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

4. RFP Loaded Chitosan Microspheres Prepared by Precipitation Methodultrasonifier, for 30 minutes. After addition of sodium sulphate, in some formulations, asolution of GA (25% w/w) was also added to evaluate the influence of cross-linking agents.Microspheres were purified by centrifugation for 15 minutes at 3000 rpm. The obtainedsediment then was suspended in water. These two purification steps were repeated twice. Allpurified particles then were lyophilized.4.1.3. Characterization of RFP-Loaded Chitosan Microspheres4.1.3.1. Particle SizingMicrospheres were analysed for their size and polydispersity index on Nano-ZS (Nanoseries,Malvern Instruments), based on photon correlation spectroscopy, at a scattering angle of 90°and temperature of 25°C. Each measurement was the result of 12 runs. Measurements werecarried out for both fresh and freeze-dried samples.Before counting, the samples were diluted with a 0.05% (w/v) tween 80 water solution inorder to prevent precipitation during the measurements. Results are the means of triplicateexperiments.4.1.3.2. Surface Charge (Zeta-Potential)The surface charge of the microspheres was determined with Nano-ZS (Nanoseries, MalvernInstruments). The measurements were carried out in an aqueous solution of KCl 0,1N.Immediately before the determinations, microspheres were diluted with KCl solution. Themeasured values were corrected to a standard reference at temperature of 20°. Results are themeans of triplicate experiments.4.1.3.3. Particles MorphologyThe Optical Microscopy (OM) (Zeiss Axioplan 2), was used for the determination of theshape of RFP loaded chitosan microspheres. A small drop of microspheres suspension wasplaced on a clean glass slide. The slide containing RFP loaded chitosan microspheres wasmounted on the stage of the microscope and observed.For scanning electron microscopy (SEM) several drops of the microsphere suspension wereplaced on an aluminum stub having previously been coated with adhesive. The samples wereevaporated at ambient temperature until completely dried, leaving only a thin layer ofparticles on the stub. All samples were sputter coated with gold-palladium (Polaron 5200, VGMicrotech,West Sussex, UK) for 90 seconds (2.2 kV; 20 mA; 150–200A°) under an argonatmosphere. The SEM (Model 6300, JEOL, Peabody, NY) was operated using an accelerationvoltage of 10 kV.55