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Gallstone Formation Inspired Bimetallic Supra-Nanostructures for Computed Tomography Image Guided Radiation Therapy Soojeong Cho, Wooram Park, Hacksung Kim, Jacob Jokisaari, Eric W Roth, Sungsik Lee, Robert F Klie, Byeongdu Lee, and Dong-Hyun Kim ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b00908 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 11, 2018

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ACS Applied Nano Materials

Gallstone Formation Inspired Bimetallic Supra-Nanostructures for Computed Tomography Image Guided Radiation Therapy Soojeong Cho†, Wooram Park†, Hacksung Kim‡,§, Jacob R. Jokisaari∞, Eric W. Roth║, Sungsik Lee∇, Robert F. Klie∞, Byeongdu Lee∇*, Dong-Hyun Kim†,O*

†

Department of Radiology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA

‡

Chemical Sciences and Engineering Division, Argonne National Laboratory, Argonne, Illinois 60439, USA

§

Center for Catalysis and Surface Science, Northwestern University, Evanston, Illinois 60208, USA Department of Physics, University of Illinois at Chicago, Chicago, Illinois 60607, USA

∞ ║

NUANCE/QBIC, Chemistry of Life Processes Institute, Northwestern University, Evanston, Illinois 60208, USA

∇X-ray O

Science Division, Argonne National Laboratory, Argonne, Illinois 60439, USA

Robert H. Lurie Comprehensive Cancer Center, Chicago, Illinois 60611, USA

*Corresponding Authors: Byeongdu Lee ([email protected]) and Dong-Hyun Kim ([email protected])

Key words: gallstone; bimetallic; nanoparticles; cholate; nanocomplexes; CT imaging; radiosensitizing; radiotherapy

Abstract Inspired by the gallstone formation mechanism, we report a fast one-pot synthesis of high surface area bimetallic hierarchical supra-nanostructures. As gallstones are generated from metal-cholate complexes, cholate bile acid molecules with Au/Ag metal precursors formed stable nanocomplexes aggregated with metal Au ions and preformed ~ 2 nm silver halide nanoparticles before the reduction. When a reducing agent was added, the metal-cholate nanocomplexes quickly formed noble bimetallic hierarchical supra-nanostructures. The morphology of bimetallic supra-nanostructures could be tailored by changing the feeding ratio of each metal precursor. In situ synchrotron SAXS measurement with a custom designed reaction cell showed a two-step growth and attachment behavior towards hierarchical supra-nanostructures from the gallstone formation inspired metal-cholate nanocomplexes in a 60-second reaction. Additional WAXS, XANES, in situ FT-IR and high-resolution STEM investigations subsequently revealed the mechanism for the evolution of bimetallic hierarchical supra-nanostructures. The gallstone formation inspired synthesis mechanism can be universally applied to other metals, for example Pt-Ag and Pd-Ag bimetallic nanostructures. Finally, the synthesized high surface area bimetallic supra-nanostructures demonstrated significantly enhanced X-ray CT imaging contrast and radiosensitizing effect for a potential image guided nanomedicine application. We believe that our synthetic method inspired by gallstone formation and understanding represent an important step towards the development of hierarchical nanoparticles for various applications.

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1. INTRODUCTION Gallstones are growing within the gallbladder out of bile components and can lead to gallstone diseases related with modern diet.1-2 Gallstones are solid fractal-tree-like structures that form from bile cholesterol and bilirubin in the gallbladder.3-4 Although the mechanisms of gallstone formation are still unclear, it has known that bile acids and interaction with divalent metal ions plays important roles in gallstone formation.5-7 Bile salts are steroidal compounds with the active hydroxyl and carboxylic acid groups on the concave hydrophilic face and three methyl groups on the convex hydrophobic face.8 In gallbladder, bile salts exist mainly as simple micelles or mixed micelles structures and there are also various divalent metal ions existing in bile.8-9 The interaction between bile salts and different metal ions influence on the aggregation behavior of bile salts, and would be one of the considerable important factors in periodic, fractal, and/or hierarchical gallstone formation (Scheme 1).5 Here, inspired by the macromolecular structure of metal ion-bridgedcholesterol forming gallstones (Scheme 1), we synthesized hierarchical bimetallic supra-nanostructures using cholate, metal ions and reducing agent. The advent of high-performance multifunctional nanoparticles and high-yield one-pot synthetic procedures is still considerably critical.10 Metallic nanoparticles have been intensively utilized in a wide spectrum of applications like diagnostic imaging agents, sensing, drug delivery platforms, and catalysts.11-12 The metallic nanoparticles showing computed tomography (CT) contrast properties and radiosensitizing properties improve therapeutic efficacy by guiding the drug delivery.13-14 Among metallic nanoparticles, those with hierarchical nanostructures, as compared to spherical nanoparticles, are thought to be advantageous because their hyper-branched or dendritic structures can provide a larger number of available active sites and surface atoms per unit area.15 The available pool of hierarchical metallic nanoparticles, however, remains limited especially for nanomedicine applications. Recently, bio-templates or environmental-friendly synthetic methods using DNA strands,16 enzymes,17 microorganisms,18-19 and plants20 or plant extracts21 have received considerable attention for the synthesis of anisotropic and hierarchical morphologies22-23. However, unfortunately, there is a lack of versatility in the shape control that can be achieved. In the case of demanddriven synthesis for hierarchical metallic nanostructures, the detailed crystal growth mechanism is often overlooked, which limits their understanding and potential expansion to more advanced applications. In this work, the metal ions-cholic acid supra-molecular structures forming gallstones were used for synthesizing noble hierarchical bimetallic supra-nanostructures. The gallstone inspired metal-bridged-cholic acid was prepared in an aqueous solution of hydrochloroauric acid (HAuCl4·3H2O), silver nitrate (AgNO3) and sodium cholic acid. Then, the addition of lascorbic acid reducing agent readily initiated a fast growth of bimetallic supra-nanostructures. In situ synchrotron smalland wide-angle X-ray scattering (SAXS/WAXS), in situ liquid phase FT-IR, X-ray absorption near edge structure (XANES) and high resolution TEM analysis revealed a stepwise growth mechanism of bimetallic supra-nanostructures in the metal ion-bridged-cholesterol complex. We investigated that our synthetic method and crystal growth mechanism can be universally applied to other metals, and a promising potential of our hierarchical supra-nanostructures for imageguided nanomedicine applications was demonstrated.

