chitosan and plga microspheres as drug delivery ... - UniCA Eprints

6. RFP Loaded PLGA Microspheres Prepared by Solvent Evaporation Methodµg/mL streptomycin was used as the growth media. The cells that form the monolayers wereharvested with trypsin (0.25%) centrifuged at low speed (1600 g, 4 min), resuspended in freshmedium and plated at a concentration of 2 x 10 5confluence on tissue culture dishes for 3 to 4 days.cells/dish. The cells were grown to6.1.6.1. MTT AssayFor dose-dependent studies, cells were treated with RFP alone and RFP-loaded PLGAmicrospheres at different concentration in RFP. The effect of RFP in microspheres on theviability of cells was determined by [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide] MTT assay (256). The dye is reduced in mitochondria by succinic dehydrogenase toan insoluble violet formazan product. A549 cells (105 cells/well) were cultured on 24-wellplates with 500 µl of medium for 24 hours, with and without the tested compounds. Then 50µl of MTT (5 mg/ml in PBS) were added to each well and after 2 h, formazan crystals weredissolved in DMSO. Absorbance at 580 nm was measured with a spectrophotometer. On thebasis of this assay IC50 values were obtained in three independent experiments for eachformulation. In all assays three different concentrations were used. In order to evaluatechanges in viability caused by the tested compounds, living cells as well as those in early andlate stages of apoptosis and necrosis were counted. All other methods were also carried outafter 24 h incubation. The data in this study were expressed as mean ± S.D.6.1.7. Statistical AnalysesAll experiments were repeated at least three times. Results are expressed as means ± standarddeviation. A difference between means was considered significant if the p value was less thanor equal to 0.05.6.2. Result and Discussion6.2.1. Preparation of RFP-Loaded PLGA MicrospheresSolvent evaporation method is the most popular technique of preparing PLGA microparticles.It involves emulsifying a drug-containing organic polymer solution into a dispersion medium.Depending on the state of drug in the polymer solution and the dispersion medium, it can befurther classified into oil in water (o/w), water in oil (w/o), and water in oil in water (w/o/w)double emulsion method. The o/w method was used in this work. For this technique, drug isdissolved or dispersed in a solution of the polymer in a water-immiscible and volatile organicsolvent (DCM). This dispersion is emulsified into an aqueous phase. The organic solvent then90