br Preparation of PMDIs PMIs PMDs and DINPs br
2.2. Preparation of PMDIs, PMIs, PMDs, and DINPs
The synthesis process is shown in Fig. 1. PLGA nanoparticles con-currently entrapping DOX and ICG (DINPs) were created by the oil-in-water emulsion solvent evaporation method. In brief, 60 mg PLGA, 1 mg DOX, and 1 mg ICG were solubilized in 4 mL of mixed solvent (methanol: dichloromethane = 1:3, v/v). The organic phase was added into 8 mL of 1% PVA solution and then sonicated for 5 min at 400 W in an iced bath. The emulsion was stirred overnight at 37 °C to remove the organic solvent and to uniformly disperse nanoparticles. The particle suspensions were subjected to centrifugation for 25 min at 16,000 g. DINPs were then washed three times with equal volumes of purified water.
Platelet membrane-coated nanoparticles were prepared as pre-viously reported . Briefly, fresh mouse blood was centrifuged at 200 g to achieve PRP and then suspended in an equal volume of ACD solution (acid-citrate-dextrose). Next, the sample was centrifuged at 800 g to obtain the platelets. After lysis, the mixture of nanoparticles and platelet membrane was sonicated and then stirred for 12 h. Thereafter, a polycarbonate membrane containing 200 nm pore size was used for extruding the mixture 10 times using an extruder (Antos Nano Technology Co., Ltd.). The same treatments were applied in the preparation of PMIs or PMDs with or without ICG or DOX.
2.3. Characterization of PMDIs, PMIs, and PMDs
The polydispersity index (PDI), size, size distribution, and zeta po-tential of the NPs were examined using the Zetasizer instrument; the tests were performed in triplicate. The shape and size of the nano-particles was observed on a JEM-2100s instrument. The concentrations of ICG and DOX encapsulated in the NPs were measured by a Varioskan Flash multimode microreader.
Fig. 1. Preparation process of nanoplatelets and their synergistic eﬀects against breast cancer metastasis by active targeting of circulating tumor E64 and combi-nation treatment of chemotherapy and photothermal therapy.
2.4. Western blotting for the key proteins in PM, PMIs, PMDs and PMDIs
The total cellular protein was extracted using a protein extraction kit (Dingguo, China). The extracted proteins were separated using SDS-PAGE electrophoresis. After electrophoresis, the gel was treated with Coomassie blue staining. Extraction of protein for western blot was performed as described above. The proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad). This was fol-lowed by a blocking step for 1 h with 5% skim milk, and then the membrane was incubated with the primary antibody against P-selectin (Proteintech) overnight at 4 °C using GAPDH as the control. Finally, the membrane was incubated with the secondary antibody for 1 h at room temperature.
For this experiment, the formulation was incubated in 30 mL release media at 37 °C. The release environment was treated with and without laser irradiation. At previously selected time-points, the dialysate was used to evaluate the absorbance of DOX, while an equivalent volume of fresh PBS was replaced.
2.6. In vitro photothermal eﬃciency
Briefly, PBS, ICG solutions, PMI, and PMDI with an equivalent ICG concentration of 50 μg/mL were mixed in 1 mL centrifuge tubes. Next, they were treated with an 808 nm laser at 1.0 W cm−2 for 10 min. An infrared thermal imaging camera (Fotric 226) was used to measure the highest temperature in the four samples.
Human breast cancer cells (MCF-7, MDA-MB-231, and CAMA-1) and human cervical cancer cells (Hela) were cultured in high glucose DMEM medium containing streptomycin (100 μg/mL), 15% FBS, and penicillin (100 units/mL). The culture conditions for the cells were 5% CO2 at 37 °C.
2.8. The expression of CD44 on the cell surface
MDA-MB-231, CAMA-1, Hela, and MCF-7 cells were trypsinized and then collected by centrifugation for 5 min at 200 g. Subsequently, the cells were washed three times and resuspended in PBS. Four kinds of cells were poured into two 4 mL centrifuge tubes at a density of 106 cells/tube. Then, 20 μL mouse anti-human CD44 monoclonal anti-body was added into each sample of tumor cells in the experimental group, and an equal volume of CD44 isotype control antibody was added to the control group. Cells were incubated at room temperature for 30 min, washed and analyzed by FACSCalibur flow cytometer (n = 3).
2.9. Adhesion of platelets to cancer cells
MCF-7, CAMA-1, Hela, and MDA-MB-231 cells were inoculated in 96-well plates and 30 mm confocal dish at a density of 2 × 104 and 3 × 104 cells/well, respectively. They were then incubated at 37 °C for 24 h. The extraction of platelets was performed as described above. The platelets were collected, washed with ACD solution and then cen-trifuged at 1280 g for 15 min. This process was repeated three times, and then the cells were resuspended in DMEM basic medium without serum. Next, the platelets were incubated with DiD (4.5 μg/mL) for 30 min in an incubator at 37 °C. Later, the platelets were washed with ACD solution three times and resuspended in DMEM basic medium. Thereafter, the DiD-labeled platelets were added into the culture plates and co-incubated for 4 h (cell: platelet = 1:3000). The non-adherent platelets were washed oﬀ by DMEM basic medium three times and then