Gallstone-Formation-Inspired Bimetallic Supra-nanostructures for Computed-Tomography-Image-Guided Radiation Therapy

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: CT imaging; bimetallic; cholate; gallstone; nanocomplexes; nanoparticles; radiosensitizing; radiotherapy.

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 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 (R_g) 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.

Figure 2.

(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.

Figure 3.

(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.

Figure 4.

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.

Figure 5.

(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).

Scheme 1.

Fractal/Hierarchical Gallstone Formation from Metal–Bile Acid Complexes