The effect of microfluidization of protein-coated liposomes on protein distribution on the surface of generated small vesicles

Tetanus toroid and immunoglobulin G (IgG), model proteins for vaccines and targeting ligands respectively, were covalently coupled to preformed dehydration-rehydration vesicles (DRV) to produce vesicles with surface-bound proteins (DRV-protein) or to preformed small unilamellar vesicles (SUV) which were used to generate DRV with bound protein [(SUV-protein)DRV]. Of the amount of protein employed for coupling (1 mg), 13.8-45.1% was recovered with the liposomes, depending on the type of preparations and the proteins used. Microfluidization of similar DRV-protein or (SW-protein)DRV for up to 10 cycles led to the formation of smaller vesicles (98-136 nm diameter) which, however, had modestly reduced (estimated as 8.8-21.7%) bound proteins, again depending on the type of preparation and protein used. Treatment of DRV-protein and (SUV-protein) DRV with proteinase revealed that 32.9-45.6% of the total bound protein was exposed on the liposomal surface. With microfluidized liposomes, the proportion of surface-exposed protein increased to 63.1-76.2%. Incubation of intact and microfluidized DRV-IgG and (SUV IgG) DRV with a protein A-Sepharose 4B CL gel confirmed the presence of IgG on the liposomal surface (47.1-68.4 and 80.5-82.1% of total bound protein respectively). These studies were supplemented with freeze-fracture electron microscopy of (SUV-toxoid)DRV which demonstrated the presence of protein particles (up to 3; 12-14 nm diameter) on the surface of both intact and microfluidized individual liposomes.