4.1 Materials
Cobalt nitrate, potassium hexacyanoferrate(III) and sodium citrate were purchased from Sigma-Aldrich (USA). 4T1 cells were gained from American Type Culture Collection (USA). Dulbecco's Modified Eagle Media (DMEM), Fatal Bovine Serun (FBS), Phosphate Buffered Saline (PBS) and 4% paraformaldehyde were purchased from Gibco (USA). A ROS Assay Kit was purchased from Beyotime (China). A LIVE/DEADTM Cell Imaging Kit was obtained from Thermo Fisher Scientific (USA). A 24-well transwell plate was obtained from Corning (USA). Primary antibodies against MMP9, CDH-1 and GAPDH were obtained from ABclonal Technology (China). The secondary antibody was obtained from Abcam (USA).
4.2 Preparation of PBA
The synthesis of PBA was based on a precipitation method with slight modification.[41] The first step was the preparation of solution A and solution B: solution A was a mixture of 0.03 mol L-1 cobalt nitrate and 0.045 mol L-1 sodium citrate, while solution B was a 20 mL potassium hexacyanoferrate(III) (0.02 mol L-1) solution. Then, solutions A and B were mixed by stirring for 1 min, and the obtained mixture was aged at room temperature for 18 h. Finally, the final PBA was obtained after centrifugation with water and ethanol.
4.3 Characterization of PBA
Scanning electron microscopy (SEM) (S-4800 FESEM, Hitachi, Japan) was used to investigate the surface of PBA. More information about the morphology and internal structure of PBA was provided by transmission electron microscopy (TEM) (JEM 2100F, JEOL, USA) and energy-dispersive X-ray spectroscopy (EDS) was presented by a Thermol UltraDry EDS system attached to the TEM instrument. The crystal structure of PBA was characterized by X-ray diffractometry (XRD) (X’Pert Pro MPD, PANalytical, Netherlands). The absorption spectrum was recorded by a UV-vis spectrophotometer (UV-1800, Shimadzu, China). MRI was obtained by a 3.0 T MR imaging instrument. Photoacoustic tomography (PAT) was collected on a multispectral optoacoustic tomography scanner (MSOT, iThera Medical, Germany).
4.4 Photothermalperformance
To investigate the photothermal properties of PBA, different concentrations of PBA (0.1 0.2, 0.5, 1, 2 mg mL-1) were prepared and treated with 808 nm irradiation at various powers (0.1, 0.5, 1, 2 W cm-2) for 6 min. The real-time temperature of sample was measured by an FLIR thermal camera. A laser on/off experiment was used to estimate the photothermal stability of PBA. The whole process included five continuous cycles, each of which was under 808 nm irradiation at 2 W cm-2 for 600 s in the laser on condition, and then turned the laser off until the solution had cooled down to room temperature naturally. Furthermore, the photothermal conversion efficiency (η) of PBA was calculated.[12]
Tmax: maximum equilibrium temperature for PBA solution
Tmax,control: maximum equilibrium temperature for PBS
I: the incident laser power
A808: absorbance of the PBA at 808 nm
m: mass of products
C: heat capacity of solvent
t: a series of constant cooling time corresponding of PBA solution
T: constant cooling temperature of PBA solution
Tamb: the ambient temperature of the surroundings
4.5 Extracellular·OH generation experiment
Three milligram PBA was added into 3 mL water, and then 20 μg mL-1 methylene blue (MB) and 100 μM H2O2 solutions were added. The ·OH generation was measured through the absorbance at 665 nm.
PBA was mixed with 10 mM o-phenylenediamine (OPD) and 100 μM H2O2 solution. The ·OH generation was measured through the absorbance at 452 nm.
4.6 Release of ferrous ions
The 2,2’-bipyridine was used to monitor ferrous ions release of PBA. The mixed solution which contained 6 mM 2,2’-bipyridine and 1 mg mL-1 PBA was prepared and the absorbance was detected at 520 nm after 12 h.
