Abstract
Amifostine is a cytoprotectant drug used as an adjunct in cancer chemotherapy and radiation therapy. A simple and sensitive HPLC method was developed and validated for the assay of Amifostine. The method consisted of 95:5 (v/v) water and acetonitrile containing 0.05M heptane sulphonic acid (pH 3.5). The separation was achieved on a Luna 5μ C8 column (250 X 4.6 mm) and the effluents were monitored at 210 nm. The assay validation parameters evaluated include linearity, precision, accuracy and sensitivity. Amifostine loaded PLGA particles were prepared by multiple emulsion solvent evaporation method. Chitosan nanoparticles were prepared by tripolyphosphate precipitation method. The particle size and the surface charge were determined with a zetameter. The surface morphology was evaluated by scanning electron microscopy (SEM). The drug loading, entrapment efficiency and in vitro release from the particles in phosphate buffer was determined. The samples were analyzed by HPLC method previously described. The physical state of the drug in the formulation was analyzed by Differential Scanning Calorimetry (DSC). The cellular uptake and cytotoxicity of the nanoparticles was determined in MDCK and CACO-2 cell lines. The retention time of Amifostine was found to be 11 min. The standard curve was linear in the concentration range of 15.25-1000 μg/ml with an R2 value greater than 0.999. The RSD values for the within-day and day-to-day precision ranged from 1.89–5.77 % and 2.17–11.65 %, respectively. The accuracy of the method was found to be in the range of 95.4 to 104.9%. The size of the nanoparticles ranged from 287 to 925 nm. The PLGA nanoparticles had a negative surface charge (-13 to -25 mV) and the chitosan nanoparticles had a positive surface charge (1.16 to 26 mV). SEM pictures revealed spherical particles with a smooth surface. The drug loads were found to be 1.5% and 4.45% for PLGA particles and 1.61% and 6% for chitosan particles. For a theoretical drug load of 2% (w/w) PLGA nanoparticles the encapsulation efficiency was found to be 75%. However for a 10% drug loaded PLGA nanoparticles encapsulation efficiency was 44.5%. For 2% and 10% (w/w) drug loading chitosan particles the encapsulation efficiency was 80.5% and 60%,respectively. The cumulative percentage release of 2% Amifostine PLGA and Chitosan nanoparticles was 50% and 40% whereas the percentage release of 10% Amifostine PLGA and Chitosan was 85% and 55%, respectively at the end of 8 hrs. Amifostine was present in the formulation in a molecularly dispersed state. Cellular uptakes were dependent on the cell types with CACO-2 cells displaying a higher uptake than MDCK cell line. Chitosan nanoparticles showed a higher uptake than both the solution and PLGA nanoparticles in both the cell lines. The cytotoxicity profiles indicate that 10% loaded chitosan formulation and chitosan blank formulation show toxicity to both MDCK and CACO-2 cell lines 72 and 96 hours post treatment. A simple and sensitive HPLC assay was developed and validated for the analysis of Amifostine. A nanoparticulate delivery system was developed and characterized using biodegradable polymers like chitosan and PLGA for the radioprotectant drug amifostine.