br Preparation of Mn chelated BPNs PDA PEG PEITC
3.9. Preparation of Mn2+ chelated BPNs-PDA-PEG-PEITC/DOX
Adding MnCl2 solution (0.5 mg/mL) in BPNs-PDA-PEG-PEITC/DOX dispersion (0.5 mg/mL). After 6 h stirring, the unbound excess Mn2+ was removed through centrifugation. Inductively coupled plasma-mass spectrometry (ICP-MS) was used to measure the concentration of Mn2+ in BPNs-PDA-PEG-PEITC-Mn/DOX.
3.10. Photothermal properties of BPNs-PDA-PEG-PEITC
Monitoring the temperature changes of BPNs-PDA-PEG-PEITC with different concentration under the NIR laser (808 nm, 1.2 W/cm2) was used to construct photothermal heating curves. The temperature mea-surement was used an NIR camera (MAG32, Shanghai Magnity Electronics Co., Ltd). The optical source was used a fiber-coupled continuous semiconductor diode laser (PSU-H-LED, Changchun New Industries Optoelectronics Technology Co., Ltd).
3.11. Hemolysis and the shape of RBCs
By the centrifugation of whole blood (5 mL, with anticoagulation) at 3000 rpm for 15 min could acquire RBCs. PBS was used to dilute the
RBCs (VPBS:VRBCs = 9:1). Afterward, BPNs-PDA-PEG-PEITC or BPNs-PDA-PEG-PEITC/DOX were confected into different concentration dis-
persion (2 mL) by PBS, and then the dispersions were incubated with 0.5 mL of RBCs suspension. Two mL of deionized water and PBS were mixed with 0.5 mL of RBCs suspension, respectively, served as positive and negative controls. Keeping all mixtures maintain at 37 °C for 3 h. After the mixtures centrifuged at 3000 rpm for 15 min, the optical density (OD) of supernatant at 541 nm was measured by microplate reader (BioTek synergy 2). The hemolysis rate was calculated with the
where ODsample , ODpositive, and ODnegative was the OD of the sample, po-sitive control, and negative control, respectively. The observation of
3.12. In vitro clotting time tests
APTT, PT, and TT, of BPNs-PDA-PEG-PEITC and BPNs-PDA-PEG-PEITC/DOX at different concentrations were measured using a coagu-lation instrument (Sysmex CA-1500 Sysmex Corp., Japan) .
In dark, 10 mL of DPBF solution (in ethanol, 20 μg/mL) was mixed with BPNs-PDA-PEG-PEITC (15 μg/mL) stirred for 120 min. Light source was used a 660 nm continuous semiconductor diode laser (Q-LINE Electronics & Technology Co., Ltd.). Taking the mixture at dif-ferent time points for UV measurements.
3.14. Intracellular generation of ROS
The MCF-7/ADR KIN59 (1 × 105 cells/well) were cultured in 24-well plate for 1 day. The culture medium was discarded and the fresh culture medium containing BPNs-PDA-PEG-PEITC was added. Four hours later, adding H2DCFHDA into the medium incubated for further 0.5 h. The cells were exposed at 660 nm laser (0.5 W/cm2) for 10 min. At last, PBS was used to wash the cells and fluorescent microscope was used to observe the cell morphology .
3.15. In vitro release behaviors
The release behaviors were studied at 37 °C in PBS (pH = 7.4, 6.8 and 5.0). Firstly, 2 mg of BPNs-PDA-PEG-PEITC/DOX was dissolved in 4 mL PBS, and the dispersion was sealed in a dialysis bag (molecular weight cutoff 14,000). Afterwards, putting dialysis bag in 15 mL PBS, the solution outside dialysis bag was collected 1 mL at given time to measure the DOX release amount. In order to study the NIR irradiation-triggered release of DOX, BPNs-PDA-PEG-PEITC/DOX solution was ir-radiated with NIR irradiation (1.2 W/cm2) at special time, and the other steps were performed in a similar way.
3.16. Intracellular DOX release
The MCF/ADR cells (5 × 105 cells/well) were seeded into 6-well plate incubated for 1 day. Then the cells were treated with BPNs-PDA-PEG-PEITC/DOX (15 μg/mL) for 2 h. To study the NIR-triggered in-tracellular DOX release, some wells were irradiated with NIR irradia-tion (1.2 W/cm2) for 5 min. At last, PBS was used to wash the cells. The fluorescent images of cells were obtained by using confocal imaging (TI-EA1R, Nikon, Japan)
3.17. Immunoblot analysis
MCF-7/ADR were cultured in 12-well plates for 24 h. Afterwards, BPNs-PDA-PEG (10 μg/mL), BPNs-PDA-PEG-PEITC (10 μg/mL), BPNs-PDA-PEG/DOX (33 μg/mL) and BPNs-PDA-PEG-PEITC/DOX (33 μg/ mL) were incubated with cells for 16 h. The cells were lysed and cen-trifugated for collecting. The acquired protein was separated on gel, transferred to poly (vinylidene difluoride) (PVDF) membranes. The membranes were incubated with milk (5%) with TBST and were then probed with p53 and β-actin antibody. The detection of proteins was Chemical Engineering Journal 370 (2019) 387–399
using enhanced chemiluminescence reagents .
3.18. Cytotoxicity assay
MCF-7 cells and MCF-7/ADR cells at a density of 5 × 103 cells/well were seeded in 96-well plates. After culture for 24 h, the samples with different concentrations were incubated with the cells for 3.5 h. For PDT, some of the wells were irradiated with 660 nm laser for 10 min. For PTT, some of the wells were irradiated with 808 nm laser for 5 min. After further 24 h, the medium was discarded and the fresh medium together with MTT reagent (5 mg/mL) was added into every well. Four hours later, after removal of MTT-containing medium, formazan crys-tals produced by the live cells was dissolved by 150 μL DMSO. The measurements of OD values at 570 nm of different experimental wells was using microplate reader to analyze cell viability .