In vitro characterization of binding and stability of single-chain Fv Ni-NTA-liposomes

Recently, we presented a new method for the generation of single-chain Fv (scFv) immunoliposomes, which circumvents the necessity to introduce additional reactive groups in the protein. This method is based on immobilizing scFv fragments via their C-terminal hexahistidyl-tag on liposomes containing nickel-complexed dioleoyl-glycero-succinyl-nitrilotriacetic acid (NiNTA-DOGS) as an anchor lipid within the lipid bilayer. Here, we have extended this approach to various other scFv fragments and further demonstrate strong and selective binding of these liposomes to target cells in vitro. In order to evaluate suitability for in vivo applications, we investigated the influence of human plasma on stability and binding behaviour of scFv Ni-NTA-liposomes in vitro using scFv A5 directed against human endoglin (CD105) as a model antibody. We could show that the binding activity to target cells is rapidly lost in the presence of human plasma. Incorporation of polyethylene glycol (PEG) chains into the lipid bilayer did not protect against loss of binding capability. Further studies showed that loss of binding is mainly due to displacement of Ni-NTA-bound scFv fragments caused by plasma proteins. In conclusion, the system allows for a rapid and flexible generation of target cell specific immunoliposomes for in vitro applications but lacks stability for in vivo applications.