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  • br Photothermal effects of Alg PDA scaffolds br The


    2.5. Photothermal effects of Alg-PDA scaffolds
    The photothermal effect of Alg-PDA scaffolds was investigated by irradiating the scaffold in PBS with an 808 nm laser (0.5 W cm 2) for 5 min. The temperature of the scaffolds was mon-itored using an IR thermal camera (FLIR A310).
    2.6. Hemolysis assay
    The red blood SCH 58261 (RBCs) were isolated from serum by cen-trifuging the mixture containing 0.5 mL of blood sample and 1 mL of PBS solution at 4500 rpm for 3.5 min. The RBCs were washed with PBS five times and then diluted the volume to 5 mL. Afterwards, 0.3 mL of the diluted RBC suspension was added to a 2.5 mL Eppendorf (EP) tube. Then, 1.2 mL of PBS, 1.2 mL of PBS solution after soaking the Alg-PDA scaffold, and 1.2 mL of deion-ized water were added to each EP tube. The mixtures were vor-texed and kept to stand for 3 h at room temperature. Samples were then centrifuged to measure the absorbance of the super-natants at 541 nm using a multifunctional microplate detector (BioTek Instruments, Synergy H1). RBCs treated with deionized water and PBS were set as positive and negative controls, respectively.
    2.7. Photothermal effects of Alg-PDA scaffold on the viability of tumor cells in vitro
    Breast cancer cells (4T1 cells) were used to evaluate the pho-tothermal effects of the fabricated scaffolds. The following five dif-ferent groups were designed: 1) cells without any treatment (Control); 2) cells treated with laser irradiation (Laser only); 3) cells treated with the Alg scaffold plus laser irradiation (Alg (+)); 4) cells treated with the Alg-PDA scaffold but without laser irradi-ation (Alg-PDA ( )); and 5) cells treated with the Alg-PDA scaffold plus laser irradiation (Alg-PDA (+)). 4T1 cells were seeded in 96-well plates at a density of 5 103 cells per well, followed by cultur-ing in DMEM (supplemented with 10% FBS, 1% penicillin, 1% glu-tamine, and 1% sodium pyruvate) at 37 LC and 5% CO2 for 24 h. Then according to the above 5 groups, different scaffolds were placed into different wells in 96-well plates, where the scaffolds did not contact with the cells directly and a gap of approximately
    500 mm was set. Then, the scaffolds were irradiated with an 808 nm laser (0.5 W cm 2) for 5 min per well. Finally, the viability of 4T1 cells was calculated by the CCK-8 cell cytotoxicity assay. The cell viability was normalized with a control group without any treatment. Four samples were measured for each group. Further, calcein-AM and propidium iodide solution were used for live/dead cell staining, and the images were captured using a fluorescence microscope (Nikon Canada, Mississauga, Canada).
    2.8. In vivo tumor therapy of fabricated scaffolds
    Animal experiments were performed according to the experi-mental practices and standards approved by the Animal Welfare and Research Ethics Committee at Shenzhen University. 4T1 cells  (1 106 cells in 0.1 mL of PBS) were injected in each mouse (female, 4–6 weeks old) to produce subcutaneous tumors. Before surgery, all mice were injected with 5% chloral hydrate adapted to their body weight. Then, different scaffolds were sterilized and implanted into the tumor site when the tumor volume reached 200 mm3. All mice were randomly divided into five groups: group
    1: mice without any treatment; group 2: mice treated only with laser irradiation; group 3: mice implanted with alginate under laser irradiation; group 4: mice implanted with Alg-PDA without
    laser irradiation; group 5: mice implanted with Alg-PDA under laser irradiation. Laser irradiation (0.5 W cm 2, 5 min) was per-formed on the mice after scaffolds were implanted in vivo for 1 and 3 days. The body weight, tumor size, and cancer recurrence of all the mice were monitored every day. Hematoxylin and eosin (H&E) staining of the major organs was performed on healthy mice and mice treated with the Alg-PDA scaffold plus laser.