Materials
HAuCl4 and NaBH4 were purchased from Aladdin (Shanghai, China). Adenosine 5′-monophosphate monohydrate (5ʹ-AMP) was purchased from Sigma-Aldrich (USA). Phosphate buffered saline was purchased from Sangon Biotech (Shanghai, China). Dialysis bags were purchased from Shyuanye (Shanghai, China). High glucose medium was purchased from Hyclone (USA). Fetal bovine serum (FBS) was purchased from Epizyme (Shanghai, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Beyotime (Shanghai, China). The living and dead cell dyes were purchased from Solarbio (Beijing, China). The double antibody solution, collagen and trypsin were purchased from Gibco (USA). Paraformaldehyde was purchased from Biosharp (Hefei, China). BALB/Ca nude mice were purchased from Jihui Experimental Animal Breeding Co., Ltd. (Shanghai, China). All chemicals were used as received without any further purification.
Synthesis of AAu NPs
In a typical experiment, 1 mL HAuCl4 (40 mmol/L) and 1 mL 5′-AMP (0.08 mmol/mL) were added into 8 mL distilled water at room temperature. After stirring for 30 min, 0.5 mL NaBH4 (0.1 mmol/mL) was slowly added into the solution. The solution turned dark purple quickly and the product was obtained after continuous stirring for 30 min. The obtained solution was placed in a dialysis bag and dialyzed with distilled water or PBS for 3 days to obtain the AAu NPs suspension, which was stored at 4 ℃ for further characterization. The Au NPs were prepared as a control using the same procedure in the absence of AMP. Collagen-induced AAu NPs aggregates (CAAu) were obtained by adding 5 vol% 3 mg/mL collagen I colloid fluid to the obtained AAu NPs suspension.
Photothermal properties of AAu NPs
The AAu NPs suspensions with different concentrations (i.e., 8, 20, 40, 80 μg/mL) in the 96-well cell culture plate were irradiated by an 808 nm laser with a collimator (spot size 0.785 cm2) under different powers (0.5, 1, 1.5, 2, 2.5 W) for 10 min. The change of temperature was constantly recorded by an infrared camera. The temperature change of PBS was recorded as a control. Four laser on/off cycles were conducted to evaluate the photothermal stability of the AAu NPs (80 μg/mL, laser power 1.5 W). The photothermal curves of Au, AAu and CAAu samples at the same concentration of 80 μg/mL were measured upon irradiation by an 808 nm NIR laser (1.5 W) for 10 min.
In vitro photothermal cytotoxicity test
In vitro photothermal cytotoxicity tests were conducted using the MTT assay on mouse cutaneous melanoma (B16-F10) cell-line. Exponentially growing cells were seeded into 96-well plate (104 cells per well) and cultured overnight in 5% CO2 humidified incubator at 37 °C for cell attachment. After being rinsed with PBS buffer (pH 7.4), the cells were treated with 100 μL of medium containing different concentrations of AAu NPs (0, 5, 10, 20, 40 μg/mL) for 24 or 48 h at 37 °C for evaluation of the materials cytotoxicity. For in vitro photothermal effect evaluation, the cells treated with different concentrations of AAu NPs (i.e., 0, 5, 10, 20, 40 μg/mL) for 2 h were irradiated with an 808 nm laser (laser power: 1.5 W; spot size: 0.785 cm2) for 5 min. Different power densities (i.e., 0, 0.5, 1.5, 2.5 W) or irradiation time (i.e., 0, 0.5, 1, 2, 5, 10 min) were applied to the cells treated with 100 μL culture medium containing 20 μg/mL AAu NPs for dose-dependent photothermal effect evaluation. After laser irradiation, the cells were rinsed twice with PBS (pH 7.4) then the cell viability was measured using MTT assay by normalizing to control group without any treatment. To further evaluate the cell viability, the cells were stained with Calcein-AM/propidium iodide (PI) staining reagents immediately after laser irradiation and PBS cleaning. The stained cells were observed with fluorescence microscope at 494 nm (green, Calcein-AM) and 545 nm (red, PI). The results were all obtained from a representative experiment out of several biological replicates. Three parallel samples were conducted in each group.
