Ultrasmall AuâAg Alloy Nanoparticles: Protein-Directed Synthesis

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Imaging and Diagnostics

Ultrasmall Au-Ag Alloy Nanoparticles: Protein-directed Synthesis, Biocompatibility and X-ray Computed Tomography Imaging † Zhongyun Chu, Lina Chen, Xiaoshuang Wang, Qingye Yang, Qi Zhao, Chusen Huang, Yuankui Huang, Da-Peng Yang, and Nengqin Jia ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.8b01176 • Publication Date (Web): 01 Jan 2019 Downloaded from http://pubs.acs.org on January 2, 2019

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ACS Biomaterials Science & Engineering

Ultrasmall

Au-Ag

Protein-directed

Alloy

Synthesis,

Nanoparticles:

Biocompatibility

and

X-ray Computed Tomography Imaging † Zhongyun Chua, Lina Chenb, Xiaoshuang Wangb, Qingye Yanga, Qi Zhao a, Chusen Huanga, Yuankui Huangb, Da-Peng Yang*c, Nengqin Jia*a aThe

Education Ministry Key Laboratory of Resource Chemistry, Shanghai Key

Laboratory of Rare Earth Functional Materials, Department of Chemistry, Shanghai Normal University, 100 Guilin Road, Shanghai 200234, China. bJingzhou

Central Hospital, The Second Clinical Medical College, Yangtze

University, 60 Jingzhong Road, Jingzhou District, Jingzhou City, Hubei Province 434020, China. cCollege

of Chemical Engineering and Materials Science, Quanzhou Normal

University, 398 Donghai Street, Quanzhou City, Fujian Province 362000, China * Corresponding author E-mail: [email protected]; [email protected] Abstract The ultrasmall sizes of nanoparticles have attracted significant attention for potential application in the fields of catalysis and nanomedicine. Herein, we reported on the green preparation and X-ray computed tomography (CT) imaging of ultrasmall bimetallic bovine serum albumin-directed gold-silver (Au-Ag@BSA) nanoparticles (2–4 nm) using BSA as a stabilizing and template-directed agent. Further, the effects of synthesis condition were systematically explored to prepare products by adjusting the different molar ratios of Au/Ag. The resulting Au-Ag@BSA nanoparticles exhibited the spherical shape, well-dispersed ability, as well as long-term room-temperature stability. The cytotoxicity effects of Au-Ag@BSA nanoparticles on A549 and MCF-7 cells were compared with those of individual Ag nanoparticles, and

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the results indicated lower cytotoxicity effect by Au-Ag@BSA nanoparticles. Furthermore, the in vivo toxicity of Au-Ag@BSA nanoparticles was investigated in the early-stage zebrafish embryos. The results indicate that there are not any obvious changes of survival and hatching percentages at multiple growth stages (4-120 hpf) even a high level of Au-Ag@BSA nanoparticles (up to 80 mM), revealing the good biocompatibility. Interestingly, a rational design of Au/Ag molar ratio (3:2) surprisingly possessed the enhanced CT performances compared to the Au nanoparticles and iohexol. Accordingly, this study highlights a new prospect in the green preparation of ultrasmall alloy nanomaterials with good biocompatibility and will be of great interest in developing CT contrast agent, catalyst as well as drug delivery carrier. Keywords: Au-Ag@BSA nanoparticles, green synthesis, biocompatibility, enhanced CT imaging, zebrafish embryos

Introduction In recent years, molecular imaging has made a great progress in the field of theranostics, and has established itself as a crucial part of it. This is primarily because of its ability to perform functional imaging, and hence providing comprehensive information on the anatomy with greatly promoted the sensitivity as well as the specificity of the imaging signal. Currently, the main imaging methods that are being widely used for clinical purposes include X-ray computed tomography (CT), positron emission tomography (PET), ultrasound (US), magnetic resonance (MR), and single photon emission computed tomography (SPECT). 1-5 CT has been notably a powerful imaging tool in medicine that can image tissues at deep penetration with excellent resolution.6-10 Contrast agents have a prominent influence on the enhanced output of CT imaging. Until now, the approved contrast agents for clinical purposes have been mostly the iodinated small molecules. Despite its success, there is still some room for improvement in some of the aspects such as a short circulation time in blood, weak surface modification ability and the potential side effects.11 Taking the various factors into consideration, an ideal contrast agent in


