**1. Introduction**

Anisotropic gold nanoparticles are of interest in physics, chemistry, and optics, due to their unique chemical and physical properties [1–5]. In addition, they are biocompatible and less toxic [6–9]. Recently, multi-branched gold nanoparticles (GNS) attracted much attention because of their absorption in the NIR region of the electromagnetic spectrum [10]. These applications are based on the LSPR phenomenon, caused by excitation with electromagnetic radiation of the NPs [11]. The characteristics of the LSPR band, including the width, peak position, and intensity, are directly related to particles size, morphology, and properties of capping agents [12]. Depending on the nature of the GNS, the absorption spectrum is composed of a weak band at 520–550 nm and a broad band between 700–1100 nm [10]. The band at the shorter wavelengths is attributed to the transverse plasmon resonance, whereas the longitudinal component is at the longer wavelengths. Due to the shape of the anisotropic particles, there are contributions to the plasmon resonance spectrum from both the transverse and longitudinal directions, with the latter being

**Citation:** Huynh, P.T.; Nguyen, G.D.; Thi Le Tran, K.; Minh Ho, T.; Lam, V.Q.; Ngo, T.V.K. Rapid and Green Preparation of Multi-Branched Gold Nanoparticles Using Surfactant-Free, Combined Ultrasound-Assisted Method. *Processes* **2021**, *9*, 112. https://doi.org/10.3390/ pr9010112


Received: 5 November 2020 Accepted: 5 January 2021 Published: 7 January 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

predominant, as the core size decreased and the length of the arms increased. However, in circumstances where the core diameters of the particles are longer than the arms, the band broadening could be due to particle aggregation, where the neighbor core plasmon resonance interact. Highly branched gold nanoparticles with small cores are characterized by broad and red-shifted LSPR maximum, with very weak or absent bands between 520–550 nm.

Multi-branched gold nanoparticles or gold nanostars are usually synthesized by the seed-mediated method, in the presence of anionic or cationic surfactants [13,14]. They take the role of shape-directing and capping agents to drive structural growth and prevent the aggregation of gold nanoparticles. The presence of surfactants result in cellular toxicity and they are also difficult to remove before the particles are used [15,16]. Many reports of surfactants-free GNS in recent years [17,18] are mainly based on seed-mediated protocols. The use of sodium borohydride, hydrazine, or trisodium citrate in seed-mediated synthesis of GNS, results in cytotoxicity and they are harmful to the environment.

Recently, green chemistry has become a popular trend in a variety of fields, as it offers a number of advantages, including safety, energy efficiency, and the production of less toxic waste [19–21]. In green synthesis of nanoparticles, people evaluate and select new nontoxic reductants, protecting agents, innocuous solvents from nature (leaf extract, microorganism, or nature polymer) to replace toxic materials [22,23], and develop advanced and energy-efficient techniques such as microwave and sonochemical methods [24,25].

Sonochemical effects are caused by acoustic cavitation in liquids with the creation and collapse of bubbles. The collapse of the cavitation bubbles is more rapid than thermal transport and generates "localized hot spots", which have a temperature of 5000 K, pressures of about 2000 atm and cooling rates of more than 109 K/s [26]. The advantages of using the sonochemical approach include the production of high purity, uniform shape, high yields, and cost effective synthesis of nanoparticles.

Several one-pot synthesis techniques of multi-branched gold nanoparticles were studied [27–29]. The advantages of these procedures are facile and free-surfactant synthesis but just short-branches gold nanostars were formed (less than 10 nm branches). Sonochemical synthesis is a popular method for spherical nanoparticles [30]. However, it is rarely studied using ultrasound assist to synthesize gold nanostars, except reports of Badilescu et al. [31]. In this work, we introduce a rapid and green preparation of multi-branched gold nanoparticles using surfactant-free and seedless combined ultrasound (US) assisted protocol. Chitosan (CS) was used as a stabilizer while hydroquinone (HQ) was used as the reducing agent. CS is a polysaccharide derived from shrimps, crabs, and other crustaceans. Due to its many advantageous properties such as biocompatibility, nontoxicity, low-cost, biodegradability, and antimicrobial agent, CS has a number of commercial and biomedical uses. Additionally, HQ has a variety of uses as a reducing agent that is soluble in water. Furthermore, HA was used to replace a traditional reducing agent, ascorbic acid, which could tune the adsorption of anisotropic gold nanoparticles towards the far NIR region [32]. Additionally, sonochemical or ultrasound assistance was applied to control the size and shape, and enhance the stability of GNS.

