Cell viability assay A549 cells were counted and seeded in 96-well plates at a density of 0.5 × 104 cells per well and incubated overnight to allow cell attachment. The cells were incubated with drug-loaded PLA-PCL-TPGS nanoparticle suspension, thiolated check details chitosan-modified PLA-PCL-TPGS nanoparticles, and Taxol® (Bristol-Myers
Squibb, New York, USA) at 0.25, 2.5, 12.5, and 25 μg/ml equivalent paclitaxel concentrations and blank thiolated chitosan-modified PLA-PCL-TPGS nanoparticles with the same amount of nanoparticles for 24, 48, and 72 h, respectively. At the determined time, the formulations were replaced with fresh DMEM containing MTT (5 mg/ml), and the cells were then incubated for additional 4 h. MTT-containing medium was aspirated off, and 150 ml of DMSO was added to dissolve the formazan crystal formed by living cells. The absorbance at 570 nm was measured by a microplate reader (Model https://www.selleckchem.com/products/gsk923295.html 680, Bio-Rad Laboratories, Hertfordshire, UK). Untreated cells were taken as a control with 100% viability, and cells without the addition of MTT were used as blank to calibrate the spectrophotometer to zero absorbance. IC50 values (concentration required to reduce cells viability by 50% as compared to the control cells) for each sample was calculated by curve fitting of the cell viability data. The results are expressed as mean ± SD of one representative experiment performed
C646 solubility dmso in triplicate, Bay 11-7085 and the experiments were performed three times. Ex vivo study The everted sac method was chosen for the measurement of transportation of paclitaxel across the intestine barrier. It was carried out according
to the slightly modified method that was described previously , as follows. First, a section of about 5 cm of the jejunum was removed from a male rat under ketamine (50 mg/kg) and chlorpromazine (10 mg/kg) anesthesia and washed with Krebs-Ringer bicarbonate solution of pH = 7.4. This section was then gently inverted with a glass rod, and a tube was inserted in one side of the section and tied securely with tape. The other side of the intestine was tied, and 1 mL Krebs-Ringer bicarbonate solution was poured through the hypodermic needle in the tube. The gut sac was placed in a medium saturated with 95% O2, 5% CO2, and contained the test sample in Krebs-Ringer bicarbonate solution at 37°C. The test samples used include: (1) paclitaxel (1 mg) as Taxol®, and (2) thiolated chitosan-modified PLA-PCL-TPGS nanoparticles (equivalent to 1 mg of paclitaxel). In absorption studies, an O2 and CO2 mixture was bubbled into the intestinal mucosa to obtain intestinal peristaltic movement. At certain periods of time, 0.5-mL samples were drawn from inside the intestine and replaced with the same volume of fresh medium. The amount of transported paclitaxel in the samples was measured by the HPLC method. Statistical analyses Data were presented as the mean ± SD.