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CT imaging would be the one that has 1) low toxicity, 2) large X-ray attenuation coefficient, 3) low cost, and 4) small size. To date, a variety of nano-based materials such as Au, Pt, Bi2S3 and two-dimensional (2D) nanomaterials have been extensively investigated as contrast agents.12-18 Compared to monometallic counterparts, bimetallic materials are found to have better performances in the corresponding synergetic effects in the fields of electronics, optics, fluorescence, and catalysis. Quite a few studies have focused on the developments and applications of different alloys such as AuAg, AgPd, AuPt, and PtNi nanoparticles.19-25 Particularly, great efforts have been invested to the preparation and applications of Au-Ag alloy, and a few of the research groups have also investigated the possible mechanism of the changes in the properties brought about by the incorporation of Ag.26-34 For example, Shi35 and co-workers had proposed the bimetallic Au-Ag nanoparticles bearing a “silver effect” for the application in catalysis. Composition dependent bimetallic nanoclusters like those of the Au-Ag nanoclusters have luminescent properties and have been fabricated by introducing Ag. This was found to modulate the optical properties (as studied by Zhu27 and co-workers) and enhance the electrochemiluminescence behaviour of Au-Ag nanoclusters (as studied by Wang30 and co-workers).28-29,32 More interestingly, the introduction of Ag leads to the regular change (increase and decrease) of light intensity with “silver effect” compared to Au nanoclusters prepared by Wang’s group.32 Noteworthy, Hu’s group36,37 have made great contributions to the application of alloys and bimetallic composites in the field of molecular imaging. Driven by the diverse behaviour of the nanomaterials observed in the fields of catalysis and optics, we made an attempt to synthesize an Au-based CT contrast agent with enhanced effect. These nanomaterials are often synthesized using the traditional approaches like that of physical vapour deposition, mechanical grinding, pyrolysis or microwave assisted method. However, it should be noted that these procedures demand harsh experimental conditions like high temperature or pressure, along with organic solvents. Furthermore, in order to extend the application of the obtained



nanomaterials for biomedical purposes, further modifications are indispensable. Thus, the development of a simple, environmentally friendly method requiring only mild experimental conditions is highly desirable and expected to draw a considerable attention. Recently, biotemplates have been extensively used to prepare nanoparticles, nanoclusters, and nanowires using the principles of biomineralization.35 Among them, protein-based template was favored by scholars thanks to its green and mild conditions. Furthermore, it significantly improves the biocompatibility, and reduced economic costs.6,38-43 At present, BSA as a biomacromolecule has become a popular biological template for the synthesis of metal nanomaterials owing to its easy availability, comparative cheapness, outstanding stability and the presence of various functional groups on its surface. The certainty of a newly synthesized material for biomedical applications cannot really be completed without in vivo studies. In this context, Zebrafish (Danio rerio), a tropical freshwater fish is a great choice in the research of biology and medicine due to its 87% homology with human genes.44-46 Several characteristics render it as an alternative animal model organism for further toxicity test. For instance, it has an efficient hatching speed of about 72 hours, its small size and the transparent body make it convenient for observations; and it is quite cost effective and has lesser space requirements. Based on these, zebrafish is frequently utilized as an attractive and predictive animal model for assessing the toxicity of nanomaterials47,48 This is quite evident from the detailed study on the toxicity of the halloysite nanotubes.49 Besides, zebrafish embryos were also applied to investigate the imaging capacity and toxicity mechanism of Ag nanoclusters with blue fluorescence.50 Herein, we report a series of BSA-directed Au-Ag alloy nanoparticles (AANPs) for their prospective application in CT imaging (Scheme 1). It is worth mentioning that the method proposed in this study has some advanced features as compared to the traditional approaches, thereby making it an attractive alternative 1) AANPs with a narrow size distribution and ultrasmall size (2–4 nm) are extremely monodisperse, 2) they have extremely good haemocompatibility and much lower in vivo and in vitro


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cytotoxicity compared to Au-0, 3) AANPs at the molar ratio of 3:2 (Au-60) has a neat X-ray absorbance in comparison to iohexol, Au-100, Au-0 and other alloy nanoparticles, 4) the presence of functional groups in BSA allows the surface modifications to be achieved more easily, 5) they are more economical and have a better CT performance compared to Au-100 nanomaterials. All the aforementioned features suggested that AANPs could act as a potential enhanced contrast agent candidate for CT imaging.

