**3. Results and Discussion**

In general, colloidal synthesis of Au nanostructures requires reductants and capping agents. In the current procedure, the Au nanostructures were readily synthesized by reacting HAuCl4 and EDTA tetrasodium salt in an aqueous solution at room temperature. The EDTA tetrasodium salt played a dual role as a reducing agen<sup>t</sup> and as a ligand, thus allowing for simple synthesis without any additives. After the reaction, the products were washed with water to remove any byproducts and unreacted reagents. More details are provided in the experimental section.

Figure 1a shows a representative TEM image of the Au nanostructures prepared using a 1:8 molar ratio of HAuCl4 to EDTA tetrasodium salt. The image presents the formation of spherical Au nanocrystals with an average diameter of 11 nm. A HR-TEM image is shown in the inset of Figure 1a, indicating the polycrystallinity of the Au nanocrystals with pronounced lattice fringes of 0.235 nm, corresponding to the (111) planes of a face-centered-cubic (fcc) Au crystal structure. Decreasing the molar ratio of HAuCl4 to EDTA tetrasodium salt induced shape control, resulting in branched forms (see Figure 1b) at 1:6 and nanowire networks (see Figure 1c) at 1:4. When the molar ratio was below 1:3, severely aggregated Au particles were observed because of the insufficient capping agent. Based on these observations, the formation of the Au nanostructures is summarized in Scheme 1, illustrating that the morphology of the Au nanostructures is easily controlled by adjusting the EDTA tetrasodium salt concentration. High concentrations of EDTA tetrasodium salt produced only spherical Au nanocrystals. Decreasing the molar ratio of HAuCl4 to EDTA tetrasodium salt resulted in the formation of non-spherical Au nanostructures, such as branched forms and nanowire networks.

**Figure 1.** Transmission electron microscopy (TEM) images of Au nanostructures prepared using different molar ratios of HAuCl4 to ethylenediaminetetraacetic acid (EDTA) tetrasodium salt: (**a**) 1:8, the inset is the high-resolution TEM (HR-TEM) image of panel (**a**); (**b**) 1:6; (**c**) 1:4; (**d**) Absorption spectra of Au nanostructures in panels (**<sup>a</sup>**–**<sup>c</sup>**).

**Scheme 1.** Schematic illustration of morphology control by adjusting the molar ratio of HAuCl4 to EDTA tetrasodium salt.

The absorption spectra of Au nanostructures, with three kinds of morphologies, are presented in Figure 1d, clearly displaying the shape-dependent surface plasmon resonance behaviors. Spherical Au nanocrystals (see red curve (a) in Figure 1d) exhibit one absorption peak at 570 nm, resulting from the transverse mode, while the transverse band becomes broader in the branched Au nanostructures (see purple curve (b) in Figure 1d). The most intriguing activity was observed when using a 1:4 molar ratio of HAuCl4 to EDTA tetrasodium salt (see black curve (c) in Figure 1d). Two distinct optical behaviors were identified in the absorption spectrum of the Au nanowire networks. The peak at 530

nm is attributed to transverse surface plasmon resonance (TSPR), and a broad absorption band over the NIR spectra regime was observed, owing to the longitudinal surface plasmon resonance (LSPR).

The Au nanowire networks showing absorption in the NIR regime were further investigated, as described below. The low-magnification TEM image of the Au nanowire networks in Figure 2a reveals that the wires have considerable lengths of a few micrometers. HR-TEM images of the Au nanowire networks are displayed in Figure 2b,c, indicating the dominant (111) lattice planes, in agreemen<sup>t</sup> with the fast Fourier transform (FFT) image shown in the inset. Moreover, these images provide insight into the crystallinity of the Au nanowire networks and their growth characteristics, acquired by the coalescence of Au particles and subsequent deposition of Au atoms, as suggested in previous studies [21,22,74].

The XRD pattern of the Au nanowire networks is presented in Figure 2d, showing that all characteristic peaks are indexed to the (111), (200), (220), (311) and (222) planes of the fcc Au crystal structure without any other phases, which also supports the TEM analysis.

