Determination of encapsulation efficiency of tetanus toxoid in microsphere and liposome drug delivery systems by two different spectroscopic methods

Spectrophotometric methods are amongst the most important methods used for determination of encapsulated proteins in particulate drug delivery systems. In Bradford method, which is used frequently, some interfering agents could affect the precise determination of proteins. In such a conditions, other methods with less interactions, such as spectrofluorimetry, can be used. Tetanus toxoid (TT) was conjugated with fluorceinisothiocianate (FITC), as a fluorescent agent. Buffer salts and unconjugated FITC were separated by dialysis and ultrafiltration. Alginate microspheres and liposomes encapsulated with fluorescent TT were prepared by emulsification-internal phase gelation and solvent evaporation, respectively. encapsulated TT in microspheres and liposomes was determined by Bradford and spectroflourimetric methods. Mean encapsulation efficiency of FITC-TT in microspheres was obtained to be 38.33 ± 6.11 and 21.33 ± 2.1, by Bradford and fluorimetry methods, respectively (P<0.01). In Bradford method, the blank microspheres showed high absorbances which could be caused by interaction from sodium alginate polymer. But in the fluorimetric method the blank microspheres showed little absorbances, so this method is more reliable. Mean encapsulation efficiency of FITC-TT in liposomes was determined by indirect method, as 46.33 ± 6.23 and 56 ± 3, by Bradford and fluorimetry methods (P<0.05). In Bradford method, supernatant of blank liposomes showed high absorbances which could be rooted from interaction of free lipids. But in the fluorimetry method supernatant of the blank liposomes showed little emission, so this method is more reliable. In determination of encapsulation efficiencies of proteins in microspheres and liposomes, when spectrophotometric methods due to interfering agents, could not be utilized precisely, spectrofluorimetric method can be preferred.