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. Author manuscript; available in PMC: 2021 May 27.
Published in final edited form as: ACS Appl Nano Mater. 2018 Aug 6;1(9):4602–4611. doi: 10.1021/acsanm.8b00908

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 †,#,*
PMCID: PMC8157682  NIHMSID: NIHMS1556802  PMID: 34056557

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 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 small-angle X-ray scattering measurement with a custom-designed reaction cell showed two-step growth and attachment behavior toward hierarchical supra-nanostructures from the gallstone-formation-inspired metal cholate nanocomplexes in a 60 s reaction. Additional wide-angle X-ray scattering, X-ray absorption near-edge structure, in situ Fourier transform infrared, and high-resolution scanning transmission electron microscopy 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 computed tomography imaging contrast and radiosensitizing effect for a potential image-guided nanomedicine application. We believe that our synthetic method inspired by gallstone formation and understanding represents an important step toward the development of hierarchical nanoparticles for various applications.

Keywords: gallstone, bimetallic, nanoparticles, cholate, nanocomplexes, CT imaging, radiosensitizing, radiotherapy

Graphical Abstract:

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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 is known that bile acids and the interaction with divalent metal ions play 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 the gallbladder, bile salts exist mainly as simple micelles or mixed-micelle structures, and various divalent metal ions also exist in bile.8,9 The interaction between bile salts and different metal ions influences the aggregation behavior of bile salts and would be one of the considerably important factors in periodic, fractal, and/or hierarchical gallstone formation (Scheme 1).5 Here, inspired by the macromolecular structure of metal-ion-bridged cholesterol-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, compared to spherical nanoparticles, are thought to be advantageous because their hyperbranched 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, biotem-plates or environmentally 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 morphologies.22,23 However, unfortunately, there is a lack of versatility in the shape control that can be achieved. In the case of demand-driven synthesis for hierarchical metallic nanostructures, the detailed crystal growth mechanism is often overlooked, which limits their understanding and potential expansion to more advanced applications.

Scheme 1.

Scheme 1.

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Fractal/Hierarchical Gallstone Formation from Metal–Bile Acid Complexes

In this work, the metal ion–cholic acid supramolecular structures forming gallstones were used for the synthesis of noble hierarchical bimetallic supra-nanostructures. The gallstone-inspired metal-bridged cholic acid was prepared in an aqueous solution of hydrochloroauric acid, silver nitrate, and sodium cholic acid. Then, the addition of an L-ascorbic acid reducing agent readily initiated fast growth of bimetallic supra-nanostructures. In situ synchrotron small- and wide-angle X-ray scattering (SAXS/WAXS), in situ liquid-phase Fourier transform infrared (FT-IR), X-ray absorption near-edge structure (XANES), and high-resolution transmission electron microscopy (HRTEM) 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 image-guided 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, MO). Sodium cholic acid (CA) was purchased from Pierce (Thermo Scientific, Rockford, IL). All of the chemicals were analytical-grade reagents and were used without further purification. Milli-Q water was used during the study.

2.2. Preparation of Metallic Supra-nanostructures.

A 10 mL aqueous solution of CA (0–4 mM) was prepared in a 20 mL vial with a magnetic stick stirrer. While the solution was stirred at 350 rpm, 1 mL of a HAuCl4·3H2O solution (0.625–20 mM) and 150 μL of an AgNO3 solution (10–30 mM) were subsequently added. Then, 150 μL of a 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 an ethanol dispersion by three times repetition of centrifugation at 12000 rpm for 15 min and redispersion in fresh ethanol. The detailed concentration of the reagents is described in Table S6.

2.3. Characterization.

Synthesized samples were characterized by transmission electron microscopy (TEM; Tecnai Spirit G2, 120 kV, FEI and JEM-2100 FasTEM, JEOL) and scanning transmission electron microscopy (STEM; HD-2300 Dual EDS Cryo STEM, Hitachi) for analysis of the morphologies and elemental mapping. The hydrodynamic size of the sample was measured with a Zetasizer Nano ZSP (Malvern Inc.).