When the solution containing the isolated Au nanowire networks was kept at room temperature for approximately one day, the color gradually changed from black to deep red, implying the transformation of the Au nanowire networks into spherical Au nanoparticles. The shape instability of nonspherical Au nanostructures has also been observed in previous works [21,22,42,43] and explained by the thermodynamic instability of the nonspheres [42,43]. To prevent the transformation of the Au nanowire networks, a stabilizing agen<sup>t</sup> exhibiting stronger binding, the mPEG-SH ligand, was used. mPEG-SH stabilized the Au nanowire networks and remained intact without any color change over one month, as shown in Figure S1 in the Supplementary Materials. The absorption spectra and TEM image in Figure S1 also support that mPEG-SH coating stabilize Au nanowire networks without morphological change.

**Figure 2.** Characteristics of Au nanowire networks: (**<sup>a</sup>**,**b**) TEM images with different magnifications, the inset is the fast Fourier transform (FFT) image obtained from the panel (b); (**c**) HR-TEM images; (**d**) X-ray diffraction (XRD) pattern, red vertical lines indicate peak positions and relative intensities of bulk Au.

To monitor the temporal evolution of the Au nanowire networks during the reaction time of 1 h, aliquots withdrawn at different reaction stages were analyzed, as shown in Figure 3. As seen in the TEM images, the length and diameter of the wires gradually increased as the reaction time progressed, implying that the Au nanowire networks were formed by depositing the Au monomer at the joints between the assembled Au nanocrystals. The absorption spectra of aliquots taken at different reaction stages are also shown in Figure S2, indicating that only one broad feature was seen at the beginning stages (e.g., 15 min). After 25 min, two distinct peaks were clearly observed, due to the TSPR and LSPR modes. This evolution shows that Au nanowire networks formed in nearly 25 min, in good agreemen<sup>t</sup> with the TEM analysis (see Figure 3f).

**Figure 3.** TEM images of the samples at different stages of the reaction: (**a**) 2 min; (**b**) 5 min; (**c**) 10 min; (**d**) 15 min; (**e**) 20 min; (**f**) 25 min; (**g**) 45 min; (**h**) 60 min.

Several kinds of control experiments were performed to understand the effects of synthetic conditions. Without the addition of the EDTA tetrasodium salt, no Au nanostructures were formed. This supports the fact that the four carboxylic groups of the EDTA tetrasodium salt reduce the Au3+ species, which is in agreemen<sup>t</sup> with previous works [71–73]. It is noteworthy that EDTA tetrasodium salt introduced strong basic conditions, thus eliminating the need for additional steps (see Table S1 in the Supplementary Materials), whereas adjusting the pH by adding a suitable base was necessary for the preparation of the Au nanostructures in previous studies [72,75,76].

In addition, the addition of other EDTA salts was attempted for the synthesis of Au nanostructures. Under similar conditions such as temperature and concentration, the use of EDTA and EDTA disodium salt led to the formation of micrometer-sized and submicrometer-sized Au aggregates, respectively, as shown in Figure S3, revealing that the four deprotonated carboxyl groups (COO−) of EDTA tetrasodium salt as a tetradentate group are essential for allowing effective growth on the nanoscale.

NIR absorption is crucial for many biomedical applications because biological tissues, blood, and water show low absorption in this wavelength range [77,78]. The cytotoxicity of the Au nanowire networks coated with mPEG-SH was evaluated to demonstrate their applicability as photothermal therapeutic agents. The MTT cell proliferation assay is presented in Figure S4, indicating that more than 80% of the U87MG cells survived up to 1.27 mM (250 μg/mL) of the Au concentration. A more detailed procedure is presented in the Experimental Section.

Figure 4 represents the images of the U87MG cells without and with the Au nanowire networks after the NIR laser irradiation at 980 nm for 10 min, clearly revealing cell damage at the center of the laser beam for cells with an incubation of the Au nanowire networks, while the control cells remained intact. The dead cells were stained blue by 0.4% trypan blue. This observation clearly demonstrates the effectiveness of the Au nanowire networks in NIR photothermal therapy by selective killing of cancer cells, because of the local heating generated by their effective NIR absorption. Because of their relatively large size and irregular shape, the Au nanowire networks may not be suitable for animal study by systemic delivery. However, due to the good photothermal effect by NIR light, they can be

used as a local heat generator to regulate cellular activity by attaching them to the cell membrane or biochip surface [79].

**Figure 4.** Optical microscope images of cancerous glioblastoma cells (U87MG) cells incubated (**a**) without; (**b**) with the Au nanowire networks, after irradiation for 10 min with a 980-nm near-infrared (NIR) CW diode laser and subsequent staining with 0.4% trypan blue.
