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

7. RFP Loaded PLGA Coated Chitosan Microspheres Obtained by WSD Methodsurface with the mucoadhesive polymer chitosan. RFP-encapsulated PLGA microspherescoated with chitosan were prepared by the emulsion solvent diffusion method in water (WSDMethod).The influence of chitosan concentration on microparticle size distribution, entrapmentefficiency (E%), nebulization efficiency (NE%) and leakage upon nebulization was studied.The toxicity of chitosan microspheres were evaluated by using A549 alveolar epithelial cells.7.1. Materials and Methods7.1.1. MaterialMedium molecular weight chitosan with a deacetilation grade of about 87% was, PLGA(75:25, mol. Wt 85,2 kDa), rifampicin (RFP), acetic acid, poly-vinyl alcool (PVA) wereprovide by Sigma Aldrich Chemie (Germany).mucin (type I-S) was purchase from Sigma Aldrich.103Lyophilised bovine submaxillary glandsThe A549 epithelial alveolar cell line (passage 31) was a kind gift from Dr. Ben Forbes(School of Pharmacy, Kings College, London) and these cells were cultured in Ham’s F12-Kmedium (Sigma Aldrich), supplemented with 10% fetal bovine serum (Gibco BRL LifeTechnology, Grand Island, NY, USA), 100 µg/ml penicillin G (Sigma Aldrich), and 100µg/ml streptomycin sulphate (Sigma Aldrich ) at 37°C in a humidified 95% air/5% CO2environment. Cultures (monolayers in tissue culture flasks, 75 cm 2 ) were fed with freshmedium every 48 h.All other reagents were of the highest grade commercially available. Water was always usedin demineralised form.7.1.2. Preparation of RFP-Loaded PLGA Coated Chitosan MicrospheresAlso in this case WSD Method was used. Chitosan was dissolved in 50 ml of acetic acidbuffer solution at pH 4.4, PVA (1%) was also dissolved in this buffer. The PLGA (100 mg)and RFP (2mg/ml) were dissolved in 5 ml of DCM which was poured into 50 ml of aqueouscoating polymer solution prepared beforehand at 2 ml/min under stirring at 600 rpm usingUltraturrax® at room temperature. The PVA dissolved in the aqueous solution of coatingpolymer prevented aggregation of the emulsion droplets and sticking of the polymers to thepropeller shaft during agitation. The entire dispersed system was then centrifuged (4500 rpm15 min) and the sediment was resuspended in distilled water. This process was repeated andthe resultant dispersion was then subjected to freeze-drying overnight.