Systemically Administered Melittin-Driven Lipid Nanoparticle And Its Anti-Melanoma Effect

Melittin, as the major component of bee venom, is a kind of amphiphilic peptide with a-helix structure. Melittin has been applied broadly in the treatment of various diseases, it has anti-inflammatory, analgesic, anti-bacterial, anti-viral, anti-radiation and anti-hypertensive effect and so on. In addition, melittin has an unique advantage in cancer treatment, it is able to make pores on the plasma membrane and induce cell lysis, thus avoiding tumor drug resistance. However, due to the disadvantage of its hemolytic effect, non-specific cellular lytic activity and rapid degradation in blood, seriously impeded the application of melittin in clinical cancer therapy. Therefore, it is still a big challenge on how to safely and effectively deliver melittin to the tumor issue without any toxicity.In this research, we established a method to deliver melittin based on HDL-mimicking peptide phospholipid scaffold, efficiently solved the hemolytic problem of melittin in cancer therapy, increased its cycle half-life and made melittin specifically released in tumor region, achieved the following results:(1) Creating a novel melittin-driven functional hybrid peptide, named a-melittin, in which the melittin peptide is conjugated to the C-terminus of an apoA-I mimicking peptide with a-helix structure (a-peptide). a-Melittin has stronger affinity for phospholipid and ability of forming a-helix structure while retaining the properties of melittin (spontaneously forms multimers and lyses cells). Compared with a-peptide, a-melittin has stronger size-control ability, it can control the size of nanoparticles made up with phospholipids to around10nm and even smaller, meanwhile with a high encapsulation efficiency (>80%).(2) The a-melittin-based lipid nanoparticles (a-melittin-NPs) synthesized with a-melittin, realized safe and efficient delivery of melittin in vivo. The nanoparticles have good dispersion with14nm size, and are able to load hydrophobic molecules in the core. a-Melittin-NPs are very stable, can keep nano-structure unchanged in physiological solution such as serum and plasma etc. Depend on the design of melittin connected to C-terminal of a-peptide and strong interaction between a-melittin and phospholipid layer, a-melittin-NPs effectively shield the positive charge of melittin (21.33±1.64mV), display a kind of natural nanoparticles (2.45±0.56mV). a-Melittin-NPs obviously decreased the hemolytic and non-specific effect of melittin. In vitro hemolysis results showed the erythrocytes lysis effect of a-melittin-NPs significantly decreased, they only induced about8%hemolysis even with a high peptide concentration of50μM. The cell proliferation results also showed the IC50value of a-melittin-NPs (11.26±.37μM) significantly increased while compared to melittin (1.71±0.04μM). By using the continuously fluorescent confocal imaging, we found the permeability of cell membrane changed, thus cell contents were leaked out, nuclears broke down and at last cells were lysed after a-melittin-NPs interacted with melanoma cells. In addition, the results of annexin V/PI staining and flow cytometric analysis showed a-melittin-NPs can mainly induce cells death by necrosis, also induced apoptosis in a few of cells. Finally, a-melittin-NPs were intravenously injected into mice bearing melanoma tumors. After4times therapy, the growth of melanoma was significantly blocked, the inhibition rate was82.8%compared with control group. Meanwhile, no side effects of treatment were found through the hemanalysis and biochemical analyses of blood samples, and through histopathologic analyses of major tissues from tumor-bearing mice.(3) Developed ultrasmall simultaneously carrying paclitaxel and melittin nanoparticles ((PTX-OL)a-melittin-NPs), they have synergistic anti-melanoma effect. We found a-melittin-NPs released melittin to cell membrane system including plasma membrane, and the hydrophobic dye molecules in the core were directly released into the cytoplasm when a-melittin-NPs interacted with tumor cells by using fluorescent confocal microscopy. Therefore,(PTX-OL)a-melittin-NPs are able to exert the anti-tumor effect of paclitaxel and melittin with two different therapeutic mechanisms. The cell proliferation results showed (PTX-OL)a-melittin-NPs have stronger ability to lyse tumor cells, with a44.8%IC50value of a-melittin-NPs. In addition, the results of annexin V/PI staining and flow cytometric analysis showed (PTX-OL)a-melittin-NPs can induce10-20%more cells death by necrosis compared with a-melittin-NPs. Moreover,(PTX-OL)a-melittin- NPs also showed enhanced inhibition effect on tumor growth in melanoma therapy in vivo.In summary, in this research we developed a kind of melittin-driven nanocarrier based on HDL-mimicking peptide phospholipid scaffold, by shielding melittin in the phospholipid monolayer. Efficiently solved the problems of melittin during transport, such as hemolysis and nonspecific activity, increased the cycle half-life. Providing a effective tool for melittin applied in cancer therapy in vivo. The method of simultaneously delivering melittin and chemotherapeutic drugs will breakthrough the limitation of single target on cancer therapy, bring new opportunities for multi-target cancer therapy.