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The encapsulation efficiency of bovine serum albumin (BSA) within liposomes and its stability in physiological conditions were determined by a specific enzyme immunoassay (EIA) developed for this purpose. BSA was encapsulated within liposomes composed of soyabean phosphatidylcholine (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 hydration or freeze-thawing methods. EIA was directly applicable to BSA encapsulated within liposomes without the usual need for sample preparation. The high sensitivity of the method allows high dilution of samples avoiding any interference with liposome formulation as was observed with high performance liquid chromatography (HPLC) method or colorimetric assay. Using this assay it was possible to evaluate that a high entrapment efficiency of BSA was obtained when the vesicles were composed of DSPC/CH/PG and prepared by the freeze-thawing method. Free BSA was stable upon incubation at 37°C for 2 h with acidic or basic buffers and in the presence of 10 mM TC, but was degraded in the presence 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 BSA entrapped in DSPC/CH/PG liposomes. When TC was added to the pancreatin, the stability of BSA (free or encapsulated in PC/CH/PG liposomes) increased, suggesting that after solubilization by TC, phospholipids rearrange forming a new structure in which BSA is protected from degradation. In conclusion, EIA might be a useful tool for the direct evaluation of the encapsulation efficiency and stability of any antigen entrapped in liposomes, without the usual need for sample preparation. |
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