12-Crown-4-based amphipathic lipid and corresponding metal cation complexes for gene therapy applications: FT-IR characterization and surface charge determination P. Bruni a, * , V. Fino a , M. Pisani a , G. Tosi a , P. Stipa a , P. Ferraris a , O. Francescangeli b a Dipartimento di Chimica e Tecnologie Chimiche. Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy b Dipartimento di Fisica e Ingegneria dei Materiali e del Territorio, Università Politecnica delle Marche, Via Brecce Bianche, 60131 Ancona, Italy article info Article history: Received 31 July 2008 Received in revised form 24 September 2008 Accepted 29 September 2008 Available online 1 November 2008 Keywords: Liposomes Gene therapy Cation coordination FT-IR spectroscopy Z potential DFT calculations abstract The new lipid 1,2-O-dioleyl-3-O-{2-[(12-crown-4)-methoxy]-ethyl}-sn-glycerol, 12C4L, has been synthe- sized. This molecule can coordinate different cations that should make the corresponding liposome a good candidate as vector of genetic material for possible applications in gene therapy. An important fea- ture of the molecule is the possibility to modulate the net surface charge of their complexes with metal cations, which is important to provide efficient DNA transfections. The molecule and its complexes with some metal cations (Mg 2+ , Ca 2+ , Mn 2+ ) have been characterized by FT-IR spectroscopy and band attribu- tions confirmed by Density Functional Theory calculations. The net surface charge has been determined by Z potential determinations. Ó 2008 Elsevier B.V. All rights reserved. 1. Introduction The design and synthesis of vectors for efficient and safe gene therapy applications is of great interest today [1,2] and cationic liposomes are the most investigated [3–7]. They consists on a hydrophilic head and a hydrophobic tail, hold together by an appropriate linker: once dispersed in water, they self-assemble with nucleic acid, giving rise to complexes (lipoplexes): the net positive charge on the surface of lipoplexes allows adhesion on the cell membrane and an efficient transfection in vitro. Never- theless, in the in vivo transfection, the introduction of such rela- tively large objects, bearing a strong positive charge, causes a dramatic perturbation of the ionic equilibrium of the cells that can compromise their correct functionality, inactivating ubiqui- tous structures [8]. Recently [9–11] we have described a new class of supramolec- ular complexes, obtained from DNA and neutral (zwitterionic) lip- ids (see Fig. 1a, for a typical structure) in the presence of divalent metal cations: the ternary complexes obtained show structures similar to the ones of lipoplexes, but have the advantage of the ab- sence of toxicity, as they are formulated with phospholipids that naturally occur in the cell membrane. Unfortunately in vitro trans- fection experiments [12], resulted in a low efficiency. It has been found that the efficiency of cationic liposomes as DNA vectors is re- lated to the entity of the net charge on the resulting complexes [13]: if it is easy to control the net charge on lipoplexes using dif- ferent ratios of cationic and neutral liposomes [14], no possibility does exist to control the amount of metal salts on the complexes of neutral liposomes with DNA and, by consequence, their net charge. In order to overcome this limit, our strategy has been to introduce a crown ether as head of an amphipathic lipid, taking advantage from its ability to coordinate metal cations [15,16] and from the finding that a strong interaction between a polyeth- yleneglycol functionalized dioleylphosphatydilethanolamine (DOPE–PEG) and metal di-cations have been demonstrated [17]. Mg 2+ and Ca 2+ , which are typical biological cations, and the transi- tion element Mn 2+ , which has a week toxicity at the low concentra- tion used, have been used in this work: in all cases it should be easy to modulate the net charge of the complexes with DNA, by differentiating the molar ratios between metal cations and crown unities. In this light we started synthesizing the new neutral lipid 1,2-O-dioleyl-3-O-{2-[(12-crown-4)-methoxy]-ethyl}-sn-glycerol 12C4L (Fig. 1b), assuming that the crown moiety can assure a suit- able interaction with metal cations, which must be strong enough to warrant the requested stability of the complex liposome–DNA before the target cells are reached; but not so strong to prevent the complex from loosing the cation and then from releasing the 0022-2860/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.molstruc.2008.09.030 * Corresponding author. Tel.:+39 071 2204721; fax: +39 071 2204714. E-mail address: p.bruni@univpm.it (P. Bruni). Journal of Molecular Structure 919 (2009) 328–333 Contents lists available at ScienceDirect Journal of Molecular Structure journal homepage: www.elsevier.com/locate/molstruc