2. EXPERIMENTAL SECTION 2.1. Materials: Hydrochloroauric acid (HAuCl4·3H2O), silver nitrate (AgNO3), l-ascorbic acid (AA), sodium tetrachloropalladate (II) (Na2PdCl4), and chloroplatinic acid hydrate (H2PtCl6·xH2O) were obtained from Sigma (St. Louis, ACS Paragon Plus Environment

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USA). Sodium cholic acid was purchased from Pierce (Thermo Scientific, Rockford, USA). All the chemicals were analytical grade reagents and used without further purification. Milli-Q water was used during the study. 2.2. Preparation of Metallic Supra-nanostructures: Aqueous solution of sodium cholic acid (0–4 mM) was prepared for 10 mL in a 20 mL vial with a magnetic stick stirrer. While stirring the solution for 350 rpm, 1 mL of HAuCl4·3H2O solution (0.625–20 mM) and 150 µL of AgNO3 solution (10–30 mM) were subsequently added. Then, 150 µL of 100 mM AA solution was added. The reaction mixture was stirred for 20 s and left undisturbed for 2 h. The nanoparticles were purified in ethanol dispersion by 3 times repetition of centrifugation at 12,000 rpm for 15 min and redispersion in fresh ethanol. The detailed concentrations of reagents were described in Table S6 in Supporting Information (SI). 2.3. Characterization: Synthesized samples were characterized with transmission electron microscopy (TEM, Tecnai Spirit G2, 120 kV, FEI and JEM-2100 FasTEM, JEOL) and scanning and transmission electron microscopy (STEM, HD2300 Dual EDS Cryo STEM, Hitachi) for the analysis of morphologies and elemental mapping. Hydrodynamic size of the sample was measured with Zetasizer Nano ZSP (Malvern Inc.). 2.4. In situ Synchrotron X-ray Analysis: X-ray scattering measurements were performed at APS 12-ID-B beamline. Xray beam with energy of 14 keV was used and scattering data were collected with Pilatus 2M and 300K for SAXS and WAXS, respectively. For ex situ SAXS measurement, a solution sample was prepared in a flow cell made of a quartz capillary with diameter of 2 mm.24 Using a syringe pump (Microlab 600, Hamilton), 100 µL of the solution sample was drawn into the capillary and was programmed to continuously flow upwards and downwards to prevent X-ray driven metal reduction. The X-ray beam was exposed for 0.2 s for 10 times to collect averaged scattering patterns. For in situ SAXS measurement, the same flow cell was used. While stirring the solution of HAuCl4, AgNO3, and sodium cholic acid in a vial, 100 µL of the solution sample was drawn and measured for the reaction time -2 s followed by injection of 150 µL of AA at the reaction time 0. Stirring continued for 20 s and SAXS/WAXS data were collected for 0.1 s. XANES measurements for Ag K-edge and Au L3-edge were performed at APS 12-BM beamline. XANES data were collected in fluorescent mode. For Ag K-edge, X-ray was injected to the plastic vial containing the sample solutions. AgNO3 and silver foil were used as reference materials. For Au L3-edge XANES, a quartz capillary set-up that was used for in situ SAXS was employed to prevent x-ray driven metal reduction. HAuCl4 and gold foil were used as reference materials. 2.5. In situ FT-IR Characterization: FT-IR spectra of sodium cholic acid and nanocomplexes in aqueous phase were obtained by a Nicolet Nexus 670 FTIR spectrometer equipped with a liquid N2-cooled Mercury Cadmium Telluride (MCT) detector using a Harrick’s Praying Mantis Diffuse Reflection attachment. The spectra were recorded by co-addition of 128 scans with a resolution of 2 cm-1. High-quality IR spectra could be obtained by using diffuse reflection (DR) mode which has not been commonly-used for studying liquid samples. A comparison of DR mode with the transmission (TR) mode and attenuated total reflectance (ATR) mode and more detailed information can be found in SI, Methods and Figure S20. 2.6. Abberation-corrected STEM/EDS characterization: Samples were collected from the original suspension, diluted 2:1 in isopropanol, sonicated for 5 minutes and drop-cast onto a lacey carbon grid. Grids were heated to ~80°C to dry, followed by Ar plasma cleaning for 10 min at 15 W (South Bay Technologies PC2000) just prior to imaging to remove hydrocarbon contamination. STEM measurements were carried out on a Cs-corrected JEOL ARM200CF equipped with a cold FEG source. STEM imaging was performed at 200 kV with a convergence angle of ~28 mrad. EDS was performed ACS Paragon Plus Environment