#### **2. Materials and Methods**

#### *2.1. Materials*

Chloroauric acid (HAuCl4xH2O, ~52% Au basis), sodium hydroxide 97%; chitosan (low molecular weight), hydroquinone 99%, phosphate buffer saline (tablet, pH 7.2–7.6), acid acetic 99% were purchased from Sigma-Aldrich, St. Louis, Missouri, US. The materials for cytotoxicity include Dulbecco's Modified Eagle Medium (DMEM), Fetal Bovine Serum (FBS), Bovine Calf Serum (BCS) were supplied by Sigma-Aldrich, Darmstadt Germany. Thiazolyl blue tetrazolium bromide (MTT), and antibiotic solution (penicillin-streptomycin) were supplied by Sigma-Aldrich, St. Louis, Missouri, US. Lysis buffer solution was purchased from Biobasic, Markham, Ontario, Canada. Normal mouse fibroblast (NIH/3T3, CRL-1658) and normal human fibroblast (BJ-5ta, CRL-4001) were ordered from ATCC, Manassas, Virgina, US. Deionized (DI) water 17.8 MΩ was used throughout the experiments.

#### *2.2. Methods*

2.2.1. Surfactant-Free Preparation of Multi-Branched Gold Nanoparticles (GNS) by the One-Step Method

First, 2 g chitosan (CS) powder was homogenized into a 98 g acid acetic 1% solution to make CS 2% solution. Next, 1 mL of HAuCl4 <sup>2</sup> × <sup>10</sup>−<sup>2</sup> M solution was added to 9 mL chitosan solution, under stirring. This mixture was pH-adjusted by acetic acid. Finally, hydroquinone (HQ) 0.1 M was mixed immediately into this mixture. The reaction solution was kept constant for 30 min at room temperature. Throughout the period of reaction, the color of the solution changed from colorless to cobalt blue, indicating that multi-branched particles had formed. The influences of conditions including pH, mass concentration of CS, hydroquinone concentration, and morphology of GNS were studied.

#### 2.2.2. Surfactant-Free Preparation of GNS Combined Ultrasound

The procedure of preparation for the combined ultrasound (US) was similar to the process above, except that the reaction solution was sonicated instead of being kept at room temperature without stirring. Sonication was carried out using the Q2000 sonicator— Qsonica, Newtown, Connecticut, US (power 1.375 watts, frequency 20 kHz). Chitosancoated GNS was synthesized at a constant frequency of 20 kHz and six different levels of amplitude (0, 20, 40, 60, 80, and 100 μm) and six time-periods (0, 2, 4, 6, 8, and 10 min), in order to investigate the effect of amplitude and sonication time on size, shape, and stability of GNS.

#### 2.2.3. Characterization

UV–Vis spectrophotometer Dynamica Halo RB-10 (Dynamica, Livingston, UK) was used to record the surface plasmon resonance (SPR) of GNS in the wavelength range of 400–1100 nm, at a scanning rate of 200 nm/min. The interaction between GNS and CS was shown by FT-IR analysis (Bruker Tensor 27, Bruker Optics, Ettlingen, Germany). All FT-IR results were obtained from the powder samples and smoothing or correction baseline was not applied. The crystal structure of GNS was determined by employing X-ray diffraction (XRD). Scanning was carried out in the 2 theta range of 20–100◦, using the X-ray diffractometer Bruker D5005 (Bruker AXS, Karlsruhe, Germany). Transmission electron microscope (TEM) analysis was examined by JEM1010-JEOL (Jeol, Tokyo, Japan). The J-Image software (NIH Image) was used to calculate the average length of branches as well as diameter of cores of GNS, based on thirty particles of each three sample from the TEM images. All analyses including UV-Vis, FT-IR, XRD, and TEM, as well as the cytotoxicity test below were carried out through separation from the same sample. The GNS solutions were sonicated before examinations and measurements.

