title: A dimethylmaleic acid-melittin-polylysine conjugate with reduced toxicity, pH-triggered endosomolytic activity and enhanced gene transfer potential.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Pharmaceutical Biotechnology, Department of Pharmacy, Ludwig-Maximilians-Universität, Butenandtstr 5-13, D-81377, Munich, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Meyer
authors.forename: Arkadi
authors.initials: A
authors.lastname: Zintchenko
authors.forename: Manfred
authors.initials: M
authors.lastname: Ogris
authors.forename: Ernst
authors.initials: E
authors.lastname: Wagner
mesh_terms: D000818:Animals; D016923:Cell Death / Q000187:drug effects; D004247:DNA / Q000378:metabolism; D011992:Endosomes / Q000187:drug effects*; D018014:Gene Transfer Techniques; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D018942:Macrolides / Q000494:pharmacology; D008299:Maleic Anhydrides / Q000737:chemistry / Q000633:toxicity*; D008555:Melitten / Q000737:chemistry / Q000633:toxicity*; D051379:Mice; D010316:Particle Size; D010455:Peptides / Q000737:chemistry; D011107:Polylysine / Q000737:chemistry / Q000633:toxicity*
chemical_list: D018942:Macrolides; D008299:Maleic Anhydrides; D010455:Peptides; D008555:Melitten; D011107:Polylysine; C007474:2,3-dimethylmaleic anhydride; C040929:bafilomycin A1; D004247:DNA; D007770:L-Lactate Dehydrogenase; D008156:Luciferases