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

6. RFP Loaded PLGA Microspheres Prepared by Solvent Evaporation Methodcondition in lungs. Freeze-dried formulations were suspended in 500 ml of the dissolutionmedium, the amount of microspheres was varied in order to kept constant the amount of drug(25 mg). The experiments were carried out at 37 ± 0.3°C at a rotation speed of 100 ± 2 rpm. Ameasure of 1 ml samples were withdrawn at appropriate time intervals and centrifuged at10000 rpm. Supernatants were diluted suitably with acetonitrile and absorbance of theresulting solution was measured at 485 nm. The residue (after centrifugation) was redispersedin 1 ml of the fresh dissolution medium and replaced back into the dissolution apparatus. Thecumulative amount of RFP was obtained from the calibration curves of RFP in acetonitrile.The stock standard solution of RFP (2 mg/ml) was prepared by dissolving the drug inacetonitrile and storing at 4°C. A standard calibration curve was built up by using standardsolutions prepared by dilution of the stock standard solution with acetonitrile.6.1.5. Mucoadhesive Studies6.1.5.1. Adsorption of Mucin on PLGA MicrospheresBradford colorimetric method was used to determine the free mucin concentration in order toassess the amount of mucin adsorbed on the microspheres and its effect on the assessment ofmucoadhesive behavior of chitosan microspheres.Standard calibration curves were prepared from 2 mL of mucin standard solutions (0.1, 0.25,0.5, 0.75, and 1 mg/2 mL). After adding Bradford reagent, the samples were incubated at37°C for 20 minutes and then, the absorbance of the solution was recorded at 595nm in a UVspectrophotometer. Triplicate samples were run. All the samples were determined with thesame procedure.The evaluation of mucoadhesive properties was carried out by preparing mucin aqueoussolution with different concentrations (0.025, 0.1, and 0.5 mg/mL). PLGA microspheres (20mg) were dispersed in the above mucin solutions, vortexed, and shaken at room temperature.Then, the dispersions were centrifuged at 4000 rpm for 10 minutes, and the supernatant wasused for the measurement of the free mucin content. The mucin content was calculated fromthe standard calibration curve.6.1.6. Cell CultureThe human A549 alveolar epithelial cell line shows similar features as type II alveolarepithelial cells. The cells were grown as monolayers in 35 mm tissue culture dishes incubatedin 100% humidity and 5% CO2 at 37°C. HAM’S medium containing 365 mg/L L-glutamine,supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin, and 10089