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using an Oxford XMAX100TLE with a thermoelectrically cooled, windowless silicon drift detector. STEM images were analyzed using Digital Micrograph (Gatan Inc.) and ImageJ software packages. 2.7. Synthesis of metal nanostructures with Pt and Pd ions: Aqueous solution of sodium cholic acid (2 mM) was prepared for 10 mL in a 20 mL PPE vial with a magnetic stick stirrer. While stirring the solution for 350 rpm, 1 mL of H2PtCl6 solution (5 mM) and 150 µL of AgNO3 solution (10 mM) were subsequently added for Pt-Ag supra-nanoparticles. Then, 150 µL of 100 mM AA solution was added to reduce metal ions. The reaction mixture was stirred for 20 s and left undisturbed for 2 h. The same procedure was repeated with 1 mL of Na2PdCl4 (5 mM) in the place of H2PtCl4 for Pd-Ag supra-nanostructures. The synthesized nanoparticles were purified in ethanol dispersion by 3 times repetition of centrifugation at 12,000 rpm for 15 min and redispersion in fresh ethanol. 2.8. Image-guided nanomedicine applications of bimetallic supra-nanostructures: Available in SI. 3. RESULTS and DISCUSSION 3.1. Gallstone Formation Inspired One-pot Synthesis of Bimetallic Supra-nanostructures. Figure 1a shows a representative hierarchical supra-nanostructure synthesized from an aqueous solution of HAuCl4·3H2O (0.22 mM)/AgNO3 (0.13 mM) and sodium cholic acid (CA, 1.8 mM) with the addition of l-ascorbic acid (AA, 100 mM) at room temperature. By simply controlling the Au/Ag molar ratios of 0.9~6.8:1, we could synthesis various shapes of supra-nanostructures (Figure S1). As shown in Figure 1, our representative hierarchical suprananostructures are overall spherical in shape but are composed of a lot of linear hair-like branches, as similar with fractal structures of bile acid precipitates5, 25. The average diameter of supra-nanostructures and the thickness of branches were 124±15.5 nm and 6.9±1.8 nm, respectively. We found that both Au and Ag precursors are required to form the suprananostructures, and absence of any of the two or CA led to only spherical or irregular thorny metal nanostructures (Figure S2). Elemental mapping images confirmed the supra-nanostructures are composed of both Au and Ag elements (Fig. 1b). 3.2. In situ Synchrotron Small Angle X-ray Scattering (SAXS) Study. To understand the growth mechanism of the Au-Ag bimetallic hierarchical supra-nanostructures from the metal-cholate solution, we performed in situ SAXS experiments using our custom designed reaction cell (Figure S3). A 100 µL aliquot of the solution in a quartz capillary was sampled for each measurement and afterwards returned it to the reaction cell. The SAXS experiment revealed two-step growth behavior. As soon as AA reducing agent was added into the solution, SAXS intensity in the high q region increased until about 33 s, indicating formation of spherical nanoparticles. Their growth in this time period was demonstrated in the changes of the particle size, or the radius of gyration (Rg), and SAXS invariant Q value (marked as (i) in Fig. 1c and Fig. 1d).24, 26 Volume of individual nanoparticles was increased 40% as calculated from increase of Rg and invariant Q values (SI, Note 1). We also found that the plateau Q values of in situ SAXS experiments for various Au feeds were proportional to the concentrations of the initial Au precursors, suggesting that the spherical primary particles made of Au atoms (Figure S4). After the early stage (> 33 s), both Rg and invariant Q remained constant (Fig. 1d), which indicates no more nucleation or attachment of monomeric metal Au atoms to the particles occurred. In this later stage, however, SAXS intensities in the small q region (or q < π/Rg (marked as (ii) in Fig. 1c)) increased to produce a power-law scattering, indicating fusing of the primary particles into a necklace-like structure ACS Paragon Plus Environment

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as shown in TEM images (Fig. 1a and b).27 We believe that this fusing process should be similar to the previously reported growth of twisted Pt3Fe nanorods.28 The power-law slope observed in our SAXS data suggests that the fused particles look like nanorods when viewed in a length scale of tens of nm, but these short nanorods are interconnected to make a supra-nanostructure appearing spherical in shape as shown in TEM images (Fig. 1a and b). Guinier-Porod fitting of the final stage SAXS curve29 confirms that the final morphology is a hierarchical supra-nanostructure comprised of multi-levels of hierarchy, as shown in Fig. 1e and SI, Note 2. Taken together, SAXS results suggest that primary particles formed in the early stage are connected to make a hierarchical supra-nanostructural morphology at the later stage. 3.3. Metal-organic Nanocomplexes Before Reduction. We noticed that even before the injection of AA reducing agent, SAXS curves showed the existence of pre-formed nanoparticles with size of ca. 2 nm in the solution containing metal-cholate complexes of HAuCl4·3H2O, AgNO3, and CA (black curve (t = -2 s) in Fig. 1c). Upturn in the smallest q region of the SAXS pattern suggests that the particles aggregated into clusters (black curve (t = -2 s) in Fig. 1c). WAXS data obtained in situ confirmed that the pre-formed nanoparticles in the metal-cholate complex were AgCl (Fig. 2a). This was also supported by XANES scan at the Ag Kedge (Fig. 2b (left)) presenting the spectra of AgCl (at a range of 25.530–25.540 keV).30 The AgCl nanocrystals are formed presumably by a reaction of Ag+ from AgNO3 and Cl− from HAuCl4. However, Au is yet to be reduced at this point, as confirmed by a distinct white line (11.923 keV) at Au L3-edge XANES (Fig. 2b (right)). In addition, SAXS invariant Q values for these meso-structures with various Ag feeds were turned out proportional to the Ag precursor concentrations and not consistent at all to the Au precursor concentration, supporting that the preformed nanoparticles were AgCl and most Ag ions were consumed to form AgCl nanocrystals in this work (Figure S5). Indeed, scanning transmission electron microscopy (STEM) measurement showed a cluster of AgCl nanocrystals, whose overall size is ca. 70 nm (Fig. 2c). We also measured the hydrodynamic size of the clusters and found them in the range of 50–160 nm (Figure S6), which is larger than the size observed in TEM, suggesting the AgCl nanocrystals might form metal-organic nanocomplexes with amphiphilic CA molecules. CA is a steroidal compound with active hydroxyl and carboxylic acid groups on its concave hydrophilic face and three methyl groups on the other convex hydrophobic face.31 CA can selfassemble with hydrophobic molecules in aqueous solutions and strongly interact with cationic metal ions, making these CA compounds forming gallstones in gallbladder.32-33 At the same time, there is a great potential for templates to synthesize noble metal nanostructures. 3.4. FT-IR Analysis of Metal-organic Nanocomplexes. To characterize pre-formed metal-cholate nanocomplexes with Au ions and AgCl nanoparticles in solution, we performed high-resolution liquid FT-IR analysis34. We could reveal that Au ions weakened the hydrogen bonding between CA and water, and thereby facilitate hydrophobic aggregation of CA with preformed AgCl nanocrystals. With references samples of a powder and aqueous solution of CA, IR spectra of the nanocomplexes in the solution were characterized by a significant blue-shift of hydrogen bonded OH stretching from 3378 to 3478 cm-1 by 100 cm-1 and weaker in its intensity due to the diminished dipoles in the nanocomplexes (‘blue’ in Fig. 2d and Figure S7).35 Weakening of the hydrogen bonding in CA could be caused by Au ions and AgCl nanocrystals. Firstly, Au ions can coordinate with oxygen atoms in the R2CH-OH unit of CA. A weakening of the C-O bond of the R2CH-OH unit by interaction with Au ions was confirmed ACS Paragon Plus Environment