2.4. In Situ Synchrotron X-ray Analysis.

X-ray scattering measurements were performed at the Advanced Photon Source (APS) 12-ID-B beamline. An X-ray beam with an energy of 14 keV was used, and scattering data were collected with Pilatus 2 M and 300 K 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 a diameter of 2 mm.24 Using a syringe pump (Microlab 600, Hamilton), 100 μL of the solution sample was drawn into the capillary and programmed to continuously flow upward and downward to prevent X-ray-driven metal reduction. The X-ray beam was exposed for 0.2 s 10 times to collect the averaged scattering patterns. For in situ SAXS measurement, the same flow cell was used. While the solution of HAuCl4, AgNO3, and CA was stirred in a vial, 100 μL of the solution sample was drawn and measured for a reaction time of 2 s, followed by the injection of 150 μL of AA at a reaction time of 0 s. Stirring was 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 the APS 12-BM beamline. XANES data were collected in fluorescent mode. For Ag K-edge, X-ray was injected into the plastic vial containing the sample solutions. AgNO3 and Ag foil were used as reference materials. For Au L3-edge XANES, a quartz capillary setup that was used for in situ SAXS was employed to prevent X-ray-driven metal reduction. HAuCl4 and Au foil were used as reference materials.

2.5. In Situ FT-IR Characterization.

FT-IR spectra of CA and nanocomplexes in aqueous phase were obtained by a Nicolet Nexus 670 FT-IR spectrometer equipped with a liquid-N2-cooled mercury cadmium telluride detector using Harrick’s Praying Mantis Diffuse-Reflection attachment. The spectra were recorded by the coaddition 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 the study of liquid samples. A comparison of the DR mode with the transmission- and attenuated-total-reflectance modes and more detailed information can be found in the Methods section and Figure S23.

2.6. Abberation-Corrected STEM/Energy-Dispersive Spectroscopy (EDS) Characterization.

Samples were collected from the original suspension, diluted 2:1 in isopropyl alcohol, sonicated for 5 min, 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 field-emission-gun source. STEM imaging was performed at 200 kV with a convergence angle of ~28 mrad. EDS was performed 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.

An aqueous solution of CA (2 mM) was prepared for 10 mL in a 20 mL PPE vial with a magnetic stick stirrer. While the solution was stirred at 350 rpm, 1 mL of a H2PtCl6 solution (5 mM) and 150 μL of an AgNO3 solution (10 mM) were subsequently added for Pt–Ag supra-nanoparticles. Then, 150 μL of a 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 place of H2PtCl4 for Pd–Ag supra-nanostructures. The synthesized nanoparticles were purified in an ethanol dispersion by 3 times repetition of centrifugation at 12000 rpm for 15 min and redispersion in fresh ethanol.

2.8. Image-Guided Nanomedicine Applications of Bimetallic Supra-nanostructures.

These are available in the Supporting Information.

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 CA (1.8 mM) with the addition of 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 supra-nanostructures are overall spherical in shape but are composed of a lot of linear hairlike branches, similar to the fractal structures of bile acid precipitates.5,25 The average diameter of supra-nanostructures and the thickness of the branches were 124 ± 15.5 and 6.9 ± 1.8 nm, respectively. We found that both Au and Ag precursors are required to form the supra-nanostructures, and the absence of any of the two or CA led to only spherical or irregular thorny metal nanostructures (Figure S2). Elemental mapping images confirmed that the supra-nanostructures are composed of both Au and Ag elements (Figure 1b).

Figure 1.

Figure 1.

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(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 CA (1.8 mM)) with a reducing agent, AA (100 mM). (inset) Electron diffraction pattern identifying the crystalline phase of the supra-nanostructures. (b) High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and elemental mapping (Ag and Au) images of supra-nanostructures. (c) In situ SAXS patterns for 60 s including before (t = −2 s) and after (t > 0 s) the addition of AA. (d) Time-dependent radius of gyration (Rg) and invariant Q values. (e) Guinier–Porod fitting of the SAXS curve obtained in a sample of supra-nanostructures. (inset) Schematic illustration of a structural model consisting of multilevel hierarchical structures confirmed by the Guinier–Porod fitting of the final stage SAXS curve.