Scheme 1. Schematic illustration of the fabrication and application of Au-Ag@BSA nanoparticles.

Experimental Materials and Reagents All the reagents used in our experiment were of analytical grade. Gold(III) chloride solution (HAuCl4), silver nitrate (AgNO3), bovine serum albumin (BSA), osmium tetroxide (OsO4), glutaraldehyde, ethylcarbodiimide hydrochloride (EDC), hyaluronic acid sodium salt (HA), dimethyl sulfoxide (DMSO), fluorescein isothiocyanate (FITC), 2-morpholineethanesulfonic acid (MES), N-hydroxysuccinimide (NHS), paraformaldehyde (4%) and sodium borohydride (NaBH4) were obtained from Sigma-Aldrich (USA). Nuclear stain Hoechst 33258 and Cell counting kit-8 (CCK-8) were obtained from Beyotime Biotechnology Co., Ltd. Lung adenocarcinoma cell line



(A549) and breast adenocarcinoma cell line (MCF-7) were purchased from the cell bank of the Chinese Academy of Science. Cell culture agents such as Dulbecco's Modified Eagle Medium (DMEM), RPMI Media 1640, trypsin, phosphate buffer solution (PBS) and fetal bovine serum (FBS) were purchased from Gibco. The blood samples used in the experiments were kindly provided by Jingzhou Central Hospital. All the experiments were performed using Milllipore water (Z≥18.2 MΩ) wherever required. Measurements Measurements of the zeta potential and the hydrodynamic size distribution were performed using Malvern Zetasizer Nano ZS90. The synthesized nanoparticles were visualized on a JEOL JEM-2100 transmission electron microscope (TEM). UV-visible (UV-vis) absorption spectra were recorded on a Varian Cary-Eclipse 500 spectrophotometer. The measurement of the electronic states of the metals in the samples was carried out by X-ray photoelectron spectroscopy (XPS) on a Perkin-Elmer PHI 5000C ESCA. An inverted microscope combined with fluorescence (Olympus, IX71, Japan) was used to observe the cells and zebrafish embryos. Synthesis of Au-Ag@BSA Nanoparticles The molar ratio of Au:Ag has been a key parameter in obtaining the desired characteristics of the AANPs. The nanoparticles were synthesized at a series of molar ratios viz. A (0:5), B (2:3), C (1:1), D (3:2), F (9:1), G (5:0), and for convenience, each of these ratios will be represented as Au-0, Au-40, Au-50, Au-60, Au-90, and Au-100, respectively in the present study. The protocol for synthesis at all the ratios are same, and hence, only the specifications used in the synthesis of D (Au-60 or Au:Ag=3:2) is described in details as an example. To begin with, an aqueous solution of BSA (3 ml, 26.1 mg/mL) was put into a 20 mL glass vial and aqueous HAuCl4 (1.2 mL, 30 mM) and AgNO3 solutions (0.8 mL, 30 mM) were added to it simultaneously with continuous stirring (650 rpm, 10 min). This was followed by the quick injection of pre-cooled NaBH4 solution (1.51 M, 1 mL). Finally, the mixture was stirred for 5 h. The by-products and the excess inorganic salts were removed by dialysis. The resultant solution was lyophilized and re-dissolved in PBS (pH = 7.4) for further use


