Phosphatidylcholine liposomes covalently coupled with immunoglobulin fragments were prepared by REV method and characterized by dynamic light scattering, absorbance, and calcein release.
Fab' antibody fragments were covalently combined with preformed large unilamellar vesicles(LUV) using a sulfhydryl-reactive phospholipid derivative N-[4-(p-maleimidophenyl) butyl] phosphatidylethanolamine (MPB-PE). A highly efficient reaction between the sulfhydryl group on each Fab' fragment and the maleimide moiety of MPB-PE molecules incorporated at a low concentration in vesicle bilayers led to the formation of a quite stable Fab'-vesicle linkage. Coupling ratio was 250㎍ of Fab'/μmol of vesicle phospholipid.
The size of large unilamellar vesicles by REV method may depend on the type of phospholipid and its solubility in the organic solvent, the interfacial tension between aqueous buffer and organic solvent, and the relative amounts of water phase, organic solvent, and phospholipids. Dynamic light scattering yielded the increase of the size of the vesicles as increasing the concentration of lipid employed and the ratio of cholesterol to the lipid. But apparently the size does not seem to be sensitive to the Fab', coupled to the vesicle.
The effects of bile salts (BS) on the stability of liposomes were investigated, observing the light absorbance of vacant liposomes. Absorbance of liposomes decreased depending on the BS/lipid ratio because of the solubilization of lipid molecule in bilayer and the formation of mixed micelles. At very low BS concentrations, BS/lipid aggregates are formed in the outer vesicles monolayer, while increasing BS concentrations mixed micelles are formed. The solubility of Fab' vesicle had lower than that of MPB-PE vesicle by BS.
The stability of liposomes were investigated as a function of time and temperature, by observing absorbance of vacant liposomes and calcein release from entrapped liposomes. As time elapsed the turbidies of Fab' vesicle and MPB-PE vesicle did not change, but the liposomes manufactured by sonication method had large change of turbidity. Such phenomena may be attributed to the disturbance of the bilayer characteristics. The fusion of liposomes was not affected by the coupling of Fab' but was affected by the method of the manufacture of liposomes.