Numerous techniques have been developed to prepare immunoliposomes based on the nucleophilic reactivity of free amine groups of proteins or peptides. One of the most popular and commonly used methods is to covalently couple free carboxylic groups to primary amines through activation of the carboxyl groups with EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide). EDC, which is a so-called zero-length crosslinking agent, reacts with the carboxyl to form an amine reactive intermediate (O-acylisourea). The produced O-acylisourea can be easily displaced by nucleophilic attack from the primary amino groups in the reaction mixture. However, this intermediate is unstable and hydrolyzed in aqueous solutions. In order to prevent the intermediate hydrolysis, sulfo-NHS (N-hydroxysulfosuccinimide) is added to EDC to produce a significantly more stable and more soluble active intermediate (NHS ester).

Consequently, the immunoliposomes are prepared by a two-step coupling procedure: first, activating the free carboxyl group of the linker lipid incorporated in the liposomes with EDC and sulfo-NHS, and then covalently conjugating the antibodies to the lipids through displacement of sulfo-NHS groups by antibody amines, as depicted below. EDC/sulfo-NHS coupling reactions are highly selective and highly efficient, and the biological activity of the protein or peptide is preserved.

ImmunoFluor™-Succinyl is a PEGylated product. For other amine reactive (PEGylated and non-PEGyalated products) and also ImmunoFluor™ products suitable for other types conjugation methods see here.