3.2. In Situ Synchrotron 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 afterward returned to the reaction cell. The SAXS experiment revealed two-step growth behavior. As soon as the AA reducing agent was added into the solution, the SAXS intensity in the high q region increased until about 33 s, indicating the 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 Figure 1c,d).24,26 The volume of the individual nanoparticles was increased 40% as calculated from the increase of Rg and invariant Q values (Supporting Information 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 are made of Au atoms (Figure S4). After the early stage (>33 s), both Rg and invariant Q remained constant (Figure 1d), which indicates no more nucleation or attachment of monomeric metal Au atoms to the particles occurred. In this later stage, however, the SAXS intensities in the small q region [or q < π/Rg (marked as (ii) in Figure 1c)] increased to produce a power-law scattering, indicating fusion of the primary particles into a necklace-like structure, as shown in the TEM images (Figure 1a,b).27 We believe that this fusion 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 nanometers, but these short nanorods are interconnected to make a supra-nanostructure appearing spherical in shape, as shown in the TEM images (Figure 1a,b). The Guinier–Porod fitting of the final-stage SAXS curve29 confirms that the final morphology is a hierarchical supra-nanostructure comprised of multilevels of hierarchy, as shown in Figure 1e and Supporting Information Note 2. Taken together, the 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 injection of the AA reducing agent, the SAXS curves showed the existence of preformed nanoparticles with a size of ca. 2 nm in the solution containing metal cholate complexes of HAuCl4·3H2O, AgNO3, and CA [black curve (t = −2 s) in Figure 1c]. The upturn in the smallest q region of the SAXS pattern suggests that the particles aggregated into clusters [black curve (t = −2 s) in Figure 1c]. WAXS data obtained in situ confirmed that the preformed nanoparticles in the metal cholate complex were AgCl (Figure 2a). This was also supported by the XANES scan at the Ag K-edge [Figure 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) in the Au L3-edge XANES spectrum [Figure 2b (right)]. In addition, SAXS invariant Q values for these mesostructures with various Ag feeds turned out to be proportional to the Ag precursor concentrations and not consistent at all with the Au precursor concentration, supporting the theory 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 (Figure 2c). We also measured the hydrodynamic size of the clusters and found them to be in the range of 50–160 nm (Figure S6), which is larger than the size observed in TEM, suggesting that 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 self-assemble with hydrophobic molecules in aqueous solutions and strongly interact with cationic metal ions, making these CA compounds form gallstones in the gallbladder.32,33 At the same time, there is great potential for templates to synthesize noble-metal nanostructures.

Figure 2.

Figure 2.

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(a) WAXS pattern of the preformed metal cholate nanocomplexes before reduction with AA. The 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 CA (green), aqueous CA (red), and nanocomplexes in an 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), and 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); (8) C–O stretching in C–OH (1078 and 1046 cm–1). (e) Schematic illustration of metal binding to preformed metal cholate nanocomplexes, as suggested by FT-IR spectra.

3.4. FT-IR Analysis of Metal–Organic Nanocomplexes.

To characterize preformed metal cholate nanocomplexes with Au ions and AgCl nanoparticles in solution, we performed high-resolution liquid FT-IR analysis.34 We could reveal that Au ions weaken the hydrogen bonding between CA and water and thereby facilitate hydrophobic aggregation of CA with preformed AgCl nanocrystals. With reference samples of a powder and an 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 a weakening of its intensity due to the diminished dipoles in the nanocomplexes (“blue” in Figures 2d and S7).35 Weakening of the hydrogen bonding in CA could be caused by Au ions and AgCl nanocrystals. First, Au ions can coordinate with O 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 by a red shift of the C–O stretching vibration by 6 cm–1 (from 1046 cm–1 to 1040 cm–1) in the nanocomplexes (denoted as “8” in Figures 2d and S8). This Au–O interaction also can likely induce hydrogen bonding between the H atom in CA’s C–OH and the strongly polar Cl atom in AuCl4−, resulting in the formation of a C–O–H–Cl–Au cyclic hydrogen-bonded structure, where Au is coordinated to the O atom. In addition, Au ions in the bridging bidentate, μ2b-OCO, where each carboxylate O atom from CA can be coordinated with a single metal cation (Au3+), would give more connection sites, favoring aggregation. μ2b-OCO was confirmed by the absence of C=O stretching vibrations in the nanocomplexes (denoted as “5” in Figure 2d) and the extent of difference in the values of asymmetric and symmetric COO– (carboxylate) stretching frequencies appearing in the 1650–1300 cm–1 region (denoted as “6” and “7” in Figures 2d and S9).34 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 as “3” and “4” in Figure 2d) but very small changes for CH2 symmetric and asymmetric stretching vibrations (Figure S10). The CH3 bonds (at 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). Taking all of the FT-IR data together, molecular-level coordination sites of each component in preformed metal cholate nanocomplexes are suggested, as shown in Figure 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 hydrogen-bonded structures are formed. These coordinated sites would likely become linear- and branched-connection hubs and growth points, which lead to hierarchical multibranched supra-nanostructures.

