br In vitro and in vivo
2.8. In vitro and in vivo photothermal imaging and hyperthermia monitoring
A thermal imaging camera was used to measure the photothermal effect of Chlorella AuNRs BSA-Gel in vitro and in vivo. Chlorella AuNRs BSA-Gel, Chlorella BSA-Gel, AuNRs BSA-Gel, and BSA-Gel in 1.5 mL eppendorf tubes were irradiated for 20 min with 808-nm laser (0.5 W/ cm2). To obtain photothermal images in vivo, Chlorella AuNRs BSA-Gel and BSA-Gel were injected intratumorally into 4 T1 tumor bearing mice, and the tumor regions were either not irradiated or irradiated with 808-nm laser (0.5 W/cm2) for 20 min. The temperature of tumors with adjacent Chlorella AuNRs BSA-Gel was precisely maintained around 41–42 °C by 808-nm laser irradiation (0.3–0.5 W/cm2) for 20 min. The temperature was recorded for each group of mice.
2.9. In vitro cytotoxic effects of Dox with Chlorella AuNRs BSA-Gel
The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich) assay was used to assess cell viability of 4 T1 Lipo3000 treated with various concentrations of Dox in normoxic or hypoxic cultures. 4 T1 cells were seeded in 96-well plates at 104 cells/well, pre-incubated for 24 h, and then treated with Dox (0, 0.3, 1, 3, 10 μg/mL)
for 24 h. The viability of cells at each time point was presented as a percentage of the control (PBS). To confirm the synergistic cytotoxic effect of Dox with oxygen generation of Chlorella AuNRs BSA-Gel plus 660-nm light illumination under hypoxic conditions, 4 T1 cells were treated with (i) PBS, (ii) chlorella, (iii) Dox (3 μg/mL), (iv) Chlorella AuNRs BSA-Gel (10 μL) + Dox (3 μg/mL) without 660-nm light, (v) Chlorella AuNRs BSA-Gel (10 μL) + 660-nm light (for 24 h), and (vi) Chlorella AuNRs BSA-Gel (10 μL) + Dox (3 μg/mL) + 660-nm light (for 24 h) in normoxic or hypoxic cultures, and cell viability was determined using an MTT assay.
4 T1 cells were seeded on a cover glass in each well of a 12-well plate (105 cells/well) and incubated for 24 h. The cells were treated with (i) PBS, (ii) chlorella, (iii) Dox (3 μg/mL), (iv) Chlorella AuNRs BSA-Gel (10 μL) + Dox (3 μg/mL) without 660-nm light, (v) Chlorella AuNRs BSA-Gel (10 μL) + 660-nm light (for 24 h), and (vi) Chlorella AuNRs BSA-Gel (10 μL) + Dox (3 μg/mL) + 660-nm light (for 24 h) in normoxic or hypoxic cultures. CellROX®Reagent was added to the cells at a final concentration of 5 μM and incubated at 37 °C for 30 min. Cells were washed three times with PBS and stained with DAPI. The pro-duction/distribution of reactive oxygen species (ROS) inside the cells was visualized using confocal laser scanning microscopy (CLSM; Carl Zeiss, Jena, Germany). To confirm the apoptotic activity of 4 T1 cells in each group, the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed under both normoxia and hy-poxia according to the manufacturer's instructions (Roche Diagnostics GmbH, Mannheim, Germany).
2.12. HIF-1α visualization in three-dimensional 4 T1 cell spheroids
4 T1 cell spheroids were prepared using Corning Spheroid Microplates. 4 T1 cells (50 μL, 104 cells/mL) were seeded into each well and cultured for 24 h. Spheroids were transferred to 94-well plates, and the medium was replaced with 100 μL of DMEM medium containing 1% FBS. The spheroids were treated with (i) PBS, (ii) chlorella, or (iii) Chlorella AuNRs BSA-Gel (100 μL) + 660-nm light for 24 h in normoxic or hypoxic conditions. The spheroids were fixed with 4% formaldehyde for 10 min and incubated for 5 min with 0.1% Triton x-100. Spheroids were then incubated with 1% BSA and PBST (PBS + 0.1% Tween 20) containing glycine (22.52 mg/mL) for 1 h to block nonspecific binding of antibodies. The spheroids were then incubated overnight at 4 °C with a 1/50 dilution of anti-HIF-1 alpha antibody-Alexa Fluor 488 (ABCAM), and the nuclei were stained with DAPI. Samples were observed using a confocal laser scanning microscope (CSLM).