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in the experiments. The nanoparticles with the varying compositions (Group A, B, C etc.) were prepared using the same parameters for the same period of time. X-ray attenuation measurements AANPs of the six groups at various molar concentrations (molar concentration refers to the combined amount of gold and silver components in the material, that is, Au+Ag is 5, 10, 20, 40, 60, 80 mM), and iohexol were transferred into eppendorfs for further measurements of X-ray attenuation. CT scanning was carried out via a Brilliance 64-slice system (Philips Healthcare, Andover, MA) with a slice thickness of 0.625 mm, tube voltage of 120 kV, field of view (FOV) of 25.0 cm and tube current of 80 mA. The CT signal strength was reflected by means of the commonly used HU value in the clinic, simply speaking, the larger the HU value, the more obvious the CT imaging effect. Hemolytic assay The percentage of hemolysis plays an essential role in biomedicine, and fresh human blood is a favourable medium for evaluating the same. 1 mL of blood sample was centrifuged at 2000 rpm for 10 min to obtain the raw human red blood cells (HRBCs). Subsequently, the HRBCs were rinsed five times with PBS to obtain the pure HRBCs. They were further diluted by PBS to 10 mL. 800 μL of AANPs at the concentrations of 5–40 μM was simultaneously mixed with 200 μL of the above HRBCs. The positive and the negative controls were prepared by the addition of 800 μL of DI water and PBS, respectively, to two separate aliquots of 200 μL of the HRBCs. All the samples were allowed to stand for 4 h, followed by centrifugation (12000 rpm, 5 min). The OD of the solutions at 541 nm was recorded using the UV-vis spectrophotometer, and the percentage of hemolysis of the HRBCs samples were calculated by the method reported in the previous literature.51 Cell Culture and in vitro Cytotoxicity Assay Cytotoxicity of nanomaterials is a vital factor that must be taken into consideration. The biotoxicity of the samples prepared in this study were ascertained using the A549 and MCF-7 cells. For instance, MCF-7 cells with DMEM in the presence of 10% FBS were cultured in a suitably humidified environment (37 ºC, 5% CO2). Subsequently,



the cells were seeded into 96-well plates under the invariable environment. At the logarithmic growth phase, the cell culture medium (DMEM and 1640) was removed and replaced with a fresh, 100 μL of complete medium for 24 h and 48 h respectively. Henceforth, the viability of the cancer cells was measured by a standard CCK-8 assay. Cellular uptake of Au-Ag@BSA Nanoparticles The study of cellular uptake, intracellular localization, and the distribution of AANPs in A549 cancer cells were observed by fluorescence localization tests and biological TEM. For fluorescence localization analysis, HA (84.14 mg) was first introduced into a glass beaker containing 50 mL MES buffer and it was mixed with EDC (191.5 mg) and NHS (57.25 mg) for 30 min. Then, Au-60 was injected into the mixture with vigorous agitation (650 rpm, 12 h). Thereafter, FITC was introduced and the reaction system was allowed to continue for 2 h. The preparation of the pure product Au-60-HA-FITC probe was continued until there was no fluorescence in the PBS dialysate. After the incubation with Au-60-HA-FITC (20 μM) probe for 6 h, cells were washed to remove any free material. They were then fixated with 4% paraformaldehyde, and further incubated with Hoechst 33258 to stain the nucleus for 10 min. After sufficient washing with PBS, the morphology and fluorescence intensity of the cells were examined by a fluorescent microscope with the blue and ultraviolet fluorescence channel, respectively. For the biological TEM analysis, A549 cancer cells were first fed with Au-60 (20 μM) for 24 h prior to rinsing and gathering, and then fixing with 2.5% glutaraldehyde overnight. Next, the samples were immobilized with 2% OsO4 in PBS for 2 h, followed by complete washing. Henceforth, the samples were dehydrated with increasing concentrations of ethanol (30%, 50%, 70%, 80%, 90%, 100%). Next, the obtained samples were embedded into a pure Epon resin with polymerization and a detailed slicing, which is essential prior to an observation by biological transmission electron microscope. Toxicity Studies in Zebrafish Embryos Adult zebrafish (AB line, male:female = 2:1) were fed in an aquarium set up with the parameters of 28.5 ℃ . And based on the zebrafish's spawning pattern, a simulated


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10h/14h dark/light cycle was supplemented, the behavior of spawning would be triggered when the morning light is turned on. Embryos were gathered at 4 hours post fertilization (4 hpf) and were rinsed by E3 medium. Next, the embryos were placed in a 6-well plate (20 embryos in 5 mL of E3 medium per well). They were treated with Au-60 AANPs at the concentrations of 0-80 mM (Au+Ag) for 4-120 hpf. The investigation was performed on five replicates at each concentration group. The morphology and state of the zebrafish at the development stage of 4-120 hpf were observed with an inverted microscope. The number of surviving and hatching embryos was carefully recorded and the survival rate and hatching rate were calculated with the following formula. Survival rate (%) Number of live zebrafish in each group 100% Number of zebrafish embroys

(1)

Number of hatching zebrafish in each group 100% Number of zebrafish embroys

(2)



Hatching rate (%) 