4.7 Depletion of glutathione (GSH)
5,5 -dithiobis-( 2-nitrobenzoic acid, DTNB) was added to 1 mg mL-1 PBA solution which contained 0.01 M GSH. After 12 h, the absorbance was monitored at 412 nm.
4.8 In vitro photodynamic property
1,3-diphenylisobenzofuran (DPBF) was added to PBA solution under irradiation for 0, 5, 10, 20 min and the release of 1O2 was detected through DPBF contents at 420 nm absorbance.
4.9 In vitro photothermal property
First, 4T1 cells were seeded into 96-well plates overnight and divided into four groups: PBS, PBS+NIR, PBA and PBA+NIR groups. Then, the culture media were removed by rinsing with PBS three times, and the samples were treated as follows: the PBA and PBA+NIR groups were treated with culture medium which contained 1 mg mL-1 PBA, and the PBS and NIR groups were treated with the same dosage of culture medium contained PBS. After incubated for 4 h, the media were removed, and the samples were rinsed with PBS three times, and then the PBS+NIR and PBA+NIR groups were accepted 808 nm irradiation at 2 W cm-2 for 10 min. Last, all of these groups were dyed with the LIVE/DEADTM Cell Imaging Kit which the green fluorescence exhibited living cells, while dead cells were marked red.
4.10 Intracellular reactive oxygen species (ROS) generation
The pretreatment process with 4T1 cells was similar to the LIVE/DEADTM Cell Imaging Kit experiment. Then, the ROS Assay Kit was used to detect intracellular ROS generation. The DCFH-DA probe was diluted in FBS-free DMEM (10 μmol L-1) and replaced with culture medium. After culturing for another 20 min, all groups were washed with FBS-free DMEM three times, and then NIR groups were treated with 808 nm irradiation at 2 W cm-2 for 10 min. All groups were recorded by fluorescence microscopy.
4.11 Quantitative polymerase chain reaction (qPCR) experiment
After treated with different concentrations of PBA and 100 μM H2O2, the total RNA of 4T1 cells were extracted. Then, cDNA were reversed using Evo M-MLV RT Premix Kit (Accurate Biotechnology Co., Ltd) and SYBR Green Premix Pro Taq HS qPCR Kit (Accurate Biotechnology Co., Ltd) was used for qPCR. The specific primers in this study were deigned as: mouse GAPDH forward primer: 5’-ACCCTTAAGAGGGATGCTGC-3’; mouse GAPDH reverse primer: 5’-CCCAATACGGCCAAATCCGT-3’; mouse GPx4 forward primer: 5’-GCCTGGATAAGTACAGGGGT-3’; mouse GPx4 reverse primer: 5’-ACCACACTCAGCATATCGGG-3’; mouse Nrf2 forward primer: 5’-AGATGACCATGAGTCGCTTGC-3’; mouse Nrf2 reverse primer: 5’-GCCAAACTTGCTCCATGTCC-3’; mouse TfR1 forward primer: 5’-GTGATTGTTAGAGCAGGGGA-3’; mouse TfR1 reverse primer: CTGATGACTGAGATGGCGGA-3’;mouse FTH1 forward primer: 5’-GCCGAGAAACTGATGAAGCTGC-3’; mouse FTH1 reverse primer: 5’-GCACACTCCATTGCATTCAGCC-3’; mouse FTL forward primer: 5’-CACCTACCTCTCTCTGGGCT-3’; mouse FTL reverse primer: 5’-CGCGATCGTTCTGAAACTCG-3’. Parameter setting as: pre incubation at 95 oC for 30 s then amplification at 95 oC for 1 min, 30 oC for 55 s and 97 oC for 1s, cooling at 37 oC for 30 s. The process was continued for 40 cycles. The Ct values were corrected through GAPDH Ct value.
