Effect of covalent attachment of immunoglobulin fragments on liposomal integrity.
Liposome stability during and after covalent coupling of Fab' antibody fragments was investigated. Large unilamellar vesicles containing entrapped 5(6)-carboxyfluorescein (CF) as a marker for liposomal integrity were prepared by extrusion through polycarbonate membranes. N-[4-(p-Maleimidophenyl)-butyryl]phosphatidylethanolamine (MPB-PE) was employed as a liposomal anchor for the covalent coupling of Fab' fragments. We observed that coupling of Fab' fragments to liposomes containing 5 mol % MPB-PE caused a concentration-dependent increase in size and polydispersity of the liposomes. Dependent on the concentration of the MPB-PE anchor in the membrane and the concentration of Fab' added, coupling was associated with the release of up to 95% of the entrapped CF. Rupture of the liposomes was identified as the primary mechanism of CF release during Fab' coupling. Reduction of the MPB-PE concentration to 1 mol % resulted in liposomes that were stable during and after Fab' coupling. The increased stability of these liposomes was due to the lower MPB-PE concentration and not to the lower number of attached Fab' fragments. By proper adjustment of the experimental conditions for coupling, the number of Fab' fragments attached to the 1 mol % MPB-PE liposomes could be increased without affecting the stability of the resulting liposomes. These stable liposomes, made by an extrusion method that avoids the use of organic solvents, detergents, or sonication, are therefore suitable for entrapment of labile compounds and can be used for immunotargeting or immunoassays.
D000818:Animals; D002784:Cholesterol; D004134:Dimyristoylphosphatidylcholine; D004355:Drug Stability; D005452:Fluoresceins; D007140:Immunoglobulin Fab Fragments / Q000302:isolation & purification; D007700:Kinetics; D008081:Liposomes; D010714:Phosphatidylethanolamines; D012756:Sheep
D005452:Fluoresceins; D007140:Immunoglobulin Fab Fragments; D008081:Liposomes; D010714:Phosphatidylethanolamines; C033182:N-(4-(4-maleimidophenyl)butyryl)phosphatidylethanolamine; C024098:6-carboxyfluorescein; D002784:Cholesterol; D004134:Dimyristoylphosphatidylcholine