs could elicit the highest neutrophil numbers and the most significant KC level while these inflammatory potentials of Au@Agx NPs became weaker and weaker with the Ag shell getting thinner and thinner. Hematoxylin and eosin (H&E) staining of lung sections (Figure 4e) further revealed the similar toxicological trend. These acute inflammatory responses are in good agreement with the in vitro toxicological effects, further consolidating the significantly reduced toxicity of [email protected] NPs compared with Ag NPs. In Vitro and in Vivo Biosafety Evaluation of pAu@Agx NPs in L02 and HEK293T Cells as Well as Mice through Intravenous Administration. Considering that PEGylation 4484

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Figure 6. SERS enhancement of pAu, pAu@Ag, and pAg NPs. (a) Raman spectra of DTTC, pDAu, [email protected], and pDAg NPs. (b) Bright-field, SERS, and their overlaid images of MCF7 cells exposed to DTTC or DTTC-attached NPs (equivalent to 0.2 μg mL−1 DTTC) for 24 h. Scale bar, 5 μm. (c) Photographs showing a laser beam focusing on the tumor. (d) SERS spectra of tumor site of MCF7 tumor bearing mice intravenously injected by DTTC or DTTC-attached NPs (equivalent to 50 μg DTTC/kg mice) at 24 h postinjection. All Raman spectra and images were collected with 785 nm laser excitation.

especially for liver and kidney. [email protected] NPs induced little histological alteration, which is comparable to that induced by PBS. Moreover, the body weight of mice was monitored continuously. Figure S16 shows that there was a slight increase in the body weight of mice receiving PBS or [email protected] NPs, respectively, but an obvious decrease in that by pAg NPs, further demonstrating the excellent in vivo biocompatibility of [email protected] NPs. Furthermore, normal serum biochemistry parameters and hematology analysis (Tables S7 and S8) confirmed that pAg NPs could induce increased aspartate aminotransferase (ALT) and alkaline phosphatase (AST) as well as increased blood cells count (WBC), suggesting their chronic toxicity. However, [email protected] NPs did not exhibit chronic toxicity. SERS Properties of Au@Ag NPs Both in Vitro and in Vivo. The excellent in vitro and in vivo safety of [email protected] NPs makes them useful for biomedical application. Au@Ag core−shell NPs have been reported to enable plasmon performance enhancement which is even better than pure Au or Ag NPs and can be used for SERS-based in vitro and in vivo sensing.6,7 In the present study, DTTC, a NIR Raman reporter molecule, was attached to pAu, pAg, and [email protected] NPs, respectively, to form pDAu, pDAg, and [email protected] NPs, and their SERS enhancement profiles were evaluated by Raman spectra. Figure 6a shows that DTTC alone lacked spectral features while pDAu, pDAg, and [email protected] NPs could greatly enhance the characteristic Raman peaks of DTTC, especially those at 495, 782, 844, 1132, and 1236 cm−1, where pDAu@ Ag2.4 NPs exhibit the most significant SERS enhancements. Probing living cells with Raman spectroscopic techniques can greatly contribute to the study of the molecular and cellular response of nanomaterials due to the high-sensitivity

oxidative stress responses, could protect cells against the disturbance of oxidant−antioxidant equilibrium. Once it fails to restore the redox equilibrium, the Jun kinase (JNK) and NF-κB pro-inflammatory signaling cascades would be activated, leading to the release of cytokine and chemokine.49 Interleukin 8 (IL-8) cytokine was selected as a typical cytokine to investigate the pro-inflammatory effects because it could induce neutrophil and lymphocyte chemotaxis and has been investigated to demonstrate inflammatory effect in various cells.56−58 IL-8 chemokine release assessed by ELISA assay revealed that the incremental IL-8 production was elicited by pAu@Agx NPs with the increase of Ag shell, and pAg NPs were still the most potent NPs in induction of IL-8 production in a dose-dependent manner (Figure 5e and Figure S15d). Mitochondrial membrane potential and superoxide level were probed by JC-1 and Mitosox Red fluorescence indicators, respectively. Figure 5f shows that pAg NPs could induce significant mitochondrial membrane depolarization and superoxide generation in L02 cells, and with the Ag shell getting thinner and thinner from 10.1 to 2.4 nm, pAu@Agx NPinduced mitochondrial-mediated toxicological responses became weaker and weaker and then finally unnoticeable. All of the above in vitro hierarchical oxidative stress toxicological studies confirm that pAu@Ag NPs cause low cell damage, and the thinner Ag shell can more remarkably attenuate the Ag+involved oxidative damage. In vivo biocompatibility of [email protected] NPs that show the lowest cytotoxicity among pAu@Ag NPs was further assessed in healthy female Balb/c mice by intravenous injection. At 21 day postinjection, major organs were collected for histology analysis based on hematoxylin-and-Eosin (H&E) staining (Figure 5g). pAg NPs could induce noticeable tissue damage, 4485