4.12 Tumor-bearing mouse model
BALB/c mice (4-5 weeks, female) were obtained from Guangdong Medical Laboratory Animal Center, and all animal studies followed the guidelines approved by the ethics committee of Guangdong Medical Laboratory Animal Center (B202009-1). All applicable institutional or national guidelines for the care and use of animals were followed. After adaptive feeding for one week, all mice were injected with 100 μL 3×106 4T1 cell suspension. All tumor interferences were carried out when the tumor size reached about 5*52 (length*width2) mm3.
4.13 Evaluation of the photothermal effect in vivo
When the tumor-bearing mouse models were successfully established, they were divided into PBS+NIR and PBA+NIR groups randomly, and 50 μL PBS or PBA (10 mg mL-1) was injected into the tumor site. After that, the mice were exposed under 808 nm irradiation at 1 W cm-2 for 10 min. The real-time temperature change and images of the whole mouse body were recorded on an FLIR thermal camera.
4.14 Animal experiment
The tumor-bearing BALB/c mice were divided into four groups: PBS, PBS+NIR, PBA and PBA+NIR groups. The PBS and PBS+NIR groups were injected with 50 μL PBS into the tumor site, while the PBA and PBA+NIR groups were injected with 10 mg mL-1 PBA into the tumor site at the same dosage. Both the PBS+NIR and PBA+NIR groups underwent irradiation treatment at 808 nm for 10 min. The tumor samples of each group were harvested with H&E staining after treatment for 24 h to estimate the pathological changes. Other tumor-bearing mice were monitored, and the tumor sizes were determined to calculate the tumor volumes (tumor volumes=length×width2/2). Twenty-seven days after treatment, several major organs were collected for H&E staining treated, and tumor metastasis numbers in the lung were counted and analyzed.
4.15 Antitumor metastasis potential in vitro
Transwell experiments were used to evaluate the antitumor metastasis effect of PBA. First, FBS-free DMEM was replaced with culture medium for 24 h. Then, 1×105/well 4T1 cells were incubated on the upper compartment of 24-well transwell plates with different concentrations of PBA (0, 10, 50 μg mL-1), and the lower chambers were filled with 20% FBS-culture medium at the same time. After incubation for 24 h, the cells that remained in the upper compartment were wiped with a cotton swab, while the cells that migrated through the membrane were fixed with 4% paraformaldehyde for 30 min, stained with 0.1% crystal violet and observed under a light microscope.
To further prove the antitumor metastasis effect, a western blot experiment was performed. After 4T1 cells were incubated with PBA (0, 10, 50 μg mL-1) for 24 h, the cells were rinsed with cold PBS twice and collected. Then, 8% SDS-PAGE electrophoresis was used to separate and identify total protein. After that, the targeted bands were transferred to PVDF membranes and blocked. Primary antibodies against MMP9, CDH-1 and GAPDH were added to distinguish the targeted protein at 4 °C overnight, and secondary antibodies were cultured for another 2 h at room temperature. Finally, the targeted protein was visualized with an ECL Western Blotting Detection Kit.
4.16 Hemolysis test
The fresh blood was collected and centrifuged under 2000 rpm for 5 min. Then, the 2% erythrocyte suspension was prepared. Several PBA solutions were added to 2% erythrocyte suspension at 37 °C for 1 h and recorded after 2000 rpm centrifugation.
4.17 Biodegradability of PBA
Several solvent systems were prepared to investigate the biodegradability of PBA, including distilled water, PBS, 10% FBS, 10% Bovine Serum Albumin (BSA) and Ethylene Diamine Tetraacetic Acid (EDTA). Then, 1 mg mL-1 PBA was prepared in the different solvents and placed at room temperature.
4.18 Statistical analysis
All the data in this report were collected and analyzed by IBM SPSS Statistics 20.0, and the quantitative data were organized as the mean ± standard deviation (SD). One-way ANOVA was used to investigate the comparison among groups. P<0.05 was considered a significant difference.