Melittin analogs with high lytic activity at endosomal pH enhance transfection with purified targeted PEI polyplexes Sabine Boeckle, Julia Fahrmeir, Wolfgang Roedl, Manfred Ogris, Ernst Wagner ⁎ Pharmaceutical Biology-Biotechnology, Department of Pharmacy, Ludwig-Maximilians-Universität, Butenandtstr. 5-13, D-81377 Munich, Germany Received 28 October 2005; accepted 6 February 2006 Available online 20 March 2006 Abstract Melittin–polyethylenimine (PEI) conjugates have been shown to enhance gene transfer efficiency of polyplexes due to their membrane- destabilizing properties. Inherent lytic activity at neutral pH however also provokes high cytotoxicity due to plasma membrane damage. In order to shift the lytic activity towards the endosomal membrane, several melittin analogs were designed. Acidic modification of melittin by replacing neutral glutamines (Gln-25 and Gln-26) with glutamic acid residues greatly improved the lytic activity of C-terminally linked PEI conjugates at the endosomal pH of 5. This activity correlated well with the gene transfer efficiency of polyplexes in four different cell lines. Melittin–PEI conjugates with high lytic activities at endosomal pH were then incorporated into EGF receptor-targeted and polyethylene glycol-shielded polyplexes. The resulting particles had virus-like dimension (150 nm) with a neutral surface charge and were subsequently purified by size exclusion chromatography to remove unbound toxic PEI conjugate. These purified polyplexes mediated EGF-receptor-specific gene transfer with up to 70-fold higher activity compared to the corresponding PEI polyplexes without melittin. © 2006 Elsevier B.V. All rights reserved. Keywords: Endosomal release; Melittin; Nonviral vectors; PEI; Polyplex 1. Introduction Nonviral vectors based on polyethylenimine (PEI) have reached high transfection efficiencies in vitro and also in vivo (for review, see [1,2]). The amino groups of PEI allow easy chemical modification, and bioactive moieties such as cell targeting ligands or compounds promoting intracellular delivery can be coupled to PEI. Chemical modifications of PEI poly- plexes with shielding agents such as polyethylene glycol (PEG) and/or targeting ligands allow the generation of polyplexes that mediate specific delivery of the transgene towards the desired tissue. For example, systemic targeting of tumors was recently demonstrated with ligand–PEI polyplexes targeted towards the transferrin receptor [3] or the epidermal growth factor (EGF) receptor [4]. Although these data demonstrate that specific delivery of shielded ligand-containing PEI polyplexes in vivo is feasible, such polyplexes like most other non-viral vectors still lack sufficient transfection efficiency. Beside nuclear entry, endosomal release is the major barrier limiting the gene transfer efficiency. To overcome this bottleneck, inactivated viruses [5], toxins [6], synthetic viral [7] or other membrane active peptides [8] have been applied. Such endosomolytic components are supposed to destabilize the endosomal membrane after cellular uptake of the vector particles. The membrane active peptide melittin was of particular interest in our efforts to improve PEI polyplexes, since melittin covalently attached to PEI has already been shown to successfully enhance gene transfer [9–11]. Recently we demonstrated that covalent binding of PEI to the C-terminus of melittin results in a highly potent conjugate (C- mel–PEI) which displayed a higher membrane lytic activity than free melittin [11]. We proposed that membrane destabilization by C-mel–PEI proceeds through insertion of the free N-terminus of melittin into the lipid bilayer. However, the high lytic activity of C-mel–PEI focused at pH 7, and the corresponding polyplexes induced high toxicity including cell lysis. In previous experiments polyplex-mediated gene transfer correlated with the lytic activity of membrane active peptides at endosomal pH 5 [12,13]. Therefore the aim of the current study was to shift the lytic activity of C-terminally linked melittin–PEI conjugates towards lower pH. To this end, the sequence of melittin was Journal of Controlled Release 112 (2006) 240 – 248 www.elsevier.com/locate/jconrel ⁎ Corresponding author. Tel.: +49 89 2180 77840; fax: +49 89 2180 77791. E-mail address: ernst.wagner@cup.uni-muenchen.de (E. Wagner). GENE DELIVERY 0168-3659/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2006.02.002