Advanced microscopy of star-shaped gold nanoparticles and their adsorption-uptake by macrophages

Abstract

Metallic nanoparticles have diverse applications in biomedicine, as diagnostics, image contrast agents, nanosensors and drug delivery systems. Anisotropic metallic nanoparticles possess potential applications in cell imaging and therapy + diagnostics (theranostics), but controlled synthesis and growth of these anisotropic or branched nanostructures has been challenging and usually require use of high concentrations of surfactants. Star-shaped gold nanoparticles were synthesized in high yield through a seed mediated route using HEPES as a precise shape-directing capping agent. Characterization was performed using advanced electron microscopy techniques including atomic resolution TEM, obtaining a detailed characterization of nanostructure and atomic arrangement. Spectroscopy techniques showed that the particles have narrow size distribution, monodispersity and high colloidal stability, with absorbance into NIR region and high efficiency for SERS applications. Gold nanostars showed to be biocompatible and efficiently adsorbed and internalized by macrophages, as revealed by advanced FE-SEM and backscattered electron imaging techniques of complete unstained uncoated cells. Additionally, low voltage STEM and X-ray microanalysis revealed the ultra-structural location and confirmed stability of nanoparticles after endocytosis with high spatial resolution.

Figure 1. FE-SEM imaging of gold nanostars

(A) SE Imaging at low magnification to observe monodispersity and size distribution of particles. (B) SE Imaging at high magnification revealing details of morphology and structuration of god nanostars. Sample mounted on ultra-flat silicon wafer was tilted to −32 degrees to get a detailed characterization of nanoparticle morphology.

Figure 2. Characterization of optical properties

(A) UV-Vis spectroscopy of gold seeds (red line, λ_max=518 nm) and gold nanostars (blue line, λ_max=612 nm). (B) Photon correlation spectroscopy (Dynamic Light Scattering) showing size distribution by intensity (%) of Au seeds and gold nanostars, inserted image shows vials containing nanoparticles (red: gold seeds, blue: gold nanostars).

Figure 3. Characterization of gold nanostars by a atomic resolution TEM

(A) BF-TEM. (B) Detail of atomic structure of a gold nanostar branch. (C) Interplanar distance. (D) FFT patterns of area selected at (C). (E) Detail showing direction of growing on branches and presence or organic coating.

Figure 4. Cell viability assay

Trypan blue exclusion of macrophages after 3 h of incubation (black line), and 24 h of incubation (gray line). Cell culture media was dosed with concentrated gold nanoparticles to achieve the concentrations indicated, percentage of viable cells were obtained from triplicates and normalized to control (without nanoparticles).

Figure 5. Ultra-high resolution FE-SEM imaging of complete macrophages

(A) SE Imaging of complete control cell. (B) BSE Imaging of complete control cell. (C) SE imaging of macrophage treated with moderate concentration of GNS (175 ug/ml) after 3h. (D) BSE imaging of macrophage treated with moderate concentration of GNS (175 ug/ml) after 3h. (E) SE imaging of macrophage treated high concentration of GNS (350 ug/ml) after 3h. (F) BSE imaging of macrophage treated high concentration of GNS (350 ug/ml) after 3h.

Figure 6. FE-SEM Imaging of cells treated with gold nanostars

(A) SE Imaging. (B) BSE Imaging (LABE detector). (C) BSE Imaging (YAG detector). (D) EDX mapping of Au-LA.

Figure 7. Ultrastructural location of GNS

(A) DF-STEM Imaging. (B) BF-STEM Imaging of area selected in (A). (C) X-Ray microanalysis. (D) EDX mapping of Au-LA.