POLY(ETHYLENE GLYCOL)-MODIFIED PHOSPHOLIPIDS PREVENT AGGREGATION DURING COVALENT CONJUGATION OF PROTEINS TO LIPOSOMES

Liposome aggregation is a major problem associated with the covalent a ttachment of proteins to liposomes. This report describes a procedure for coupling proteins to liposomes that results in little or no change in liposome size. This is achieved by incorporating appropriate level s of poly(ethylene glycol)-modified lipids into the liposomes. The stu dies employed thiolated avidin-D coupled to liposomes containing the t hio-reactive lipid N-(4-(p-maleimidophenyl)butyryl)dipalmitoyl phospha tidylethanolamine (1 mol % of total lipid) and various amounts of MePE G-S-POPE (monomethoxypoly(ethylene glycol) linked to phosphatidylethan olamine via a succinate linkage). The influence of PEG chain length an d density was also assessed. The presence of PEG on the surface of lip osomes is shown to provide an effective method of inhibiting aggregati on and the corresponding increase in liposome size during the covalent coupling of avidin-D. A balance between the size of the PEG used and the amount of PEG-lipid incorporated into the liposome had to be achie ved in order to maintain efficient coupling. Optimal coupling efficien cies in combination with minimal aggregation effects were achieved usi ng 2 mol % MePEG(2000)-S-POPE (PEG of 2000 MW) or 0.8 mol % MePEG(5000 )-S-POPE (PEG of 5000 MW). At these levels, the presence of PEG did no t affect the biotin binding activity of the covalently attached avidin . The ability of the resulting liposomes to specifically target to bio tinylated cells is demonstrated.