Gold chloride trihydrate (HAuCl4·3H2O), Hexadecyl trimethyl ammonium bromide (CTAB), Nitric acid (GR,65-68%) and Hydrogen peroxide solution (GR,30%) were received from Shanghai Aladdin biological technology Co. Ltd. Sodium borohydride (NaBH4) and silver nitrate (AgNO3) were received from Shanghai Lingfeng Chemical Reagent Co. Ltd. Hydrochloric acid (HCl) were received from Dongguan Dongjiang Chemical Reagent Co. Ltd. Hydroquinone were received from Energy Chemical. Bovine Serum Albumin were obtained from Sigma-Aldrich. Sodium hydroxide were received from Greagent.
RPMI 1640 Medium and Penicillin-Streptomycin were purchased from HyClone. Phosphate buffered solution (PBS) were purchased from Corning. Pancreatin were purchased from Coolaber. Foetal bovine serum (FBS) were purchased from Gibco. 4T1 cells were provided by Research Center for Biomedical Optics and Molecular Imaging in the Shenzhen Institute of Advanced Technology, Chinese Academy of Science. Dojindo Chemical Technology (Shanghai) Co., Ltd supplied Cell Counting Kit-8 for Cell proliferation and toxicity test. The Millipore ultrapure water was used throughout the experiment.
Preparation of [email protected] nanoparticles
The synthesis of gold nanorods is conducted as follows: 0.4 mL of HAuCl4(aq) (10 mM) and 10 mL of CTAB(aq) (0.1 M) were added to 23-33 µL of AgNO3(aq) (100 mM). Then, 10-30 µL of HCl (1.2 M) and 525 µL of aqueous hydroquinone (0.1 M) were added to the growth solution under gentle mixing. The color of the growth solution turned from orange to a very light yellow color. After 15 min of stirring, 10-40 µL of freshly prepared ice-cold NaBH4(aq) (10 mM) solution was injected into the growth solution. The mixture was stirred for 30 s and aged for 18 h at room temperature. The [email protected] was then washed by PBS twice.
Preparation of [email protected]
First, we add a certain amount of CTAB to adjust its concentration in [email protected] solution to 1 mM, and then ultrasound until CTAB is completely dissolved. 3 mL [email protected] is slowly added to 3 mL BSA solution bovine serum albumin (10 mg/mL), and the mixed solution is sonicated for 30 min. After centrifugation at 9,500 x r for 40 min, the supernatant was replaced with 6 mL BSA solution (5 mg/mL), and then the pH value was adjusted to 11-12 with sodium hydroxide (2 M), stirred for at least 18 h. After that, the synthesized [email protected] was centrifuged at 9,500 x r for 40 min, then washed twice with PBS, and dissolved in PBS for further use.
Characterizations of [email protected] and [email protected] Nanoparticles
The morphology analysis of gold nanorods was obtained by Beijing Zhongke Baice Co., Ltd. through Talos F200X electron microscope to obtain transmission electron microscope (TEM) images. Zetasizer Nano ZS (Malvern, UK) was used to study the size distribution and zeta potential of various nanoparticles by dynamic light scattering (DLS). The UV-Vis absorption spectrum was determined by UV-2700 Ultraviolet-Visible Spectrophotometer (SHIMADZU, Japan).
For the morphology characterization of [email protected] inside tumor, 100 µL of [email protected] (OD = 25) were injected into tumor sites with irradiation about 10 min, after that the treated tumors were collected. Non-treated tumor was collected as control. The collected tumors were incubated in 2.5% glutaraldehyde solution (Coolaber.co., Beijing, China) for transmission electron microscopy (Beijing Zhongke Baice Co., Ltd).
Measurement of the photothermal performance of [email protected] and [email protected]
The gold nanorod solution was diluted to different OD (0.5, 1, 1.5, and 2), and the PBS was used as a blank control. The gold nanorods (500 µL) were irradiated with a 1064 nm laser (Haoliangtech, Shanghai, China) at a power intensity of 0.35 to 1 W/cm2 for 30 min. The temperature was recorded with an infrared thermal imager (FLUKE TI25).
