title: C- versus N-terminally linked melittin-polyethylenimine conjugates: the site of linkage strongly influences activity of DNA polyplexes.
abstract: BACKGROUND
  One major barrier limiting the transfection efficiency of polyplexes is poor endosomal release, especially when small particles are applied. In an approach to overcome this barrier, covalent attachment of the membrane-active peptide all-(L)-melittin to polyethylenimine (PEI) polyplexes was found to enhance gene transfer efficiency.
  
  
  METHODS
  The N-terminus of natural all-(L)- or non-immunogenic all-(D)-melittin was covalently coupled to PEI. In addition, two different all-(D)-melittin conjugates were synthesized, with PEI covalently attached to either the C-terminus (C-mel-PEI) or the N-terminus of melittin (N-mel-PEI). Melittin-PEI polyplexes with particle sizes < 150 nm were generated in HEPES-buffered glucose and tested in transfection experiments. The membrane lytic activities of conjugates and polyplexes were analyzed at neutral and endosomal pH.
  
  
  RESULTS
  All-(D)-melittin conjugates mediated enhanced gene expression similar to the natural all-(L)-stereoisomer, with up to 160-fold higher luciferase activity than unmodified PEI. The site of melittin linkage strongly influenced the membrane-destabilizing activities of both conjugates and polyplexes. C-mel-PEI was highly lytic at neutral pH and therefore elevated doses of C-mel-PEI polyplexes induced high toxicity. In contrast, N-mel-PEI was less lytic at neutral pH but retained higher lytic activity than C-mel-PEI at endosomal pH. This apparently promoted better endosomal release of N-mel-PEI polyplexes resulting in efficient gene delivery in different cell lines.
  
  
  CONCLUSIONS
  The high potency of C-mel-PEI to destabilize membranes at neutral pH is presumably due to a reported destabilization mechanism proceeding through membrane insertion of the peptide. In contrast, N-mel-PEI is supposed to induce lysis by insertion-independent pore formation according to the toroidal pore model.
authors.affiliation: Pharmaceutical Biology-Biotechnology, Department of Pharmacy, Ludwig-Maximilians-Universität, Butenandtstr. 5-13, D-81377 Munich, Germany. sabine.boeckle@web.de
authors.forename: Sabine
authors.initials: S
authors.lastname: Boeckle
authors.forename: Ernst
authors.initials: E
authors.lastname: Wagner
authors.forename: Manfred
authors.initials: M
authors.lastname: Ogris
mesh_terms: D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D004247:DNA / Q000008:administration & dosage*; D004337:Drug Carriers; D011992:Endosomes / Q000737:chemistry; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000187:drug effects / Q000378:metabolism; D008081:Liposomes; D008555:Melitten / Q000009:adverse effects / Q000737:chemistry*; D051379:Mice; D010957:Plasmids; D011094:Polyethyleneimine / Q000009:adverse effects / Q000737:chemistry*; D013237:Stereoisomerism; D013552:Swine
chemical_list: D004337:Drug Carriers; D008081:Liposomes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008555:Melitten; D011094:Polyethyleneimine; D004247:DNA