melitten and 2-3-dimethylmaleic-anhydride

Melittin (MLT), as a natural active biomolecule, can penetrate the tumor cell membrane to play a role in cancer treatment and will attract more attention in future development of antitumor drugs. The main component of natural bee venom MLT was modified by introducing a pH-sensitive amide bond between the 2,3-dimethyl maleimide (DMMA) and the lysine (Lys) of MLT (MLT-DMMA). MLT and its corresponding modified peptide MLT-DMMA were used for antitumor and biocompatibility validation. The biomaterial characteristics were tested by MALDI-TOF MS,

Topics: Acids; Animals; Antineoplastic Agents; Biocompatible Materials; Cell Survival; Chromatography, High Pressure Liquid; Drug Delivery Systems; HeLa Cells; Hemolysis; Humans; Hydrogen-Ion Concentration; Larva; Maleic Anhydrides; Melitten; Neoplasms; Proton Magnetic Resonance Spectroscopy; Reproducibility of Results; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Zebrafish

Poor endosomal release is one major barrier of gene delivery. Endosomolytic polyethylenimine-melittin conjugates have shown to enhance gene transfer efficiency; however, cytotoxicity due to their general membrane-destabilizing properties limits their application. To overcome this drawback we grafted a polycation with a masked pH-responsive melittin derivate and investigated lytic activity, gene transfer efficiency and cytotoxicity of the resulting conjugate.. Melittin (Mel) was modified with dimethylmaleic anhydride (DMMAn) and covalently coupled to poly-L-lysine (PLL). The membrane lytic activity was analyzed after incubation at neutral or endosomal pH. PLL-DMMAn-Mel polyplexes were generated in HEPES-buffered glucose and tested in transfection experiments using luciferase as reporter gene. Cellular cytotoxicity was analyzed by measurement of membrane integrity and metabolic activity.. Covalent attachment of DMMAn-modified melittin to PLL resulted in a pH-responsive conjugate. No lytic activity was observed at neutral pH; after acidic cleavage of the protecting groups at pH 5 lytic activity was regained. Acute toxicity was greatly reduced (as compared to PLL-Mel or even unmodified PLL) and high gene expression levels (up to 1800-fold higher than unmodified PLL) were obtained.. Modification of the polycationic carrier PLL with DMMAn-masked melittin not only enhances gene transfer efficiency, but also strongly reduces the acute toxicity of melittin and PLL. Hence this modification might be useful for optimizing polycationic gene carriers.

Topics: Animals; Cell Death; DNA; Endosomes; Gene Transfer Techniques; Humans; Hydrogen-Ion Concentration; L-Lactate Dehydrogenase; Luciferases; Macrolides; Maleic Anhydrides; Melitten; Mice; Particle Size; Peptides; Polylysine