Design and Characterization of a Stealth Immunoliposome Encapsulating a Large Molecule Biotherapeutic (Antibody)

This thesis project will endeavor to solve the obstacles around synthesis of Immunoliposomes loaded with an antibody therapeutic. Most steps in the preparation of liposomes in most of the methods include harsh conditions that are not amenable for antibody therapeutic entrapment. These methods utilize high concentrations of organic solvents (chloroform and/or methanol) which, if in contact with an antibody at high concentrations would not provide an optimal environment for the antibodies’ tertiary structure. Additionally, preparation methods usually employ physical processes such as high-speed centrifugation, sonication, milling and emulsification in an attempt to dissolve lipid compositions in organic solvents, all of which if not optimized could render the biotherapeutic inactive. This study primarily focuses on preparation of liposomes using the Dry Film Hydration method. Multiple lipids were mixed together (ratio amounts based on the molecular weights) in absolute ethanol to make a homogeneous mixture. The mixture was first evaluated on the Nanoassemblr platform (Precision Nanosystems), which enabled small scale controlled, bottom-up, and assembly of liposomes. The mixing to get a homogenous preparation is done on cartridges that allow millisecond mixing of nanoparticle components at the nanolitre scale. This technology is user friendly and allows a quick method to evaluate different lipid compositions, different flow ratios and rates while characterizing liposome size and size distribution in order to determine the best lipid mixture/composition yielding liposomes within the range of requirement for your purposes. Once the Lipid compositions were selected, larger preparations of about 5-20mL were prepared and a thin dry film/cake was accomplished using the rotary evaporation system in vacuum. The film allowed to dry in vacuum after which it was hydrated with an antibody biotherapeutic dissolved in an aqueous medium. Further sizing of liposomes was accomplished via bath sonication and filtration of the liposomes to remove aggregated or liposome debris was done using Tangential Flow Filtration (TFF), which filters liposomes based on a cutoff membrane at a specific KiloDalton (KDa). Using Dynamic Light Scattering technology (DLS), it was realized that liposomes of distinct sizes could be synthesized ranging from 200nm – 1000nm. Sizing by sonication on the water bath and then filtration via TFF especially improved the quality of the preps ensuring a more monodispersed population of liposomes. Sonication yielded uniformly monodispersed liposomes up to a certain point upon which prolonged sonication affected the dispersity and stability of the ensuing liposomes. To prepare the targeting mAb for conjugation on the surface of liposomes in order to make Immunoliposomes, a monoclonal antibody was prepared by digesting it to make F(Ab)’2 using a commercially available kit. The targeting mAb were digested and thiolated to reduce and expose disulfide bonds, which were later, used to conjugate onto liposomes bearing chemically modified lipids. To measure biotherapeutic encapsulation efficiency, liposomes were treated with TritonX-100 to rupture them followed by an ELISA method to detect and quantify amounts of biotherapeutic after TritonX-100 treatment. Conjugation of targeting mAbs was also characterized using an ELISA method for detection of the targeting antibody in modified liposomes incubated with the thiolated mAb.