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by a red-shift of the C-O stretching vibration for 6 cm-1 (1046 cm-1 to 1040 cm-1) in the nanocomplexes (denoted ‘8’ in Fig. 2d and Figure S8). This Au-O interaction also can likely induce hydrogen bonding between H atom in CA’s C-OH and strongly polar Cl atom in AuCl4-, resulting in the formation of C-O-H---Cl-Au cyclic H-bonded structure where Au is coordinated to O atom. In addition, Au ions in bridging bidentate, µ2b-OCO, where each carboxylate oxygen from CA can be coordinated with a single metal cation (Au3+), would give more connection sites, favoring aggregation. The µ2b-OCO was confirmed by absence of C=O stretching vibrations in the nanocomplexes (denoted ‘5’ in Fig. 2d), and the extent of difference in values of asymmetric and symmetric COO− (carboxylate) stretching frequencies appearing in the 1650–1300 cm-1 region (denoted ‘6 and 7’ in Fig. 2d and Figure S9). On the other hand, AgCl nanocrystals interact differently with hydrophobic groups such as CH3 and CH in CA. The IR spectra of AgCl-free CA and the nanocomplexes showed notable spectral changes in the frequency and intensity for CH3 and CH stretching and bending vibrations (denoted ‘3’ and ‘4’ in Fig. 2d), but very small changes for CH2 symmetric and asymmetric stretching vibrations (Figure S10). The CH3 bonds (on positions 18, 19, and 21 in CA) protruding toward the nanocomplex core have a steric advantage for interaction with AgCl nanocrystals located in the core (convex cave). Taken all FT-IR data together, the molecular level coordination sites of each components in pre-formed metal-cholate nanocomplexes are suggested as shown in Fig. 2e. The carbon–rich hydrophobic face36 of amphiphilic CA interacts via CH3 bonds with AgCl nanocrystals. The other hydrophilic sites are coordinated by Au ions and then bridging bidentate and cyclic H-bonded structures are formed. These coordinated sites would likely become linear- and branched-connection hubs and growth points which lead to hierarchical multi-branched supra-nanostructures. 3.5. Crystal Growth of Bimetallic Supra-nanostructures. As shown in in situ SAXS data (Fig. 1c), the concentrated Au ions within the metal-cholate nanocomplexes were rapidly reduced within 60 s upon the addition of AA. AgCl was also reduced to Ag metals as WAXS data demonstrated that AgCl diffraction peaks in the pre-formed metal-cholate nanocomplexes disappeared after AA addition (Fig. 2a and Fig. 3a). Instead, a broad (111) peak of either Au or Ag nanocrystals was detected after the addition of AA (Fig. 3a). Since Au and Ag are hardly distinguishable in WAXS due to their similar unit cell sizes, the reduction of AgCl was confirmed by XANES. The Ag K-edge XANES for the reduced sample showed a characteristic feature of metallic Ag0 (25.550 keV; dot-lined in Fig 3b (left)) with a suppressed white line (25.520 keV; dot-lined in Fig 2b (left) and 3b (left)), indicating the reduction of AgCl to Ag0 (Fig. 2a and 3b). Reduction of Au ions to Au0 is also confirmed by a suppressed white line (11.921 keV; dot-lined in Fig. 3b (right)) and the distinct metallic feature (11.947 keV, dot-lined in Fig 3b (right)) in the Au L3-edge XANES spectra (Fig. 3b). High-resolution (HR) TEM, STEM, and EDS elemental mapping analysis were followed to observe those detail structures. It showed that the hierarchical supra-nanostructure is an assembly of multiple nanocrystals, with sizes of around 3–6 nm in diameter as characterized in SAXS analysis. Viewed from the ‘top’ of the arm, many crystallites were identified by the spacing of the fringes ((110) or (111) planes), but those were not coherent through the larger particle with a wide range of angles between crystallite boundaries. This suggests that while nucleation or fusion occurred to a greater degree along [110] and [111] directions, we cannot ascribe the growth mechanism to either epitaxial growth nor to the oriented attachment mechanism specifically, but instead it is likely more complex (Fig. 3c). High-resolution EDS elemental mapping revealed pure or Ag-rich domains or crystallites in the structure (Fig. 3d). Considering reduction potentials of AgCl (E0 (AgCl/Ag) = +0.22 V) and Au ions (E0 (Au3+ /Au) = ACS Paragon Plus Environment