Photostability of [email protected]
To test the photostability, the absorption spectra of [email protected] were measured as a function of the irradiation time. The [email protected] (OD=1) were irradiated under NIR laser (1064 nm, 0.5 w/cm2). From 0 to 10 min, the spectrum was recorded every minute. The photothermal cycle test was also carried out as the [email protected] solution (0.5 mL) irradiated with every 10 minutes laser irradiations on and off (1064 nm, 0.5 W/cm2), and the temperature change was recorded.
Murine breast cancer cell line (4T1 cells) was cultured in RPMI 1640 containing 10% FBS and 100 U/mL penicillin or 100 µg/mL streptomycin. The culture environment is 37 °C, and the humidification condition is 5% CO2.
In vitro cytotoxicity assessment of gold nanoparticles
The CCK-8 assay was used to identify the cytotoxicity of gold nanorods. 4T1 cells were pre-seeded into 96-well plates (5×103 per well) and incubated for 24 h. Subsequently, 10 μL of various concentrations of [email protected] and [email protected] were added and incubated for another 24 h. After washing with PBS twice, 10 μl CCK-8 solution was added to each well and incubated for 40 min, followed by measuring the absorbance at 450 nm with a microplate reader.
For phototoxicity, 4T1 cells were pre-seeded into a 96-well plate (5×103 per well) and incubated for 24 h, then the cells were irradiated with NIR laser (1064 nm, 0.75 W/cm2, 10 min) and further incubated for 24 h. After that, 10 μL CCK-8 solution was added into each well and incubated for a further 40 min at 37 °C. Then, a microplate reader was used to detect the absorbance of each well at 450 nm.
Construction of tumor-bearing mouse model
All BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. All animal experiment procedures were carried out following the standard procedures approved by Shenzhen Institute of Advanced Technology Committee of the Chinese Academy of Sciences. The tumor model was established by subcutaneously injecting 4T1 cells (2×106) into the back of mice. Animal studies were conducted when the tumor volume reaches approximately 100 mm3.
In Vivo Blood Circulation and Biodistribution
For circulation time measurement, first, 200 μL of [email protected] was intravenously injected into the tail vein of BALB/c mice, and then 20 μL of blood was collected at 0.25, 2, 4, 6, 8, 12, 36, and 48 h, and diluted with 30 μL PBS to obtain 50 μL blood sample. About 400 μL concentrated HNO3 (chromatographic grade) was added, the lid was tighten, and digested at 90 °C for 2 h. After cooling to room temperature, 150 μL H2O2 (chromatographic grade) was slowly dropped in, and then heated to 90 °C for 1 h without cover. In the end the solution was diluted to 5 mL by ultrapure water. The concentration of Au ion was measured by ICG-OEC after passing through 0.44 mm nylon syringe filter.
For biodistribution measure, about 200 μL [email protected] was intravenously injected into the tail vein of BALB/c mice. After 24 h, the mouse was scarificed and the heart, liver, spleen, lung, and kidney were taken and dried in an oven at 80 °C. Before digestion, each organ was weighed, 800 μL concentrated HNO3 (GR) was added, and heated to 90 °C for 2 h. After cooling to room temperature, 200 μL H2O2 (GR) was slowly added dropwise, heated at 90 °C for 1 h, and then the solution was diluted to 10 mL by ultrapure water. Finally, concentration of Au ion was measured by ICG-OEC after passing through 0.44 mm nylon syringe filter.
Photothermal treatment efficiency
In order to evaluate the thermal therapeutic effect of [email protected], the tumor-bearing mice with 4T1 tumors were randomly divided into four groups with the tumor volume about 100 mm3: (1) [email protected], (2) [email protected] + Laser; (3) Only Laser (4) Blank control. An infrared thermal imager was used to record the infrared thermal image of the tumor site. The tumor volume and body weight of mice were recorded before and after treatment, respectively. The tumor volume can be calculated according to the normal equation (volume = width2 × length/2). Two weeks later, the mice were sacrificed, and the tumors were isolated.
SPSS 16.0 statistical software was used for data analysis. The measurement data were expressed as mean ± d, the comparison between groups was done by analysis of variance, and the comparison of count data was performed by chi-square test. P < 0.05 was considered statistically significant.