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Nano Letters detection limit and selective molecule reactions of SERS.59 Using these prominent SERS features, the in vitro SERS imaging feasibility (785 nm laser) of [email protected] NPs was examined in MCF7 cancer cells that were incubated with DTTC, pDAu, pDAg, and [email protected] NPs for 24 h. Confocal Raman mapping of the single cell showed the distribution of DTTC and these NPs. Figure 6b shows the bright field images, SERS images, and their overlaid images. The cell treated with [email protected] NPs showed the most prominent SERS signals, indicating the strong in vitro SERS enhancement feature of [email protected] NPs. Furthermore, in vivo SERS-based sensing of [email protected] NPs was further evaluated in MCF7 tumor bearing nude mice that were intravenously injected with DTTC, pDAu, pDAg, and [email protected] NPs, respectively. Figure 6c shows a photograph of a Raman laser beam (785 nm) focusing on the tumor site of a mouse at 24 h postinjection, and the Raman spectra acquired on the tumor site were presented in Figure 6d. Similarly, the tumor area of mice treated with [email protected] NPs still could show the strongest SERS enhancement among these molecules or nanomaterials. Simultaneously, the Raman spectra of normal muscles showed low SERS signals (Figure S17). Substantial difference in the signal was observed between the tumor and skin. Plasmonic nanomaterials have presented attractive and unique optical properties due to their LSPRs, by which the absorption and scattering of light can be greatly enhanced. The biomedical field has greatly profited from the vast research on the properties of plasmonic nanomaterials. Regarding diagnosis, plasmonic nanomaterials have played a crucial role for breakthroughs in the development of both novel imaging techniques and bionanosensors, such as dark-field microscopy,60 reflectance confocal microscopy,61 photoacoustic imaging,62 one- and two-photon fluorescence imaging,63 and surface-enhanced Raman scattering (SERS).64,65 Regarding therapy, nonradiative properties of plasmonic nanomaterials can enable efficient conversion of light to heat, offering excellent photothermal therapy and light-controlled drug release for diseases.66,67 So far, the most commonly utilized plasmonic nanomaterials in the fields of diagnosis and therapy are still Au nanomaterials. Although Ag nanomaterials possess 10 times higher plasmonic activity than Au nanomaterials of similar size,3,68 their biomedical application is merely limited to the antibacterial or anti-inflammation field. On one hand, the susceptibility of Ag to oxidation deteriorates plasmonic activity, and on the other hand the toxicity of the released Ag+ ions also significantly limits their biomedical application in diagnosis and therapy.4,5 A surface layer can be formed on Ag NPs to prevent Ag oxidation and Ag+ ion release, however, the resulting Ag NPs still remain sensitive to oxidation and display compromised plasmonic properties.10,11 As a result, Au−Ag heterostructures such as Au@Ag, and Ag@Au and Au/Ag alloy NPs composed of Ag and Au have become an alternative in plasmon applications. However, Ag@Au NPs had limited plasmonic enhancement compared with Au@Ag NPs6,8,69 and are difficult to be prepared because galvanic reaction will occur instantaneously, resulting in hollow nanostructures.70−73 Despite some groups having successfully achieved Ag@Au NPs by retarding the galvanic reaction between Ag and HAuCl4,74−76 the experimental condition must be strictly controlled. The easily achieved Au@Ag NPs showed better SERS performance than Ag NPs and Ag@Au NPs.6−8 As a result, Au@Ag NPs were used in the manuscript. Au/Ag alloy NPs have been successfully prepared and surface plasmon