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+1.50 V),37-38 Au nanocrystals presumably formed before the reduction of AgCl. This fast process was observed in the first stage of our in situ SAXS experiment (Fig. 1c). As AgCl is reduced, Ag particles begin to nucleate and grow. Eventually their growth fronts encounter Au particles formed in the first stage and eventually interconnect them, as was observed in the second stage of in situ SAXS (Fig. 1c). During this process, some Ag ions may leach out and deposit onto Au crystallites forming mixed layers or crystallites.39 Taken all results together, the growth mechanism of the Au-Ag bimetallic hierarchical supra-nanostructure from the metal-choate complexes inspired by gallstone formation is suggested as described in a schematic illustration (Fig. 3e). In this mechanism, HAuCl4 plays two roles. It loses Cl ions to Ag ions to form AgCl nanocrystals. Au ions make CA molecules hydrophobic by hindering hydrogen bonding with water and facilitate them to form nanocomplexes with AgCl nanocrystals. The pre-formed metal-cholate nanocomplex becomes a container keeping a significant amount of Au ions and AgCl nanocrystals inside. When reducing agent, AA is added, the nanocomplex functions as a nanoreactor, wherein Au ions are reduced to form nanoparticles that are eventually connected by Ag particles as Ag ions are reduced from AgCl. Based on the suggested growth mechanism, it is expected that the morphology of Au-Ag supra-nanostructures can be tailored by changing the molar concentration ratio of each component. We indeed observed various shapes of hierarchical supra-nanostructures (Figure S1) with varying Au:Ag feed ratio. When excessive Au ions are used, more spherical particles are formed, which may have been formed outside of the metal-cholate nanocomplex container. Further discussion for the role of each component (HAuCl4·3H2O, AgCl nanocrystals, CA molecules, and AA) is found in SI, Note 3–6 and Figure S11–15. 3.6. Pt-Ag and Pd-Ag Bimetallic Supra-nanostructures Synthesis. As cholate molecules easily bridged with various multivalent metal ions, we hypothesized that the mechanism should be applicable to other metal ions in the replacement of Au as long as their reduction potentials are higher than AgCl and their counter anion is Cl−. Indeed, Figure 4 shows Pt-Ag and Pd-Ag bimetallic supra-nanostructures are synthesized using H2PtCl6 and Na2PdCl4 as precursors, respectively. We had to optimize the reaction conditions since the reduction speed of these metals are different from Au. Nevertheless, hierarchical Pt-Ag and Pd-Ag supra-nanostructures resembling the AuAg structure in both the shape and size were successfully formed. XRD data and elemental mapping images showed each Pt-Ag and Pd-Ag bimetallic nanocrystals and XPS data confirmed the presence of each element in the bimetallic suprananostructures (Figure 4 and Figure S16 and Figure S17). Our synthetic protocol with suggested mechanism should be promising for synthesizing various other bimetallic supra-nanostructures that can be used for enhanced catalytic performance through the higher surface areas and synergistic effects from the presence of two metals.40 3.7. Image-guided Nanomedicine Applications of Bimetallic Supra-nanostructures. Our developed bimetallic supra-nanostructures synthesized by the metal-cholate nanocomplexes have a promising potential especially in the field of nanomedicine. The high surface area of supra-nanostructures allows higher number of surface atoms compared to conventional spherical metallic nanoparticles in the same volume; this feature greatly contributed to X-ray computed tomography (CT) imaging contrast effect and radio-sensitizing efficacy for the potential image guided radiation therapies. In our phantom study, the CT attenuation coefficient of the supra-nanostructures was found to be 41.1 HU/mg/ml, which was significantly higher than commercially available iodine contrast (Lipiodol; 21 ACS Paragon Plus Environment


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HU/mg/ml) or spherical AuNP (27.4 HU/mg/ml) (Fig. 5a). High atomic number materials also have been shown to enhance the photoelectric and Compton effect (subsequent emission of secondary electrons) that generate reactive oxygen species (ROS) to significantly increase radiation-induced DNA damage.41 When the supra-nanostructures were exposure to a single fraction of radiation doses (1-8Gy), the radiosensitizing ROS generation was significantly enhanced compared with only radiation or conventional spherical Au nanoparticles (Figure S18).41-42 The cytotoxic ROS43 induced by the supra-nanostructures directly increased cancer cell apoptosis rate (Fig. 5b). PC-3 human prostate cancer cell clonogenic studies with different radiation doses further demonstrated a remarkable radiosensitizing dose enhancing factor (DEF) of 22.0 compared with 1.95 of DEF for spherical nanoparticles at the same concentration (Fig. 5c and SI, Note 7). The significantly high DEF value of supra-nanostructures anticipates to increase tumoricidal effects while decreasing current required excessive radiation doses in radiation therapies. The strong CT contrast and radiosensitizing effects of suprananostructures are highly promising for CT image-guided radiotherapy applications that require both imaging and targeted therapeutic roles for the cancer treatment. Here, for instance, CT image guided focused radiation therapy using the suprananostructures was demonstrated in vivo in a human prostate cancer (PC3) xenograft mouse model. Firstly, reconstructed CT body images allowed identification and coordination of the tumor region (region-of-interest (ROI)) for the targeted injection of our supra-nanostructures (Figure S21). The infusion procedures of supra-nanostructures could be directly monitored with the enhanced CT signal in short time scans (ca. 5 min/scan). This intraoperative contrast enhanced (CE)CT imaging with the supra-nanostructures permitted prompt adjustments of the amount of supra-nanostructures or injection site (catheter placement). Intra-tumoral uptake and distribution of the infused supra-nanostructures depicted with significant contrast enhancement (contrast to noise ratio (CNR)=970 HU) (Figure S21). At the same time, the suprananostructures locally distributed in the tumor significantly enhanced in vivo cancer cell killing effect in a 10 Gy single fraction of image guided local radiation therapy (Fig. 5d and Figure S22). The strong CT contrast and radiosensitizing effects of supra-nanostructures are highly promising for the image guided therapies such as image guided proton-, brachy-, external-or internal-radiotherapies or combinational multi-kinase targeting chemo-, or immune-therapies to generate synergetic impact for the treatment of cancers. 4. CONCLUSION In summary, we synthesized bimetallic hierarchical supra-nanostructures using gallstone formation inspired metalcholate complexes. The growth mechanism in the metal-cholate nanocomplexes was investigated using in situ SAXS, WAXS, imaging techniques including TEM and STEM/EDX, and spectroscopic tools including XANES and FT-IR. AgCl silver halide nanocrystals in the metal-cholate nanocomplexes were formed as soon as the two precursors of HAuCl4·3H2O and AgNO3 were mixed in cholate solution, forming metal-cholate nano-complexes. Thereby the nanocomplexes concentrate the Au ions and AgCl nanoparticles inside. Reduction of the gallstone formation inspired metal-cholate nanocomplexes by AA proceeded in two steps. Au ions were reduced to form Au nanoparticles followed by AgCl reduction to Ag nanoparticles that eventually connected the Au nanoparticles in a necklace-like structure which formed the branches of the resulting supra-nanostructures. While final morphologies varied with the Au/Ag feed ratio, they all presented hierarchically branched supra-nanostructures unless excessive Au ions were used. We also confirmed that this mechanism using the gallstone formation inspired metal-nanocomplexes could be applied to other metal elements such as Pt(II) and Pd(II) and synthesized Pt-Ag and Pd-Ag bimetallic supra-nanostructures with similar hierarchies to the Au-Ag supra-nanostructure. Finally, we demonstrated significantly enhanced performance of the supra-nanostructures ACS Paragon Plus Environment

