In addition, a flow cytometry assay was performed. selleck inhibitor A total of 5 �� 104 MKN-45 cells were seeded in a 24-well plate with 0.5 mL RPMI 1640 supplemented with 20% FBS, and allowed to adhere at 37��C with 5% CO2 for 24 hours prior to assay. The medium was then replaced with fresh medium without serum prior to adding ibuprofen-loaded PLGA NPs. After 2 hours of incubation, the cell monolayers were rinsed three times with PBS buffer to remove excess NPs, and incubated with complete medium. Cells were collected and washed twice with cold PBS. The pellet was resuspended in FACSFlow Sheath Fluid (BD Biosciences, Franklin Lakes, NJ), and analyzed with a FACSCanto II cytometer (BD Biosciences). For each sample, >20,000 cells were analyzed using FACSDiva software (Version 6.0, BD Biosciences).

Release of ibuprofen in cell lysates from NPs The release of ibuprofen from ibuprofen-loaded PLGA NPs was evaluated by gas chromatography�Cmass spectrometry. To analyse the time of ibuprofen release into the cells from ibuprofen-loaded PLGA NPs, cells were collected, washed twice with PBS, and lysed with radioimmunoprecipitation assay buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM sodium chloride, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (Sigma-Aldrich, St Louis, MO), and protease inhibitors (Sigma-Aldrich). The cells were incubated on ice for 10 minutes, and then centrifuged at 8000 �� g at 4��C for 10 minutes to pellet the cell debris. A subsequent centrifugation at 20,000 �� g was performed to collect NPs. The lysates were frozen at ?80��C until analysis.

The release of ibuprofen into the cell lysates was then quantified by gas chromatography�Cmass spectrometry. The samples were extracted in hexane and trimethylsilylated in 100 ��L N,O-bis(trimethylsilyl) acetamide (TMSA, Sigma-Aldrich) at 80��C for 45 minutes. The samples were dried under nitrogen and dissolved in 10 ��L hexane, and the supernatants were used for analysis. The samples were analyzed by gas chromatography�Cmass spectrometry on an Agilent instrument (Agilent 5890; Agilent Technologies, Santa Clara, CA) under the following conditions: capillary column (ZB-5; Phenomenex Inc, Torrance, CA); 30 m �� 0.25 mm ID; flow rate, 1 mL/min; helium as carrier gas) with a temperature program of 120��C for 3 minutes, which then increased from 100��C to 280��C at 20��C/min.

Flow cytometric analysis of DNA content For the monoparametric cell-cycle analysis, we collected the treated and control cells, washed them in PBS, and fixed them with cold 70% ethanol at 4��C for ��24 hours. Then, 2 �� 106 cells were Anacetrapib washed twice in PBS, and incubated with 2 mL of 20 ��g/mL propidium iodide (Sigma-Aldrich) in PBS containing 0.002% Nonidet P40 (Roche Applied Science, Penzberg, Germany) and 12.5 ��g/mL RNase A (Sigma-Aldrich) at RT for ��60 minutes.