3.5. Crystal Growth of Bimetallic Supra-nanostructures.

As shown in the in situ SAXS data (Figure 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 because the WAXS data demonstrated that AgCl diffraction peaks in the preformed metal cholate nanocomplexes disappeared after AA addition (Figures 2a and Figure 3a). Instead, a broad (111) peak of either Au or Ag nanocrystals was detected after the addition of AA (Figure 3a). Because Au and Ag are hardly distinguishable in WAXS, because of their similar unit cell sizes, the reduction of AgCl was confirmed by XANES. The Ag K-edge XANES spectrum for the reduced sample showed a characteristic feature of metallic Ag0 [25.550 keV; dotted line in Figure 3b (left)] with a suppressed white line [25.520 keV; dotted line in Figures 2b (left) and 3b (left)], indicating the reduction of AgCl to Ag0 (Figures 2a and 3b). Reduction of Au ions to Au0 is also confirmed by a suppressed white line [11.921 keV; dotted line in Figure 3b (right)] and the distinct metallic feature [11.947 keV, dotted line in Figure 3b (right)] in the Au L3-edge XANES spectra (Figure 3b). HRTEM, STEM, and EDS elemental mapping analysis were followed to observe those detailed structures. It showed that the hierarchical supra-nanostructure is an assembly of multiple nanocrystals, with sizes of around 3–6 nm 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 the [110] and [111] directions, we cannot ascribe the growth mechanism to either epitaxial growth or the oriented attachment mechanism specifically, but instead it is likely more complex (Figure 3c). High-resolution EDS elemental mapping revealed pure or Agrich domains or crystallites in the structure (Figure 3d). Considering the reduction potentials of AgCl [E0(AgCl/Ag) = +0.22 V] and Au ions [E0(Au3+/Au) = +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 (Figure 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 with them, as was observed in the second stage of in situ SAXS (Figure 1c). During this process, some Ag ions may leach out and deposit onto Au crystallites, forming mixed layers or crystallites.39

Figure 3.

Figure 3.

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(a) In situ WAXS pattern of the supra-nanostructures from the metal cholate nanocomplexes after reduction with 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) HRTEM image of the branches of supra-nanostructures. Primary nanocrystals (3–6 nm) are randomly attached and form chained branches of the supra-nanostructures. (d) High-resolution elemental mapping images of Ag (green) and Au (red) and merged Ag (green) and Au (red) of the branches of supra-nanostructures. (e) Schematic of the suggested mechanism of the Au–Ag bimetallic hierarchical supra-nanostructure evolution.

Taking all of the results together, the growth mechanism of the Au–Ag bimetallic hierarchical supra-nanostructure from the metal cholate complexes inspired by gallstone formation is suggested as described in a schematic illustration (Figure 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 preformed 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. On the basis of 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. A further discussion for the role of each component (HAuCl4·3H2O, AgCl nanocrystals, CA molecules, and AA) is found in Supporting Information Notes 3–6 and Figures S11–S15.

3.6. Pt–Ag and Pd–Ag Bimetallic Supra-nanostructure Synthesis.

Because 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 counteranion is Cl–. Indeed, Figure 4 shows that Pt–Ag and Pd–Ag bimetallic supra-nanostructures are synthesized using H2PtCl6 and Na2PdCl4 as precursors, respectively. We had to optimize the reaction conditions because the reduction speeds of these metals are different from that of Au. Nevertheless, hierarchical Pt–Ag and Pd–Ag supra-nanostructures resembling the Au–Ag structure in both shape and size were successfully formed. X-ray diffraction (XRD) data and elemental mapping images showed each Pt–Ag and Pd–Ag bimetallic nanocrystal, and XPS data confirmed the presence of each element in the bimetallic supra-nanostructures (Figures 4 and S16 and S17). Our synthetic protocol with the suggested mechanism should be promising for the synthesis of 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

Figure 4.

Figure 4.

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TEM images, XRD pattern, and elemental mapping images of (a–c) Pt–Ag and (d–f) Pd–Ag supra-nanostructures synthesized by our one-pot synthesis using the gallstone-formation-inspired metal–cholic acid complexes.