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both as medical CT imaging contrast and radio-catalytic agents for an image-guided radiation therapy. We believe that our synthetic approach and investigated growth mechanism of the bimetallic supra-nanostructures will be beneficial in various areas requiring high surface area metallic nanoparticles.

ASSOCIATED CONTENT Supporting Information is available free of charge on the ACS Publication website. The supporting information contains, supplementary figures, tables, notes, and methods.

Acknowledgements We acknowledge D. Lee for fruitful discussions. This work was supported by grants R21CA173491, R21CA185274 and R21EB017986 from the NCI and NIBIB, and was funded by the Chicago Biomedical Consortium (CBC) with support from the Searle Funds at the Chicago Community Trust. This work used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. This work made use of the JEOL JEM-ARM200CF in the Electron Microscopy Service (Research Resources Center, UIC). The acquisition of the UIC JEOL JEM-ARM200CF was supported by a MRI-R2 grant from the National Science Foundation [DMR-0959470].

Conflict of interest The authors declare no conflict of interest.

References 1. Sedaghat, A.; Grundy, S. M., Cholesterol Crystals and the Formation of Cholesterol Gallstones. New Engl. J. Med. 1980, 302, 1274-1277. 2. Bouchier, I. A., The formation of gallstones. Keio J. Med. 1992, 41, 1-5. 3. Lammert, F.; Gurusamy, K.; Ko, C. W.; Miquel, J. F.; Mendez-Sanchez, N.; Portincasa, P.; van Erpecum, K. J.; van Laarhoven, C. J.; Wang, D. Q., Gallstones. Nat. Rev. Dis. Primers. 2016, 2, 16024. 4. Liu, S.; Kong, X.; Xie, A.; Shen, Y.; Zhu, J.; Li, C.; Zhang, Q., A Study of the Fractal Structure of the Precipitate and the Mechanism of its Formation from the Gallbladder Bile of a Patient. Russ. J. Phys. Chem. a+ 2007, 81, 2084-2089. 5. Peng, Q.; Wu, J. G.; Soloway, R. D.; Hu, T. D.; Huang, W. D.; Xu, Y. Z.; Wang, L. B.; Li, X. F.; Li, W. H.; Xu, D. F.; Xu, G. X., Periodic and Chaotic Precipitation Phenomena in Bile Salt System Related to Gallstone Formation. Biospectroscopy 1997, 3, 195-205. 6. Weerakoon, H.; Navaratne, A.; Ranasinghe, S.; Sivakanesan, R.; Galketiya, K. B.; Rosairo, S., Chemical Characterization of Gallstones: An Approach to Explore the Aetiopathogenesis of Gallstone Disease in Sri Lanka. Plos One 2015, 10. 7. Sun, Y.; Yang, Z. L.; Shen, G. R.; Zhou, Y.; Zhou, X. S.; Wu, J. G.; Xu, G. X., Progress in the Study on the Composition and Formation: Mechanism of Gallstone. Sci. China Ser. B 2001, 44, 449-456. 8. Carey, M. C.; Small, D. M., Micelle Formation by Bile-Salts - Physical-Chemical and Thermodynamic Considerations. Arch. Intern. Med. 1972, 130, 506-527. 9. Soloway, R. D.; Trotman, B. W.; Ostrow, J. D., Pigment Gallstones. Gastroenterology 1977, 72, 167-182. 10. Terreno, E.; Uggeri, F.; Aime, S., Image Guided Therapy: the Advent of Theranostic Agents. J. Control. Release 2012, 161, 328-37. 11. Jain, P. K.; Huang, X. H.; El-Sayed, I. H.; El-Sayed, M. A., Noble Metals on the Nanoscale: Optical and Photothermal Properties and Some Applications in Imaging, Sensing, Biology, and Medicine. Acc. Chem. Res. 2008, 41, 1578-1586. 12. Dykman, L.; Khlebtsov, N., Gold Nanoparticles in Biomedical Applications: Recent Advances and Perspectives. Chem. Soc. Rev. 2012, 41, 2256-2282. 13. Ruan, J.; Wang, Y.; Li, F.; Jia, R.; Zhou, G.; Shao, C.; Zhu, L.; Cui, M.; Yang, D. P.; Ge, S., Graphene Quantum Dots for Radiotherapy. ACS Appl. Mater. Interfaces 2018, 10, 14342-14355. 14. Huang, H.; Yang, D. P.; Liu, M.; Wang, X.; Zhang, Z.; Zhou, G.; Liu, W.; Cao, Y.; Zhang, W. J.; Wang, X., pHsensitive Au-BSA-DOX-FA Nanocomposites for Combined CT Imaging and Targeted Drug Delivery. Int. J. Nanomedicine 2017, 12, 2829-2843. 15. Hao, F.; Nehl, C. L.; Hafner, J. H.; Nordlander, P., Plasmon Resonances of a Gold Nanostar. Nano Letters 2007, 7, 729-732. ACS Paragon Plus Environment