resonance property of Au/Ag alloy NPs has been widely studied,77,78 showing excellent catalytic79,80 and SERS81,82 applications. However, the electron compensation effect of Au/Ag alloy has not been widely explored, thus it is valuable to investigate the relationship between electron compensation effect and biosafety of Au/Ag alloy NPs in the future. In the present study, intrinsic electronic properties of Agbased nanomaterials were considered to regulate both the plasmonic property and the safety of Ag-based nanomaterials. An electron compensation effect from Au to Ag was utilized to achieve safer Ag-based nanomaterials with excellent plasmonic properties. The thinner Ag shell of Au@Ag core−shell NPs could attract more electrons from the Au core than the thicker one and induced more significant electron donation from the Au core to the Ag shell (Figure 3a,b). The less Ag oxidation and more efficient hole generation were found in Au@Ag NPs with the thinner Ag shell compared with the thicker Ag shell (Figure 3c−e), resulting in the lower Ag ion release (Figure 3f). Cell viability and oxidative injury assessment corroborated the highest in vitro safety of bare [email protected] or [email protected] NPs among various bare Au@Ag or pAu@Ag NPs (Figure 4a−d and Figure 5a−f), and the histological analysis of major organs of mice further supported their excellent in vivo biocompatibility through oropharyngeal aspiration or intravenous administration (Figure 4e and Figure 5g), providing opportunity for their safe biomedical application. Using SERS detection as a model, the safe [email protected] NPs exhibited more excellent in vitro and in vivo SERS enhancement for DTTC than pure Au or Ag NPs (Figure 6b,d), demonstrating their promising potential for biomedical application. In summary, a series of Au@Ag core−shell NPs with different Ag shell thickness (2.4, 5.1, 7.9, and 10.1 nm) were synthesized for optimization of electron enrichment on Ag shell. [email protected] NPs showed the most pronounced electron compensation effect as evidenced by XPS and XANES analysis, including negative shifts in Ag 3d XPS peak, positive shifts in Au 4f XPS peak, increased hole density on the Au core, and reduced Ag oxide level in the Ag shell. The Ag+ ion release as well as in vitro and in vivo toxicity assessments further confirmed the biological safety of [email protected] NPs. Meanwhile, [email protected] NPs still kept excellent plasmonic property and showed great SERS enhancement both in vitro and in vivo, exhibiting their excellent biocompatibility and advanced biomedical applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.nanolett.9b01293. Experimental section, derived parameters for the unoccupied d states, Ag species of Au@Agx and Ag NPs, hydrodynamic size, zeta potential analysis, serum biochemical analysis, whole blood analysis, size distribution, enlarged TEM, HR-TEM, XPS analysis, Au L3edge XANES spectra, Ag ion dissolution, cell viability, live/dead cell staining assay of Raw 264.7 cells treated with bare Au@Agx and Ag NPs, viability assessment, live/dead cell staining assay, cellular uptake of HEK 293T and L02 cells treated with pAu@Agx and pAg NPs, toxicological responses of pAu@Agx and pAg NPs in 4486

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Nano Letters

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HEK 293T cells, body weight of mice, SERS spectra of normal muscles of MCF7 tumor bearing mice (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel: 86-431-85262136. Fax: 86431-85262582. *E-mail: [email protected]. Tel: 86-10-88233195. Fax: 86-10-88233195. ORCID

Yanlin Feng: 0000-0003-4857-4567 Yun Chang: 0000-0003-0731-7943 Yan Cheng: 0000-0002-2471-2219 Bingbing Sun: 0000-0002-5444-5078 Liming Wang: 0000-0003-1382-9195 Chunying Chen: 0000-0002-6027-0315 Haiyuan Zhang: 0000-0003-4076-1771 Author Contributions

H.Z. and L.W. supervised the project and commented on the project. Experiments were conducted by Y.F. G.W. and B.S. performed the characterization. L.W. and C.C. conducted the XANES characterization. The manuscript was prepared by Y.F. and H.Z., L.W., and C.C. contributed to the improvement of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (21573216, 21703232, 21777152, and 11435002), Hundred Talent Program of Chinese Academy of Sciences. The work was partly supported by the Ministry of Science and Technology of China (2016YFA0203200) and the Users with Excellence Project of Hefei Science Center CAS (2018HSC-UE004). We appreciated the SSRF beamline BL14W1 and BSRF beamline 1W1B of China for their kind assistance with XANES experiments.

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