#### 2.2.4. Cytotoxicity of Chitosan-Capped GNS

NIH/3T3 were cultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 1% penicillin–streptomycin (Pen–Strep) and 10% bovine calf serum (BCS), while the BJ-5ta cells were in DMEM with 1% Pen–Strep and 10% FBS (Fetal Bovine Serum. Both cell lines were grown in a humidified incubator with 5% CO2. The cell lines were detached from the culture flasks using a trypsin (0.25%)—EDTA (0.53 mM) solution. Cell viability was evaluated using thiazolyl blue tetrazolium bromide (MTT). Both cell lines were seeded onto 96 well plates at 10<sup>4</sup> cells/well. CS-coated GNS was added to each well so that the final concentration samples were 50, 75, 100 and 125 μg/mL. The negative control subject was Lysis buffer. Cells were incubated for 24 h in an incubator (37 ◦C and 5% CO2). Then, MTT solution (0.5 mg/mL) was added to each well. The plates were incubated at 37 ◦C for 4 h to form MTT formazan. Lysis buffer (4 mM HCl) was added to each well to dissolve the crystallized MTT formazan.

The plate absorbance (OD) was read at wavelength λ = 570 nm, using a Microreader. Cell viability could be calculated and compared to the control samples, as follows:

$$\% \text{ cell viability} = \frac{OD \text{ of sample}}{OD \text{ of negative control}} \times 100\% \tag{1}$$

#### **3. Results and Discussion**

*3.1. One-Step Surfactant-Free Preparation of GNS*

3.1.1. Effect of Mass Concentration of CS

Figure 1 shows that the UV–Vis result of GNS prepared in different mass concentration of CS. According to previous reports, the absorption spectrum was a plasmon resonance peak (SPR) ranging from 500 to over 1100 nm, indicating multi-branched particle presence. The SPR absorbance of GNS shifted, depending on the size and shape [33]. An increase in length of branches led to SPR shift toward the NIR region [34], while SPR absorbance moved to blue shift, due to the short branched formations [35]. On the other hand, broadening and increasing the maximum absorbance wavelength of SPR toward the NIR region suggested a form of aggregation, due to the interaction of the spherical core of the particles with each other [36]. It was clear that the intensity of SPR increased when the mass concentration of CS was increased from the beginning, evaluating the concentration to 1%. However, the SPR absorbance moved to the blue shift and the intensity of SPR decreased when mass concentration increased to 2%. This could be related to an increase in the size of the core particles. Table 1 describes the influence of % CS to SPR and intensity of SPR of GNS. At the beginning, 0.25% CS both of the SPR and the intensity of SPR rose from 845 nm and 0.15, to 875 nm and 0.18, respectively. With the increasing % CS, although the SPR increased slightly to 878, the intensity of SPR reached the highest value at 0.33. However, both SPR dropped to 667 nm and 877 nm, when % CS increased to 2. TEM images of GNS are shown in Figure 2. The prepared GNS at 1% CS (Figure 2a) had an average core of 57.33 ± 5.91 nm, and long, sharp branches with an average length of 44.32 ± 9.27 nm. Meanwhile at 1.5% CS, the average length of the branches decreased to 20.71 ± 8.57 nm (Table 2).

**Figure 1.** Absorption spectra of GNS prepared in various mass concentrations of CS: (**a**) 0.25%, (**b**) 0.50%, (**c**) 1.0%, (**d**) 1.5% and (**e**) 2.0%.


**Table 1.** The influence of mass concentration of chitosan to surface plasmon resonance and intensity absorbance.

**Figure 2.** TEM images of GNS prepared in 1.0% (**a**) and 1.5% CS (**b**).

**Table 2.** The influence of mass concentration of chitosan on the morphology, average core and branches of GNS.