Page 10 of 17

16. Ma, X.; Huh, J.; Park, W.; Lee, L. P.; Kwon, Y. J.; Sim, S. J., Gold Nanocrystals with DNA-Directed Morphologies. Nat. Commun. 2016, 7, 12873. 17. Willner, I.; Baron, R.; Willner, B., Growing Metal Nanoparticles by Enzymes. Adv. Mater. 2006, 18, 1109-1120. 18. Nair, B.; Pradeep, T., Coalescence of Nanoclusters and Formation of Submicron Crystallites Assisted by Lactobacillus Strains. Cryst. Growth Des. 2002, 2, 293-298. 19. Konishi, Y.; Ohno, K.; Saitoh, N.; Nomura, T.; Nagamine, S.; Hishida, H.; Takahashi, Y.; Uruga, T., Bioreductive Deposition of Platinum Nanoparticles on the Bacterium Shewanella Algae. J. Biotechnol. 2007, 128, 648-653. 20. Sharma, R. K.; Gulati, S.; Mehta, S., Preparation of Gold Nanoparticles Using Tea: A Green Chemistry Experiment. J. Chem. Educ. 2012, 89, 1316-1318. 21. Philip, D., Biosynthesis of Au, Ag and Au-Ag Nanoparticles Using Edible Mushroom Extract. Spectrochim. Acta A 2009, 73, 374-381. 22. Lu, Y.; Liu, G. L.; Kim, J.; Mejia, Y. X.; Lee, L. P., Nanophotonic Crescent Moon Structures with Sharp Edge for Ultrasensitive Biomolecular Detection by Local Electromagnetic Field Enhancement Effect. Nano Letters 2005, 5, 119124. 23. Martin, C. R., Membrane-Based Synthesis of Nanomaterials. Chem. Mater. 1996, 8, 1739-1746. 24. Kwon, S. G.; Krylova, G.; Phillips, P. J.; Klie, R. F.; Chattopadhyay, S.; Shibata, T.; Bunel, E. E.; Liu, Y.; Prakapenka, V. B.; Lee, B.; Shevchenko, E. V., Heterogeneous Nucleation and Shape Transformation of Multicomponent Metallic Nanostructures. Nat. Mater. 2015, 14, 215-223. 25. Shen, Y. H.; Xie, A. J.; Zhang, J. Z.; Dong, T., Fractal Patterns of Precipitates Observed in Boar Bile. Physica. B 2003, 337, 281-286. 26. Roe, R. J., Methods of X-ray and Neutron Scattering in Polymer Science. Oxford University Press: New York, 2000; p xiv, 331 p. 27. Li, T.; Senesi, A. J.; Lee, B., Small Angle X-ray Scattering for Nanoparticle Research. Chem. Rev. 2016, 116, 11128-11180. 28. Liao, H.-G.; Cui, L.; Whitelam, S.; Zheng, H., Real-Time Imaging of Pt3Fe Nanorod Growth in Solution. Science 2012, 336, 1011-1014. 29. Hammouda, B., A new Guinier-Porod Model. J. Appl. Crystallogr. 2010, 43, 716-719. 30. Veronesi, G.; Aude-Garcia, C.; Kieffer, I.; Gallon, T.; Delangle, P.; Herlin-Boime, N.; Rabilloud, T.; Carriere, M., Exposure-Dependent Ag+ Release from Silver Nanoparticles and its Complexation in AgS2 Sites in Primary Murine Macrophages. Nanoscale 2015, 7, 7323-7330. 31. Madenci, D.; Egelhaaf, S. U., Self-Assembly in Aqueous Bile Salt Solutions. Curr. Opin. Colloid In. 2010, 15, 109-115. 32. Luo, J. T.; Chen, Y. L.; Zhu, X. X., Invertible Amphiphilic Molecular Pockets Made of Cholic Acid. Langmuir 2009, 25, 10913-10917. 33. Huang, W. D.; Hu, T. D.; Peng, Q.; Soloway, R. D.; Weng, S. F.; Wu, J. G., EXAFS and FTIR Studies on the Binding of Deoxycholic-Acid with Copper and Zinc Ions. Biospectroscopy 1995, 1, 291-296. 34. Schweitzer, N. M.; Hu, B.; Das, U.; Kim, H.; Greeley, J.; Curtiss, L. A.; Stair, P. C.; Miller, J. T.; Hock, A. S., Propylene Hydrogenation and Propane Dehydrogenation by a Single-Site Zn2+ on Silica Catalyst. ACS Catal. 2014, 4, 1091-1098. 35. Arunan, E.; Desiraju, G. R.; Klein, R. A.; Sadlej, J.; Scheiner, S.; Alkorta, I.; Clary, D. C.; Crabtree, R. H.; Dannenberg, J. J.; Hobza, P.; Kjaergaard, H. G.; Legon, A. C.; Mennucci, B.; Nesbitt, D. J., Defining the Hydrogen Bond: An Account (IUPAC Technical Report). Pure Appl. Chem. 2011, 83, 1619-1636. 36. Calabresi, M.; Andreozzi, P.; La Mesa, C., Supra-Molecular Association and Polymorphic Behaviour in Systems Containing Bile Acid Salts. Molecules 2007, 12, 1731-1754. 37. Au, L.; Lu, X. M.; Xia, Y. N., A Comparative Study of Galvanic Replacement Reactions Involving Ag Nanocubes and AuCl2- or AuCl4-. Adv. Mater. 2008, 20, 2517-2522. 38. Wang, J. L.; Chen, F. Y.; Jin, Y. C.; Johnston, R. L., Highly Active and Stable AuNi Dendrites as an Electrocatalyst for the Oxygen Reduction Reaction in Alkaline Media. J. Mater. Chem. A 2016, 4, 17828-17837. 39. Zhou, S.; Li, J.; Gilroy, K. D.; Tao, J.; Zhu, C.; Yang, X.; Sun, X.; Xia, Y., Facile Synthesis of Silver Nanocubes with Sharp Corners and Edges in an Aqueous Solution. ACS Nano 2016, 10, 9861-9870. 40. Liu, X. W.; Wang, D. S.; Li, Y. D., Synthesis and Catalytic Properties of Bimetallic Nanomaterials with Various Architectures. Nano Today 2012, 7, 448-466. 41. Pronschinske, A.; Pedevilla, P.; Murphy, C. J.; Lewis, E. A.; Lucci, F. R.; Brown, G.; Pappas, G.; Michaelides, A.; Sykes, E. C. H., Enhancement of Low-Energy Electron Emission in 2D Radioactive Films. Nat. Mater. 2015, 14, 904907. 42. Al Zaki, A.; Joh, D.; Cheng, Z. L.; De Barros, A. L. B.; Kao, G.; Dorsey, J.; Tsourkas, A., Gold-Loaded ACS Paragon Plus Environment

