A Green Method to Synthesize Size-Controllable Gold Nanostars for Photothermal Therapy and Photoacoustic Imaging ^†

Abstract

Photothermal therapy (PTT) is a novel therapy for cancer treatment which is based on the conversion of photon energy into heat (>43 °C), and photoacoustic imaging (PAI) is a new bioimaging method for diagnosing and monitoring cancer. To enhance the impact depth of PTT and the signal of PAI, near-infrared (NIR)-absorbing photothermal agents are usually used. The development of novel NIR-absorbing photothermal agents with excellent properties, such as a high stability under long-term irradiation, strong absorption in the NIR range, and excellent biocompatibility, is needed in modern biomedicine. Previous research has proved that gold nanostars (AuNSs) have promising potential applications in photo-based therapies owing to their strong absorption in the NIR range and strong photothermal effects. However, the reported methods to synthesize AuNSs are complicated and toxic, which can limit its practical application. In this work, we proposed a new environmental strategy to synthesize AuNSs by using chitosan and vitamin C. Chitosan plays multiple roles, acting as stabilizing, shape-directing, and size-controllable agents in this method for the first time. The obtained AuNSs show strong NIR absorption and biocompatibility toward non-cancerous and cancerous cell lines. The in vitro tests proved the high efficiency of the obtained AuNSs in both PTT and PAI.

1. Introduction

Photoacoustic imaging (PAI) [1,2] and photothermal therapy (PTT) have been the focus of many research groups recently [3,4]. In most cases, the external agents have been utilized to enhance the performances of PTT and PAI. AuNPs are the most frequently used photothermal agents owing to their unique surface plasmon resonance characteristics [5]. AuNSs are a potential candidate for PTT, owing to their high absorption in the NIR region (700 to 1870 nm) [6]. Additionally, AuNSs can work as photoacoustic agents for PAI [7]. To overcome the disadvantages of traditional methods (i.e., complicated [8,9] and toxicity [8]) to synthesize AuNSs, we proposed an environment method for the synthesis of AuNSs by using non-toxic materials. We used vitamin C for reducing the Au(III) to Au(0) and chitosan (CS) for stabilizing the AuNSs. The size of the resulting nanoparticles ranged from 111 to 250 nm. The in vitro test evidenced that AuNSs are promising agents for both PTT and PAI.

2. Materials and Methods

2.1. Materials

CS (50 to 190 kDa, 75–85% deacetylation), gold chloride (HAuCl₄), hydrochloric acid (HCl, 37%), L-ascorbic acid (vitamin C), dimethyl sulfoxide (DMSO), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were bought from Sigma Aldrich (St. Louis, MO, USA). The cell culture materials including Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), antibiotics, trypsin, and phosphate-buffered saline (PBS) were bought from HyClone (South Logan, UT, USA).

2.2. Synthesis of AuNS

To obtain 0.01 M HAuCl₄ soluti on, 157.5 mg HAuCl₄·3H₂O was added to 25 mL DW. The difference of CS was added to distilled water to obtain a range of concentrations of CS. Then, 1 mL of 0.01 M HAuCl₄ was added to the CS–vitamin C solution. AuNS mass was determined by centrifugation and washing with distilled water.

2.3. Characterization

The shape of AuNSs was visualized under a field-emission transmission electron microscope (FETEM, JEOL JEM-2010 Microscope, Tokyo, Japan). The UV–Vis absorption of the AuNS solution was recorded by using UV–Vis spectroscopy (Thermo Biomate 5 Spectrophotometer). The size distribution of samples was studied by an electrophoretic light-scattering spectrophotometer (ELS-8000, OTSUKA Electronics Co., Ltd., Osaka, Japan). An 808 nm NIR laser with continuous-wave (CW) pulse from Hi-Tech Optoelectronics Co. (Beijing, China) was used in all photothermal-related experiments.

2.4. In Vitro PTT

A 6-well plate was prepared with a density of 6 × 10⁵ cells/well. The four groups of the cells were the control (no treatment), NIR laser, AuNS, and AuNS + NIR laser groups. Then, 60 µg/mL of the AuNS solution was added to cells in groups III and IV and incubated further for 4 h. Photothermal treatment experiments were conducted on groups II and IV with NIR laser (1.5 W/cm², 5 min). The AO/PI staining was applied to visualize the live/dead cells after the photothermal experiment.

2.5. In Vitro PAI

The solutions of AuNSs with a range of concentrations (30, 15, 7.5, 3.75, 1.87, and 0.93 µg/mL) were loaded into the PTFE tube and the phantoms were placed in a degassed water chamber for PAI [10].

3. Results and Discussion

3.1. TEM and UV–Vis Absorption Spectra

The TEM images are shown in Figure 1. The AuNSs that were obtained from the experiments with 0.05% to 0.4% (m/v) CS had elongated and sharp tips; meanwhile, the AuNSs from 0.7% to 0.9% (m/v) CS had short and unsharp tips. The nanoparticle sizes of AuNSs ranged from 111 to 225 nm. As shown in Figure 2, the AuNS solution from the 0.05% to 0.4% (m/v) CS experiments had absorption peaks from 685 to 750 nm and strong broadband absorption in the NIR region. Meanwhile, the AuNSs from 0.7% to 0.9% (m/v) CS experiments had an absorption peak at 605 nm and weak absorption in the NIR region.

3.2. In Vitro PTT

Breast cancer cells, MDA-MB 231, which was chosen as the cancer cell model, were divided into four groups: control, NIR laser, AuNS, and AuNS + NIR laser. The trypan blue staining method was used to discriminate the dead and damaged cells. As shown in Figure 3, only the cells in the AuNS + NIR group were blue, which indicates the dead and damaged cells. An NIR laser or AuNSs alone did not cause damage to the cells.

3.3. In Vitro PAI

AuNSs were loaded into the PTFE tubes. The amplitude of the signal was increased when the AuNS concentration increased; meanwhile, the control tube with PBS did not show any PA signals (Figure 4). The PAI with AuNSs has potential for diagnosing and monitoring tumors.

4. Conclusions

In this paper, we have reported a novel and green strategy to prepare AuNSs; in this method, vitamin C was used as a green reducing agent and CS was used as a shape-directing agent. The AuNSs have sizes from 111 to 250 nm with peak absorptions in the NIR range. AuNSs plus the NIR laser effectively killed MDA-MB-231 cancer cells, proving their use in PTT. Furthermore, the strong PA signals of the AuNSs also confirmed the potential of AuNS-based PAI.