In vivo antitumor experiment of Au–AMP
Female BALB/Ca mice, 5 weeks old, were used in the in vivo experiment. The animal experiments were approved by the Experimental Animal Ethics Committee of Shanghai Tenth People’s hospital and all animal operations were in accord with institutional animal use and care regulations. The primary tumor model was established by subcutaneous injection of B16-F10 melanoma cells (2 × 106) into the right axillary of nude mice. Once the tumor volume reached about 60 mm3, the tumor-bearing mice were randomly divided into 3 groups (n = 4), including (1) PBS; (2) AAu; and (3) AAu + Laser. Then, the three groups of mice were intratumorally injected with 500 μL of PBS or AAu NPs (80 μg/mL). After 10 min of intratumoral injection, the third group of mice were irradiated with an 808 nm laser (laser power: 1.5 W; spot size: 0.785 cm2) for 10 min every 3 days (the nanoparticles were injected every 3 days before laser irradiation). Tumor volume and body weight were measured every 2 days. Tumor volume was calculated by the following equation: tumor volume = (length * width2)/2. Ten days later, the mice were euthanized, and the tumors and the organs were collected for weighing, H&E staining, Ki67 staining and inductively coupled plasma-optical emission spectrometry (ICP) analysis.
Calculation of photothermal conversion efficiency
The photothermal conversion efficiency (η) of AAu NPs is calculated using the following formula:
$$\upeta =\frac{hA\Delta {T}_{max}-{Q}_{s}}{I(1-{10}^{-{A}_{\lambda }})},$$
(1)
where h is the heat transfer coefficient, A is the surface area of the container, ΔTmax is the temperature change at the maximum steady-state temperature, I is the laser power, Aλ is the absorbance of AAu NPs at 808 nm, QS is the heat related to the light absorption of the solvent, which is independently measured as 25.2 mW by Yanlan Liu et al. (2013b).
In order to get hA in the formula (1), a dimensionless parameter θ is introduced as follows:
$$\theta =\frac{\Delta T}{\Delta {T}_{max}}.$$
(2)
Therefore, hA can be determined by applying the linear time data of cooling cycle versus −lnθ, as shown in the following:
$$t\, = \,\frac{{\sum\limits_{i} {m_{i} } \,c_{{p,i}} }}{{hA}}\,\ln \theta$$
(3)
The photothermal conversion efficiency (η) of AAu can be calculated by substituting hA value into the formula (1).
Material characterization
The morphology of the AAu was observed using a transmission electron microscope (TEM, Hitachi H‑800, Japan). Confocal laser scanning microscopy (CLSM, FV1000, Olympus Corporation, Japan) was used to observe the self-assembly of AAu NPs within extracellular matrix of tumor cells. The dynamic light scattering (DLS) of AAu in ultrapure water was measured using a Particle Size Analyzer (Zetasizernano, Malvern, UK). UV–Vis–NIR absorption spectra were recorded on a UV–Vis–NIR spectrometer (UV-3600, Shimadzu, Japan). X-ray powder diffraction (XRD) patterns were measured with an X’Pert PRO MPD powder diffractometer. Infrared absorption spectra are measured using an infrared spectrometer (FTIR-7600, Lambda Scientific, Australia). Thermal images were recorded by a near-infrared thermal imaging camera (FLIRTM A325SC camera) after irradiation using an 808 nm near-infrared laser equipment (Shanghai Connect Fiber Optics Company, China).
Statistical analysis
All data were expressed as mean ± standard deviation (SD). SPSS statistical software version 26 (IBM, Armonk, NY) was used for one-way ANOVA to determine the multiple comparisons between groups. **P < 0.01 or *P < 0.05 are significant differences.
