Genuine DNA/polyethylenimine (PEI) Complexes Improve Transfection Properties and Cell Survival PATRICK ERBACHER a , THIERRY BETTINGER b , EMMANUEL BRION c , JEAN-LUC COLL d , CHRISTIAN PLANK e , JEAN-PAUL BEHR c and JEAN-SERGE REMY c, * a Polyplus Transfection, Faculty of Pharmacy, B.P. 24, F-67401Illkirch, France; b Bracco Research, Department of formulation. CH-1228 Plan-les-Ouates, Geneva, Switzerland; c Genetic Chemistry, Faculty of Pharmacy, UMR 7514 CNRS/Universite ´ Louis Pasteur, F-67401, Illkirch, France; d Groupe de recherche sur le cancerdu poumon (GRCP), Equipe mixte INSERM 9924, Institut Albert Bonniot, F-38706 La Tronche, France; e Institute of Experimental Oncology, TU Mu ¨nchen, Munich, Germany Polyethylenimine (PEI) has been described as one of the most efficient cationic polymers for in vitro gene delivery. Systemic delivery of PEI/DNA polyplexes leads to a lung-expression tropism. Selective in vivo gene transfer would require targeting and stealth particles. Here, we describe two strategies for chemically coupling polyethylene glycol (PEG) to PEI, to form protected ligand-bearing particles. Pre-grafted PEG–PEI polymers lost their DNA condensing property, hence their poor performances. Coupling PEG to pre-formed PEI/DNA particles led to the expected physical properties. However, low transfection efficacies raised the question of the fate of excess free polymer in solution. We have developed a straightforward a purification assay, which uses centrifugation-based ultrafiltration. Crude polyplexes were purified, with up to 60% of the initial PEI dose being removed. The resulting purified and unshielded PEI/DNA polyplexes are more efficient for transfection and less toxic to cells in culture than the crude ones. Moreover, the in vivo toxicity of the polyplexes was greatly reduced, without affecting their efficacy. Keywords: Transfection; Non-viral vector; Polyethylenimine; PEG; Gene delivery INTRODUCTION The cationic polymer polyethylenimine (PEI) has been described as one of the most potent non-viral gene delivery vectors in an increasing number of in vitro, as well as in vivo studies (Abdallah et al., 1995; Boussif et al., 1995; Goula et al., 1998a; Coll et al., 1999; Aoki et al., 2001). PEI is used for systemic delivery (Goula et al., 1998a; Bragonzi et al., 2000; Zou et al., 2000), and found that, linear PEI (L-PEI) was much more potent than branched PEI (B-PEI). However, ca. 95% of transgene expression was found in the lungs (Goula et al., 1998a; Bragonzi et al., 2000). The mechanism by which this “non-specific targeting” occurs is still under investigation (Goula et al., 2000; Chollet et al., 2001). As intravenous injection should be a route of choice for delivering genes in many therapeutic applications, several attempts, including ours, have been made to target in vivo ligand-derivatized PEI/DNA polyplexes to several organs or cell types (Erbacher et al., 1999b; Li et al., 2000). Although we were able to show that ligand- modified PEI/DNA polyplexes transfect the corresponding receptor-expressing cells in vitro, with efficacies much higher than those of native polymers (Kircheis et al., 1997; Zanta et al., 1997; Erbacher et al., 1999b), none of these formulations showed positive results following intra- venous injection. It is generally accepted that polyplexes for i.v. delivery should possess two well-defined physico- chemical properties: (i) a size below 100 nm, in order to facilitate extravasation and diffusion within organs (Goula et al., 1998b); (ii) a neutral/negative surface charge, to prevent RES clearance (Plank et al., 1996; Ogris et al., 1999). This implies to replace electrostatic interactions with the cell surface interactions, and to coat the complexes with a protective hydrophilic shell. Polyethylene glycol (PEG) is the most commonly used polymer for this purpose (Ogris et al., 1999; Finsinger et al., 2000). We previously showed that polyethylene glycol grafting to branched PEI was interfering with proper DNA conden- sation into compact particles (Erbacher et al., 1999a). To circumvent this problem, we describe here a post- grafting strategy, in which PEG or ligand–PEG residues are conjugated to preformed B- or L-PEI/DNA spherical polyplexes. Their physical characteristics (i.e. morphology, size and surface charge) were as anticipated, yet low transfection efficacies raised the question of excess free ligand–PEG–PEI polymer molecules that would compete with the polyplexes for receptor binding. ISSN 1061-186X print/ISSN 1029-2330 online q 2004 Taylor & Francis Ltd DOI: 10.1080/10611860410001723487 *Corresponding author. E-mail: remy@bioorga.u-strasbg.fr Journal of Drug Targeting, May 2004 Vol. 12 (4), pp. 223–236