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, evaluate the efficacy of the Percoll gradient method to separate AM from free MS, RIF-MS (MS3 formula) were added to cells at 4°C and immediately processed to separation on Percoll gradient

. For, After vortexing and centrifugation, 800 µl of the DCM phase was collected, evaporated under N 2 flux and 1 ml of the mobile phase for the "RIF-MS" samples and 100 µl for the "RIF-solution" samples were added. Samples were analyzed by reversed-phase HPLC with UV detection (333 nm wavelength). RIF standards were prepared in 1 mL DCM, evaporated under N 2 flux and reconstituted with 1 ml of the mobile phase. Calibration curves were linear (r 2 > 0.995) within the 0.25 to 8 µg/ml RIF concentration range. The chromatographic system consisted of Waters 717 Autosampler, cell pellets were thawed and the remaining water was evaporated under N 2 flux at 45°C. One ml of dichloromethane (DCM) and 50 µl of water were added to the samples

, After 1 hr at 37°C under 90-95% relative humidity and 5% (v/v) CO 2 atmosphere, 80 µg of blank microspheres, of RIF-MS (MS1, MS2, MS3) or of their equivalent amount of RIF (5.6, 13.6 and 31.2 µg respectively) were added to the cells for 4 hrs, Cell viability was then assessed using the Celltiter 96 aqueous solution from Promega

. Li, PLGA Resomer ® RG 502 H with the 50:50 ratio of PGA:PLA and low molecular weight. It was selected for its well-known biocompatibility (Shive and Anderson, 1997) and its fastest degradation rate among Resomer® PLGA grades. Microspheres of 1.6 µm diameter made from Resomer ® RG 502 H were shown by SEM to be extensively degraded within one week in vitro at 37°C in buffer, pH 7.4 (Díez and Tros de Ilarduya, 2006), which should avoid lung accumulation on the basis of 2 or 3 administrations a week for several months. Sucrose palmitate was used as a surfactant as an alternative to nonbiodegradable PVA. Sucrose esters, a group of non-ionic surfactants of low toxicity and good biodegradability and biocompatibility, are increasingly used in the formulation of biodegradable and biocompatible drug delivery nanosystems, RIF-loaded microspheres RIF-MS were prepared by the simple emulsion solvent evaporation method using as constitutive polymer, 2011.

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