Page 11 of 17 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Polymeric Micelles for Computed Tomography-Guided Radiation Therapy Treatment and Radiosensitization. ACS Nano 2014, 8, 104-112. 43. Ott, M.; Gogvadze, V.; Orrenius, S.; Zhivotovsky, B., Mitochondria, Oxidative Stress and Cell Death. Apoptosis 2007, 12, 913-22.

Scheme 1. Fractal/hierarchical gallstone formation from metal-bile acid complexes.



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Figure. 1. (a) TEM images of supra-nanostructures synthesized from an aqueous solution of gallstone formation inspired metal-cholate complexes (HAuCl4·3H2O (0.22 mM)/AgNO3 (0.13 mM) and sodium cholic acid (CA, 1.8 mM)) with a reducing agent, l-ascorbic acid (AA, 100 mM). (inset) The electron diffraction pattern identifies crystalline phase of the supra-nanostructures. (b) High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and elemental (Ag and Au) mapping images of supra-nanostructures. (c) In situ SAXS patterns for 60 s including before (t = 2 s) and after the addition of AA (t > 0 s). (d) Time dependent radius of gyration (Rg) and invariant Q values. (e) GuinierPorod fitting of SAXS curve obtained in a sample of supra-nanostructures. (inset) Schematic illustration of a structural model consists of multi-level hierarchical structures confirmed by the Guinier-Porod fitting of the final stage SAXS curve.


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Figure 2. (a) WAXS pattern of the pre-formed metal-cholate nanocomplexes before the reduction with l-ascorbic acid. Reference peaks of AgCl, Ag2O, and Ag are indicated on the bottom. (b) Normalized Ag K-edge and Au L3-edge XANES spectra of the nanocomplexes. Dashed vertical lines mark the position of the white-line maximum and an arrow indicates a characteristic peak position presenting the spectra of AgCl. (c) HAADF-STEM and merged Au (red), Ag (cyan), and Cl (pink) elemental mapping images of the nanocomplexes. (d) FT-IR spectra of solid cholic acid (CA) (green), aqueous CA (red), and nanocomplexes in aqueous solution (blue): (1) t-OH (3550 cm-1), (2) hydrogen bonded OH vibration (3380– 3480 cm-1), (3) asymmetric CH3 stretching (2973 cm-1), asymmetric CH2 stretching (2943 cm-1), symmetric CH3 stretching (2909 cm-1), symmetric CH2 stretching (2868 cm-1), (4) methylene C-H stretching (2850 cm-1), (5) C=O stretching vibration, (6) symmetric COO (1561–1572 cm-1), (7) asymmetric COO (1407–1413 cm-1), and (8) C-O stretching in COH (1078 and 1046 cm-1). (e) Schematic illustration of metal binding to pre-formed metal-cholate nanocomplexes suggested by FT-IR spectra.



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Figure 3. (a) In situ WAXS pattern of the supra-nanostructures from the metal-cholate nanocomplexes after the reduction with l-ascorbic acid (AA). The reference peaks of Au and Ag are indicated on the bottom. (b) Normalized Ag K-edge and Au L3-edge XANES spectra of the supra-nanostructures. (c) A high resolution (HR)-TEM image of the branches of suprananostructures. Primary nanocrystals (3~6 nm) are randomly attached and forming chained branches of the suprananostructures. (d) HR elemental mapping images of Ag (green) and Au (red), and merged Ag (green) and Au (red) of the branches of supra-nanostructures. (e) Schematics of suggested mechanism of the Au-Ag bimetallic hierarchical suprananostructures evolution.


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Figure 4. TEM images, X-ray diffraction (XRD) pattern, and elemental mapping images of (a, b and c) Pt-Ag and (d, e and f) Pd-Ag supra-nanostructures synthesized by our one-pot synthesis using the gallstone formation inspired metalcholic acid complexes.



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Figure 5. (a) A linear relationship between CT numbers and concentrations of supra-nanostructures (supra-NS), spherical nanoparticles (spherical NP), and Lipiodol→ (oil-based radio-opaque CT contrast agent). The inset shows the concentration-dependent CT-contrast images of supra-nanostructures (1st row), spherical NP (2nd row) and Lipiodol→ (3rd row). (b) Flow cytometry results showing apoptotic cell death for control and samples with a single fraction of each 0, 4 and 6 Gy radiation after 24 h incubation. FACS analysis using FITC Annexin-V and propidium iodide (PI) staining. (c) Radiation dose dependent surviving fraction of cells treated with supra-nanostructures (red), spherical NP (blue), and nontreatment (black). (Supporting Information, Note7 and Figure S20). (d) A comparison of apoptotic area (%) of tumors from each animal groups (radiation + supra-NS, radiation only, supra-NS only and non-treated control; human prostate cancer (PC-3) xenograft mice model, radiation: single dose of 10 Gy). (each group n=6, *P

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