Results and Discussion Synthesis and Characterization of Au-Ag@BSA nanoparticles The preparation and application of Au-Ag@BSA nanoparticles are explicitly shown in Scheme 1. BSA is an affordable multifunctional protein that has a unique molecular space structure and great number of functional groups on its surface. In the current work, BSA is found to behave not only as a biotemplate, but also as a stabilizer. Owing to its unique features, BSA was naturally selected as the platform, with HAuCl4 and AgNO3 serving as the source of Au3+ and Ag+. The UV-vis absorption curves with the appearance of a single one plasmon absorption (Figure 1) showed an obvious red shift with the change in the composition of silver, indicating the existence of diverse materials (i.e. different alloy nanoparticles). Additionally, the composition of the alloy particles was determined by ICP-OES. In all the cases (Table 1), the molar ratio of Au:Ag in the product is slightly smaller than



the corresponding feed molar ratio. Table 1. Feed and practical molar ratio of Au/Ag in alloy product.

Figure 1. Regular UV-vis absorption spectrum of the prepared AANPs.

X-ray attenuation behavior of Au-Ag@BSA nanoparticles In order to further assess the CT imaging performance of the alloys, the X-ray attenuation behaviour of the prepared Au-0 nanoparticles, Au-100 nanoparticles and other silver composition-dependent AANPs were compared with that of iohexol. In the present work (Figure S1 and Figure 2), a regular increase in the brightness of the CT images and an enhancement of the HU values could be observed with increasing concentrations of nanomaterials and iohexol. In principle, brighter the image, the higher will be the HU values and more significant will be the CT effect. It is quite clear from Figure 2b that the HU values, and hence the contrast effect of 80 mM


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AANPs are remarkably better than the same concentration of iohexol. Meanwhile, the HU values of all the alloys were considerably higher than the Au-0 nanoparticles. Interestingly, the HU values increased up to the composition of Au-60, and then further decreased with an increasing composition till Au-100. The HU value of Au-60 is almost twice as high as that of iohexol at the concentration of 80 mM, and approximately equal to 1.3 times that of Au-100. It is also observed that Au-0 has a CT effect similar to that of iohexol. The CT contrast effect could be strengthened with the suitable introduction of an Ag+ precursor till Au-60. Beyond this, however, an increasing amount of Ag+ led to a decrease in the HU values till Au-100. In other words, AANPs could attain the best X-ray attenuation coefficient at the Au/Ag ratio of 3:2 (Au-60). Based on the experimental results and related works on the process of formation of gold and silver alloys,28,29,32 a plausible mechanism of the adjustable CT performance was speculated. During the formation of the alloy, AuCl4– and Ag+ undergo a co-reduction process, i.e. the previously formed gold catalysed the formed silver deposits, followed by the co-reduction reaction. That may also be a possible reason for the gold and silver components in the product are different from the feed ratio. In accordance with this, it is expected that the CT performances could be greatly improved with a defined amount of Au-catalysed silver deposition. However, the CT performance may decrease by an excessive silver deposition, because of which a dip in the HU values can be observed after a particular Au:Ag ratio (3:2). Hence, the synthesised AANPs could qualify as an ideal CT imaging contrast agent with adjustable contrast ability.



Figure 2. (a) HU values of AANPs and iohexol in the concentration range of 5-80 mM.(b) HU values of AANPs and Iohexol at the concentration of 80 mM.

Characterization of Au-Ag@BSA nanoparticles Based on the CT performance, Au-60 was chosen for further experiments, using Au-0 and Au-100 as the controls. In addition to their UV-vis absorption properties, the morphology and particle sizes were also investigated. The hydrodynamic sizes were monitored using dynamic light scattering (DLS, Figure S2a-c), which predicted a very narrow size distribution ranging from 16 to 26 nm. The zeta potential measurements (Figure S2d-f) revealed that the BSA-stabilized alloy particles dispersed in PBS (pH = 7.4) bore a negative charge. TEM images (Figure 3a-c and Figure 3g-i) demonstrate that Au-0, Au-60, and Au-100 are evenly dispersed in an aqueous solution with a homogeneous diameter of about 2-4 nm. Not all the nanoparticles show a lattice mismatch or a structure similar to that of a nuclear shell, suggesting that they were cast in bimetallic alloys. The fact was also supported by the UV-vis absorption spectra shown in Figure 1. Meanwhile, the high-resolution TEM images (HRTEM, Figure 3d-f) reveal clear lattice fringes measuring about 0.235 nm. However, the spacing between the Au and Ag crystal planes (2.3547 Å and 2.3591 Å) is very close, because of which the crystal lattice structure of gold and silver in the alloy could not be precisely detected by HRTEM.31,52


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Figure 3. TEM images, HRTEM images and size distribution of AANPs, from left to right are Au-0, Au-60, and Au-100.

