Protection of a model protein, lactate dehydrogenase, by encapsulation in liposomes

The objective of this study was to evaluate the protective effect of entrapping a model protein, lactate dehydrogenase (LDH), inside liposomes. Besides the usual problems of stability in aqueous solution, LDH presents special challenges as it loses a considerable amount of activity during freeze-thaw cycles and freeze drying. Liposomes were prepared by modification of Bangham's method and the unentrapped LDH was separated from the encapsulated LDH by ultracentrifugation. The final liposomal preparation was analyzed for lipid content, vesicle size and protein content.; The influence of several processing parameters, such as centrifugation, sonication, and membrane filtration (shearing and adsorption), on LDH activity in water was investigated. The stability of LDH was determined in TRIS-HCl buffer at 25{dollar}spcirc{dollar} and 40{dollar}spcirc{dollar}C. LDH encapsulated in the liposomes was found to be 3 times more stable than LDH in the presence of blank liposomes or in buffer at 40{dollar}spcirc{dollar}C.; LDH, when encapsulated in liposomes, showed approximately 10 times slower degradation than when outside the liposomes, against the proteolytic activity of trypsin. This is an important finding as it suggests that it may be eventually possible to administer protein drugs orally.; Freeze-thaw studies comparing LDH encapsulated in liposomes and free in water showed that about 75% of its activity was retained when LDH was encapsulated compared to 10% when it was not. Further, an attempt was made to balance the osmotic pressure in order to increase the encapsulation efficiency. Again, when a freeze-thaw study was carried out on such liposomes, it was found that LDH encapsulated in liposomes showed better protection compared to both LDH outside the liposomes and LDH in buffer. The protection offered by liposomes during the freeze-thaw cycles was compared to that of LDH in the presence of cryoprotectants.; Karl-Fischer titrations were performed to determine the moisture content of the freeze dried product. LDH when encapsulated in liposomes and freeze-dried showed about 60% retainment of activity compared to 1% when LDH was freeze-dried simultaneously in buffer or in the presence of blank liposomes. The stability of the freeze dried product was also evaluated.