An aqueous solution of b-galactosidase protein (40 IU/ml) was prepared in PBS. Subsequently, 100 ll of each solution was slowly added to freeze-dried lipids (6 lM) and incubated for 10 min at room temperature until rehydration was completed and liposomes were formed. To remove nonencapsulated b-galactosidase, liposomes were washed three times with PBS by centrifugation at 8,000-9,0009g for 10 min at 4°C and re-suspended in 50 ll PBS. The amount of encapsulated b-galactosidase in PEG-liposomes was calculated from total amount of protein entrapped in Antigen-expressing immunostimulatory liposomes as a genetically programmable synthetic vaccine 23 liposomes. For some formulations pDNA encoding bgalactosidase was co-entrapped with the b-galactosidase enzyme at a concentration of 20 nM. The amount of encapsulated b-galactosidase was measured based its enzymatic activity.