3.7. Image-Guided Nanomedicine Applications of Bimetallic Supra-nanostructures.

Our developed bimetallic supra-nanostructures synthesized by the metal cholate nanocomplexes have promising potential, especially in the field of nanomedicine. The high surface areas of the supra-nanostructures allow a higher number of surface atoms compared to conventional spherical metallic nanoparticles in the same volume; this feature greatly contributed to the X-ray CT imaging contrast effect and radiosensitizing 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 that of the commercially available iodine contrast (Lipiodol; 21 HU/mg/mL) or spherical AuNP (27.4 HU/mg/mL) (Figure 5a). High-atomic-number materials also have been shown to enhance the photoelectric and Compton effects (subsequent emission of secondary electrons) that generate reactive oxygen species (ROS) to significantly increase radiation-induced DNA damage.41 When the supra-nanostructures were exposed to a single fraction of radiation doses (1–8 Gy), the radiosensitizing ROS generation was significantly enhanced compared with only radiation or conventional spherical Au nanoparticles (Figure S19).41,42 The cytotoxic ROS43 induced by the supra-nanostructures directly increased the cancer cell apoptosis rate (Figure 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 (Figure 5c and Supporting Information Note 7). The significantly high DEF value of supra-nanostructures predicts increasing tumoricidal effects, while decreasing current required excessive radiation doses in radiation therapies. The strong CT contrast and radiosensitizing effects of the supra-nanostructures 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 supra-nanostructures was demonstrated in vivo in a human prostate cancer (PC3) xenograft mouse model. First, reconstructed CT body images allowed the identification and coordination of the tumor region (region of interest) 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). Intratumoral uptake and distribution of the infused supra-nanostructures depicted with significant contrast enhancement [contrast to noise ratio = 970 HU;Figure S21]. At the same time, the supra-nanostructures locally distributed in the tumor significantly enhanced the in vivo cancer cell killing effect in a 10 Gy single fraction of image-guided local radiation therapy (Figures 5d and S22). The strong CT contrast and radiosensitizing effects of the supra-nanostructures are highly promising for image-guided therapies such as image-guided proton, brachy, external, or internal radiotherapies or combinational multikinase-targeting chemo- or immunotherapies to generate the synergetic impact for the treatment of cancers.

Figure 5.

Figure 5.

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(a) Linear relationship between the 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 (first row), spherical NP (second row), and lipiodol (third 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 of incubation. FACS analysis using FITC Annexin-V and propidium iodide staining. (c) Radiation-dose-dependent surviving fraction of cells treated with supra-nanostructures (red) and spherical NP (blue) and not treated (black) (Supporting Information Note 7 and Figure S20). (d) Comparison of the apoptotic area (%) of tumors from each animal groups [radiation + supra-NS, radiation only, supra-NS only, and nontreated control; human prostate cancer (PC-3) xenograft mice model, radiation: single dose of 10 Gy]. (each group n = 6; *P < 0.02). (inset) Representative images of the TUNEL-stained tumor tissues in each group (scale bar: 1 mm).

4. CONCLUSION

In summary, we synthesized bimetallic hierarchical supra-nanostructures using gallstone-formation-inspired metal cholate 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 a cholate solution, forming metal cholate nanocomplexes. Thereby the nanocomplexes concentrate the Au ions and AgCl nanoparticles inside. The 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 PtII and PdII and synthesized Pt–Ag and Pd–Ag bimetallic supra-nanostructures with hierarchies similar to those of the Au–Ag supra-nanostructures. Finally, we demonstrated significantly enhanced performance of the supra-nanostructures both as medical CT imaging contrast and radiocatalytic 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.

Supplementary Material

Supporting Information

ACKNOWLEDGMENTS

We acknowledge D. Lee for fruitful discussions. This work was supported by Grants R21CA173491, R21CA185274, and R21EB017986 from the NCI and NIBIB and funded by the Chicago Biomedical Consortium with support from the Searle Funds at the Chicago Community Trust. This work used resources of the APS, 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 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 (Grant DMR-0959470). The IR Work at ANL was supported by the U.S. DOE BES under contract DE-AC02-06CH11357 and DE-FG02-03-ER15457. H.K. acknolwedges the financial support from the R&D Convergence Program of MSIP (Ministry of Science, ICT and Future Planning) and NST (National Research Council of Science & Technology) of Republic of Korea (CRC-14-1-KRICT).

Footnotes

Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsanm.8b00908.

Supplementary figures, tables, notes, and methods (PDF)

The authors declare no competing financial interest.

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Supporting Information
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