Characterization of AnExILs and liposomes loaded with b-galactosidase and/or pDNA Cell-free protein synthesis was used to transcribe and translate the lacZ gene encoding for E. coli b-galactosidase either in bulk, inside liposomal compartments (AnExILs-IN) or adhered onto the surface of liposomes (AnExIL-ON) (For details of liposome preparation see materials & methods section). The volume-weighted mean diameter of b-galactosidase-loaded liposomes and AnExIL formulations was approximately 1.5 lm. The dose of b-galactosidase was standardized to 800 mU per formulation (see ''Materials and methods''), which corresponds to approximately 2 lg of active b-galactosidase from a commercial source (Sigma). The S30 extract used for IVTT was derived from E. coli BL21 Tuner TM strain, which is devoid of endogenous b-galactosidase. This is important to avoid interference of endogenous b-galactosidase in the experiments (Amidi et al. 2010 ). SDS-PAGE and Western blot analysis of S30 extract made from E-coli Tuner TM strain proved absence of endogenous b-galactosidase in S30 extract (Fig. 2) . Importantly, Western blot analysis confirmed that the expressed b-galactosidase was recognized by polyvalent anti-bgalactosidase antibodies and the antigenicity of b-galactosidase was preserved in IVTT mix (Fig. 2) . Systemic antibody responses after i.m. immunization in mice