Then, the medium was replaced with Dox–PEG, AG73–Dox, AG73T–Dox, or free Dox diluted with culture medium for Metformin manufacturer a final Dox concentration of 10 or 20 μg/ml. The plates were incubated for 4 h at 37 °C. The medium was removed, and subsequently, each cancer cell line was washed with PBS and then fixed with 4% paraformaldehyde for 1 h at 4 °C (293T-Syn2) or for 15 min at room temperature (colon26). For nuclear staining, the cells were treated with

DAPI for 1 h. Fluorescence images of the cells were analyzed using an FV1000-D confocal microscope (OLYMPUS, Tokyo, Japan). The cytotoxicity of liposomes was determined using the WST assay. 293T-Syn2 and colon26 suspensions (1×104 cells/well for 293T-syn2 and 3×103 cells/well for colon26) were added to Dox–PEG, AG73–Dox, AG73T–Dox, or free Dox with a serial concentration of Dox ([Dox]=1–40 μg/ml). Then, the suspension was incubated for 4 h at 37 °C in 5% CO2. After 4 h of incubation at 37 °C, the cells were washed, and fresh DMEM was added. The cells were then seeded in a 96-well plate and incubated for 48 h at 37 °C in 5% CO2. After PI3K inhibitor incubation, 10 μL of the cell-counting solution (WST-8, Dojindo Laboratories, Tokyo, Japan) was added to each well and was

incubated for 2 h (293T-Syn2) or 1 h (colon26) at 37 °C in 5% CO2. Cell viability was assessed by measuring the absorbance at 450 nm with a reference absorbance at 650 nm (Infinite M1000, TECAN, Männedorf, Switzerland). Cell viability was calculated according to the following formula: Cellviability(%)=A450(sample−blank)/A450(control−blank)×100. Colon26 cells (1×106 cells/mouse) were inoculated subcutaneously oxyclozanide in the right flank of mice. Five days after tumor inoculation (when the tumor volume reached approximately 50 mm3), HBS buffer used as a vehicle (control), Dox, Dox–PEG, or AG73–Dox was administered

via a tail vein of the mice (n=4). The injected dose of Dox in each administration was 2 mg/kg (approximately 10 μmol/kg dose of lipid). Various formulations were given every other day for a total of 5 doses. The size of tumors and the body weight of each mouse were monitored, and the tumor volume was calculated using the following equation: Tumorvolume(mm3)=longerdiameter×(shorterone)2×0.5 Colon26 cells (1×106 cells/mouse) were inoculated subcutaneously in the right flank of mice. Seven days after tumor inoculation (when the tumor volume reached approximately 100–200 mm3), DiI-labeled liposomes (lipid concentration: 10 μmol/kg) were administered via a tail vein of the mice (n=6). At 6 h after injection of the liposomes, the mice were sacrificed, and the tumors and organ tissues (heart, spleen, liver, and kidney) were dissected. These tissues were fixed in 10% paraformaldehyde substituted with 20% sucrose and then embedded in optimal cutting temperature compound (Sakura Finetech, Co., Ltd., Tokyo, Japan) and frozen at −80 °C.

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