Structural Modifications to Convert Melittin from a Cytolytic Peptide to a Stable Cargo Linker

Abstract

Melittin is a 26 amino acid peptide that comprises more than half of the dry weight of the venom of the honeybee Apis Mellifera. In previous studies, we demonstrated that melittin stably bound to the lipid membrane of perfluorocarbon (PFC) nanoparticles and served as an active anti-cancer therapeutic agent in vivo (Soman et al. J Clin Invest. 2009). Here, we report the structure modification that converts the mellittin to a cargo linker for post-formulation liposome customization. We introduced point mutations and truncations to define the lytic and membrane binding activities of melittin. For each of the mutations, lytic activity of the peptides was tested by the carboxyfluorescein fluorescence dequenching assay on carboxyfluorescein encapsulated liposomes. Among all six melittin mutations, the mutation, D1-7, with the first 7 amino acids removed (VLTTGLPALISWIKRKRQQ) dramatically reduces the melittin lytic activity. Using immobilized Giant Unilamellar Vesicles (GUV), we illustrated that at peptide:lipid ratio of 40:1, D1-7 bound to the GUV without pore forming, while at peptide:lipid ratio of 1:83, melittin already formed pores on the GUV membrane. These results suggest that D1-7 eliminates the lytic activity but retains the strong lipid membrane binding. For further confirmation, we measured liposome sizes and zeta potentials with and without D1-7 loading. Consistently, D1-7 did not affect the size of the liposome, but shifted the zeta potential (or surface charge) of the liposome towards the positive voltage range, because D1-7 is a positively charged peptide. Among numerous existing nanosystems for drug delivery, liposomes are approved by FDA for anti-cancer and gene therapy. Accordingly, this linker and/or its refinements could enhance the therapeutic potential of approved liposomal drugs by enabling flexible incorporation and cargo multiplexing through post-formulation surface editing.