Colloids and Surfaces B: Biointerfaces 68 (2009) 136–144 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb Comparison of different commercially available cationic liposome–DNA lipoplexes: Parameters influencing toxicity and transfection efficiency A. Masotti d,∗,1 , G. Mossa a , C. Cametti b,c , G. Ortaggi d , A. Bianco d , N. Del Grosso a , D. Malizia d , C. Esposito a,1 a Istituto di Neurobiologia e Medicina Molecolare del CNR, Via del Fosso del Cavaliere n. 100, I-00133 Rome, Italy b Dipartimento di Fisica, Universita’ di Roma “La Sapienza”, Piazzale A. Moro n. 5, I-00185 Rome, Italy c INFM CRS-SOFT, Unita’ di Roma1, Italy d Dipartimento di Chimica, Universita’ di Roma “La Sapienza”, Piazzale A. Moro n. 5, I-00185 Rome, Italy article info Article history: Received 16 September 2008 Accepted 17 September 2008 Available online 25 September 2008 Keywords: Cationic liposomes DNA lipoplexes Toxicity Serum effect Transfection C6 abstract Lipid–DNA complexes (lipoplexes) are widely used, since several years, as gene carriers. However, their transfection efficiency, both in vitro and in vivo, depends, in a rather complex way, on different intercon- nected parameters, ranging from the chemical composition of the lipid components to the size and size distribution of the complexes and, moreover, to the composition of the suspending medium. In this paper, we have investigated the behavior of nine different commercially available transfection agents (liposomal and non-liposomal) and their lipoplexes, at different molar charge ratios and in different experimen- tal conditions. The size and the time stability of the resulting lipoplexes were investigated by means of dynamic light scattering methods and their toxicity and transfection efficiency were assayed in vitro in a model tumor cell line (C6 rat glioma cell line). An attempt to correlate the different parameters governing the complex phenomenology observed has been made. Whereas all the formulations investigated display a low toxicity, that increases with the increase of the lipid–DNA molar charge ratio, the transfection effi- ciency markedly depends, besides the molar charge ratio, on the lipid composition and on the lipoplex size, in a rather correlated way. The aim of this work is to present, in a wide scenario, an example of the inter-correlation among the different parameters that influence the transfection efficiency of lipoplexes and to suggest the role exerted by the average size of the resulting aggregates in their overall effectiveness as carriers in gene therapy. © 2008 Elsevier B.V. All rights reserved. 1. Introduction In the last few years, cationic liposome–DNA complexes (lipoplexes) have been extensively investigated and widely used in gene therapy to deliver DNA into mammalian cells, owing to their potential advantages over viral vectors, such as their safety, versa- tility and low immunogenicity [1–4]. However, despite the recent clinical successes, the basic knowledge of the structure–activity relationship is still unsatisfactory and the optimization of these systems remains largely a result of trial and error. In order to better understand the role of the different param- eters in improving the clinical performances of these systems, a systematic in vitro study of the physico-chemical and biologi- cal properties of both liposomes and lipoplexes is crucial. These ∗ Corresponding author. Tel.: +39 06 49913341; fax: +39 06 490631. E-mail address: andrea.masotti@uniroma1.it (A. Masotti). 1 These two authors contributed equally to this work. systematic investigations are demanded by the very intricate phe- nomenology these systems undergo. In fact, it is well known that the kinetic of lipoplex formation is essentially a two-step process. First, a fast growing regime, where the typical size of the aggre- gate (of the order of 1 m) reaches an essentially steady-state value within a few minutes following the mixing of liposome suspension to the DNA solution [5]. Second, a slower growing regime, where a further increase of the aggregates occurs, leading to even larger structures (up to 2–5 m) which are completed within some hours. These aggregating processes, which ultimately govern the trans- fection efficiency, are influenced by a variety of physico-chemical parameters, such as the relative concentration of liposomes and DNA (molar charge ratio), the ionic strength and temperature of the suspending medium, the order of addition of liposome to DNA solution and “viceversa” and, finally, the mixing rate of the two components. Among these, the composition of suspending medium (presence or absence of serum for subsequent assessment of toxi- city and transfection efficiency) markedly affects the size and size distribution of the resulting complexes. 0927-7765/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2008.09.017