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

4. RFP Loaded Chitosan Microspheres Prepared by Precipitation MethodFig.4.1: Collecting Nebulised Formulations of Microspheres for Evaluating Effects UponNebulization4.1.4. Release/Stability StudiesIn vitro release of RFP from chitosan microspheres was determined using, as the releasemedia, phosphate buffer (pH 7.4) and acetate buffer (pH 4.4) in order to simulate thecondition in the lungs, and SGF (pH 1.2), in order to evaluate the stability of chitosanmicrospheres in acidic medium. Freeze-dried formulations were suspended in 500 ml of thedissolution medium, and the amount of microspheres was varied in order to kept constant theamount of drug (25 mg). The experiments were carried out at 37 ± 0.3°C at a rotation speed of100 ± 2 rpm. A measure of 1 ml samples were withdrawn at appropriate time intervals andcentrifuged at 10000 rpm. Supernatants were diluted suitably with acetonitrile and absorbanceof the resulting solution was measured at 485 nm in a UV spectrophotometer. The residue(after centrifugation) was redispersed in 1 ml of the fresh dissolution medium and replacedback into the dissolution apparatus. The cumulative amount of RFP was obtained from thecalibration curve of RFP in acetonitrile. The stock standard solution of RFP (2 mg/ml) wasprepared by dissolving the drug in acetonitrile and storing at 4°C. A standard calibrationcurve was built up by using standard solutions prepared by dilution of the stock standardsolution with acetonitrile.4.1.5. Mucoadhesive Studies4.1.5.1. Adsorption of Mucin on Chitosan MicrospheresBradford colorimetric method (255) was used to determine the free mucin concentration inorder to assess the amount of mucin adsorbed on the microspheres and its effect on theassessment of mucoadhesive behavior of chitosan microspheres.Standard calibration curves were prepared from 2 mL of mucin standard solutions (0.25, 0.5,0.75, and 1 mg/2 mL). After adding Bradford reagent, the samples were incubated at 37°C for57