Figure 4. (a) EDS analysis (a) and mapping of Au-60 (b) Ag, (c) Au, (d) C, (e) N, (f) O, (g) S, (h) combined).



The EDS measurements reveal the presence of elements such as Au, Ag, C, N, O, and S (Figure 4a), suggesting the success of the BSA-coated alloy nanoparticles. Meanwhile, the integral distribution of such elements over Au-60 is illustrated by the mapping (Figure 4b-h). The mapping images suggest a uniform distribution of Au, Ag, C, N, O, and S species in Au-60. Furthermore, the oxidation states of Au and Ag in the alloys were confirmed by the XPS measurements. The XPS spectrum in Figure 5 and Figure S3a-b showed two peaks corresponding to Ag 3d5/2 and Ag 3d3/2 with binding energies of 367.9 and 374.1 eV, respectively, indicating the coexistence of Ag (I) and Ag (0). Similarly, the binding energies at 84.4 and 87.2 eV (Figure 5 and Figure S3c-d) can be attributed to Au 4f7/2 and Au 4f5/2, thereby confirming the presence of Au (0) in the alloys.53,54 In addition, the peak at 163.3 eV as shown in Figure S4 can be assigned to the binding energy of S2p in Au-S bonding. The S might be derived from the protein in the alloys, that is, from the 35 cysteine residues present in BSA.31,32,42 The presence of S proves the fact that BSA could indeed act as a template in the synthesis of the gold and silver alloyed nanomaterials.

Figure 5. Total XPS spectrum of AANPs (a) Au-0, (b) Au-60, (c) Au-100.

Stability of Au-Ag@BSA nanoparticles A good stability of the nanomaterial is an important aspect in biological applications. In order to determine the stability of the AANPs, their behaviour was studied at different pH and temperatures. In order to do this, the nanoprobe was initially dispersed in (i) a cell culture medium and FBS for 12h (Figure S5a-c) and (ii) PBS for three time points: 12 h, 7 days and 30 days (Figure S5d-f). From left to right, the


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solutions in Figure S5 correspond to Au-0, Au-60 and Au-100. No distinct visible aggregation or precipitation could be observed in these solutions (Figure S5a-c), indicating a good colloid stability of the alloys. Nonetheless, an unusual adhesive behavior occurred on the walls of the bottle containing the Au-100 solution after 30 days (Figure S5f), while no such phenomenon occurred for Au-0 and Au-60. Thus, Au-0 and Au-60 had a wider range of colloidal stability. In addition, no appreciable changes could be seen in the UV-vis spectra after an adequate incubation of these AANPs under a series of varying pH (pH=4~9, Figure 6a-c) or temperatures (T=4~50 ºC, Figure 6d-f). Thus, the synthesized nanoparticles Au-60 and Au-0 were quite stable in terms of pH and temperatures compared with Au-100, which also paves the way for its subsequent biological applications.

Figure 6. UV-vis absorption spectrum of 5 µM AANPs in PBS at different pH (a-c) and temperatures (d-f). The images from left to right represent Au-0, Au-60, Au-100.

In vitro biocompatibility of Au-Ag@BSA nanoparticles Haemocompatibility is a crucial factor that needs to be taken care of in biological applications. Accordingly, the haemocompatibility of the AANPs was studied, using PBS and water as the positive and negative controls, respectively. Figure 7b-c shows that the percentage haemolysis of Au-60 and Au-100 is lesser than 5% even at a concentration of 40 µM, while it was 7.2% for Au-0 (Figure 7a). This was a clear



indication of the better haemocompatibility of Au-60 and Au-100 in comparison to that of Au-0. In vitro cytotoxicity assays are also equally crucial besides the haemolytic assays. Consequently, the cell viability of A549 and MCF-7 cells incubated with Au-0, Au-60, and Au-100 (0-80 µM) for 24 h and 48 h was successively estimated by the accepted standard CCK-8 analysis method. It was interesting to find that the viability of both the cell lines was close to 92% in the presence of Au-60 (Figure 8(b, e)) or Au-100 (Figure 8(c, f)). However, the viability of both the cell lines was quite low in Au-0 (figure 8(a, d)).

