USE OF AN ENZYME-IMMUNOASSAY FOR THE EVALUATION OF ENTRAPMENT EFFICIENCY AND IN-VITRO STABILITY IN INTESTINAL FLUIDS OF LIPOSOMAL BOVINE SERUM-ALBUMIN

The encapsulation efficiency of bovine serum albumin (BSA) within lipo somes and its stability in physiological conditions were determined by a specific enzyme immunoassay (EIA) developed for this purpose. BSA w as encapsulated within liposomes composed of soyabean phosphatidylchol ine (PC), cholesterol (CH), phosphatidylglycerol (PG) (molar ratio 6:3 :1) or distearoylphosphatidylcholine (DSPC), CH, PG, (molar ratio 6:3: 1). Vesicles were prepared according to either the thin lipid film hyd ration or freeze-thawing methods. EIA was directly applicable to BSA e ncapsulated within liposomes without the usual need for sample prepara tion. The high sensitivity of the method allows high dilution of sampl es avoiding any interference with liposome formulation as was observed with high performance liquid chromatography (HPLC) method or colorime tric assay. Using this assay it was possible to evaluate that a high e ntrapment efficiency of BSA was obtained when the vesicles were compos ed of DSPC/CH/PG and prepared by the freeze-thawing method. Free BSA w as stable upon incubation at 37 degrees C for 2 h with acidic or basic buffers and in the presence of 10 mM TC, but was degraded in the pres ence of a mixture of pancreatin and TC. In the presence of pancreatin alone, BSA entrapped in PC/CH/PG liposomes was less stable than the BS A entrapped in DSPC/CH/PG liposomes. When TC was added to the pancreat in, the stability of BSA (free or encapsulated in PC/CH/PG liposomes) increased, suggesting that after solubilization by TC, phospholipids r earrange forming a new structure in which BSA is protected from degrad ation. In conclusion, EIA might be a useful tool for the direct evalua tion of the encapsulation efficiency and stability of any antigen entr apped in liposomes, without the usual need for sample preparation.