Figure 7. Hemolytic percent of HRBCs incubated with AANPs at different concentrations (a) Au-0, (b) Au-60, (c) Au-100. .

Figure 8. CCK-8 assay based cell viability of A549 (a-c) and MCF-7 (d-f) cells after incubation with Au-0 (a, d), Au-60 (b, e) and Au-100 (c, f) AANPs for 24 h and 48 h. .


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Furthermore, the cytotoxicity could also be examined macroscopically by directly observing the morphology of the A549 and MCF-7 cells. Cells without any treatment were set as the control group (Figure S6a). Cells which were incubated in the presence of 40 µM AANPs for 12 h did not show any visible morphological change for Au-60 (Figure S6c) and Au-100 (Figure S6d). However, a drastic change in the cell morphology could be observed in cells treated with Au-0 (Figure S6b). Both the cytotoxicity studies pointed towards a good biocompatibility of Au-60 and Au-100, and this is also in sync with the behaviour of the Au-Ag@PEG synthesized by Cormode’s group.55 The good biocompatibility can be explained by the fact that the inclusion of gold in the silver nanoparticles increases the oxidizing power of silver, which prevents the leaching of the silver ions and makes the nanoparticles biocompatible. In vivo toxicity of Au-60 for zebrafish embryos Based on our extensive research, it was found that Au-60 has the best stability, dispersion, in vitro biocompatibility, and CT imaging ability as compared to the other proportions of the alloys as well as Au-0 and Au-100. Therefore, Au-60 has a better biological application prospect than alloys of other components. Thus, Au-60 was chosen to study the effect of the synthesized AANPs on the development, survival, hatchability, and morphology of the zebrafish embryos. Zebrafish is an ideal animal model for the assessment of phenotypic abnormalities upon the treatment of nanomaterials. Consequently, zebrafish embryos were exploited to investigate the toxicity of our synthesized nanoparticles. The toxicity of Au-60 at the concentrations of 0, 5, 10, 20, 40 and 80 mM towards zebrafish embryos was evaluated from 4 hpf to 120 hpf. The morphology of zebrafish larvae was observed after 10, 24, 48 and 96 hpf during the growth of the zebrafish. In the survival rate experiment (Figure 9), based on the survival rate statistical method (equation 1), it could be seen that almost 96% of the eggs had normally developed into zebrafish even at 80 mM Au-60 indicating that Au-60 had only a little side effect on the growth of the zebrafish.



Figure 9. Toxicities of Au-60 in zebrafish. Bar graphs depict the survival rate of zebrafish in Au-60 at (a) 24 hpf, (b) 48 hpf, (c) 72 hpf, (d) 96 hpf, and (e) 120 hpf. (f) dose–response and time course of the survival rate. . hpf and 72 hpf is also considered as a The hatching rate of embryos between 48

crucial parameter of investigation. In order to assess any destructive effect of the Au-60 nanoparticles on the hatchability, the hatching behavior of the eggs from 10 hpf to 120 hpf was observed according to equation 2. Interestingly, most of the zebrafish (95%) showed a normal hatching behaviour in every group (Figure 10). The hatching behaviour in the presence of Au-60 was almost similar to that observed in the control groups, thus suggesting that Au-60 did not impart any remarkable toxicity to zebrafish. Further, the appearance of zebrafish exposed to various concentrations of the AANPs during each incubation period was also recorded (Figure 11). A comprehensive comparison clearly depicts that there were no abnormalities or variations in the morphology of the zebrafish. Therefore, the profiles of both the survival rate and hatching clearly show that as-synthesized Au-60 is safe and non-toxic within the dose range of 80 µM.


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Figure 10. Hatching rate of Au-60 at (a) 48 hpf, (b) 72 hpf and (c) 96 hpf. (d) dose–response and time course of the hatching rate.

Targeting ability and in vitro cellular uptake of Au-60 nanoparticles Considering the excellent properties of our synthesized Au-60, we decided to investigate its ability to be endocytosed by cancer cells. It is well known that CD44 protein can be targeted with HA, meaning that HA and CD44 could act as the targeting group and receptor, respectively. Accordingly, A549 cells with the overexpression of CD44 protein were chosen to appraise the cellular uptake, since it is only slightly expressed in MCF-7 cells. In the present work, both fluorescence imaging and biological TEM were utilized to follow the trail of Au-60.



Figure 11. Morphology of zebrafish incubated with Au-60 obtained using an inverted microscope. It should be noted that at 48 hpf, the eggs coexist with the fish. .

First, A549 cells and MCF-7 cells were treated with Au-60-HA-FITC (5 μM) for 6 h at 37 ºC. After washing the cells sufficiently, Hoechst 33258 was added for nuclear fluorescent staining. Figure 12b displays that the FITC green fluorescence could be easily captured in the targeting A549 cells. At the same time, only weak green FITC signals could be observed in the MCF-7 cells, suggesting that only a small amount of nanoprobes were internalized by these cells (Figure 12e). It also shows that the surface modified material has good targeting ability to A549 cells. Furthermore, the fluorescence signal due to the uptake of Au-60 clearly depicts that the materials were mainly distributed in the cytoplasm around the nucleus.


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Figure 12. Fluorescence images of A549 cells (top row) and MCF-cells (bottom row) incubated with Au-60-HA-FITC. The images from left to right represent Hoechst 33258 fluorescence, FITC fluorescence and the overlay of the above fields.The scale bars in the images (a−c) are 50 μm. .

Figure 13. TEM images of A549 cells: blank control group (a-c) as a comparison, test group incubated for 24h (d-f). (b), (c) and (e), (f) were local enlarged views of the highlight area in (a) and (d), respectively.



Additionally, in order to further confirm the distribution of the nanoparticles, the cells were visualized in a biological TEM. The TEM images in Figure 13d-f are accompanied by chromatic aberration in the cells, however, such a feature, could not be found in the control group (Figure 13a-c), indicating the distribution in lysosomes.56 Thus, the fluorescence localization and the intracellular absorption experiments point towards a remarkable targeting performance and also establish the fact that AANPs could be absorbed inside the cells. Conclusions In summary, utilizing BSA as a biotemplate, we have successfully designed a novel, green, environmentally friendly, and stable ultrasmall CT imaging contrast agent with a mean size of 2-4 nm. The contrast agent with adjusted performance has a low cytotoxicity, and is also lysosome-targeted. Compared to Au-100, the alloyed Au-60 synthesized in this study is expected to save material costs. Besides, alloyed Au-60 also attenuate the biological toxicity compared to Au-0 within the same time period. The novel functional alloy nanomaterial is expected to have a promising future in CT imaging. The study of the detailed mechanism of the characteristics imparted by the Au-Ag@BSA alloy nanoparticles in enhancing the X-ray computed tomography performance is on-going in our research group. Author Information Corresponding Author:*E-mail: [email protected]; [email protected] Conflict of Interest: The authors declare no conflict of interest. Acknowledgments This study was supported by Shanghai Science and Technology Committee (17070503000, 18dz2308700), National Natural Science Foundation of China (21373138, 81472001), Program for Changjiang Scholars and Innovative Research Team in University (IRT_16R49), International Joint Laboratory on Resource Chemistry (IJLRC), Shanghai Government (18DZ2254200) and Science and Technology Innovation Foundation for College Students from Shanghai Normal University.


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Supporting Information Supporting Information is available. Supporting Figure S1 included the CT images of AANPs and Iohexol . Supporting Figure S2 included the hydrodynamic size distribution and zeta potential of AANPs. Supporting Figure S3 included the XPS spectrum of Au and Ag. Supporting Figure S4 included the XPS spectrum of S element. Supporting Figure S5 included the pictures of alloy liquid at different time. Supporting Figure S6 included the morphology of A549 and MCF-7 cells. References 1.

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For Table of Contents Use Only

Ultrasmall

Au-Ag

Protein-directed

Alloy

Synthesis,

Nanoparticles:

Biocompatibility

and

X-ray Computed Tomography Imaging † Zhongyun Chua, Lina Chenb, Xiaoshuang Wangb, Qingye Yanga, Qi Zhao a, Chusen Huanga, Yuankui Huangb, Da-Peng Yang*c, Nengqin Jia*a

Table of content


Ultrasmall AuâAg Alloy Nanoparticles: Protein-Directed Synthesis

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