Published: February 21, 2011 r2011 American Chemical Society 497 dx.doi.org/10.1021/bc100537r | Bioconjugate Chem. 2011, 22, 497–509 ARTICLE pubs.acs.org/bc Cationic Amphiphiles with Fatty Acyl Chain Asymmetry of Coconut Oil Deliver Genes Selectively to Mouse Lung Voshavar Chandrashekhar, † Marepally Srujan, † Rairala Prabhakar, † Rakesh C. Reddy, † Bojja Sreedhar, ‡ Kiran K. R. Rentam, § Sanjit Kanjilal, † and Arabinda Chaudhuri* ,† † Division of Lipid Science and Technology, ‡ Inorganic and Physical Chemistry Division and § Pharmacology Division, Indian Institute of Chemical Technology, Hyderabad 500 007, India b S Supporting Information ’ INTRODUCTION Clinical success of gene therapy continues to remain critically dependent on the use of safe and efficient gene transfer reagents. Gene delivery reagents, popularly known as transfection vectors, are broadly classified into two types: viral and nonviral. Viral vectors are, in general, efficient in delivering genes into body cells. However, viral vectors suffer from numerous biosafety- related disadvantages including adverse inflammatory and im- munogenic responses, insertional mutagenesis through random integration into the host genome, and so forth. 1 On the other hand, because of their superior biosafety profiles, nonviral vectors such as cationic liposomes, 2-7 cationic polymers, 8 dendri- mers, 9,10 and so forth hold therapeutic promise. Among these arsenals of nonviral gene transfer reagents, the distinguishing features of cationic liposomes include their less immunogenic nature, robust manufacture, ability to deliver large pieces of DNA, and ease of handling and preparation techniques. 11,12 A number of structure-activity studies have demonstrated in the past that the gene delivery efficiencies of cationic amphiphiles crucially depend upon molecular architectures of the hydrophobic alkyl chain lengths 13-15 and the nature of polar headgroups, 16-19 as well as on the nature of linker and spacer functionalities used in covalent tethering of the polar headgroups and the nonpolar tails of cationic amphiphiles. 20,21 In 2006, in a thought-provoking structure-activity study, Koynova et al. demonstrated dramati- cally superior (about 50-fold) in vitro gene transfer efficiency of a synthetic cationic amphiphile with asymmetric hydrocarbon chains, namely, oleoyldecanoyl-ethylphosphatidylcholine (C18:1/ C10-EPC) to that of its structurally very similar saturated asym- metric counterpart stearoyldecanoyl-ethylphosphatidylcholine (C18:0/C10-EPC). 22 In this report, it was demonstrated that liposomal compositions of C18:1/C10-EPC containing two de- fined asymmetric hydrocarbon chains (oleoyl and decanoyl) formed a pronounced nonlamellar phase which, in turn, played a dominant role in imparting high membrane fusogenicity and thereby enhanced transfection properties to C18:1/C10-EPC. 22 More recently, structure-activity studies by Koynova et al. 23 and Nantz et al. 24 have convincingly demonstrated the dramatic influe- nce of hydrophobic domain asymmetry in modulating the gene transfer efficacies of synthetic cationic amphiphiles. These impor- tant recent structure-activity findings prompted us to design cationic amphiphiles possessing natural fatty acyl chain asymmetry of food-grade coconut oil. We reasoned that, if the mixture of fatty Received: December 1, 2010 Revised: January 21, 2011 ABSTRACT: Recent structure-activity studies have revealed a dramatic influence of hydrophobic chain asymmetry in enhan- cing gene delivery efficacies of synthetic cationic amphiphiles (Nantz, M. H. et al. Mol. Pharmaceutics 2010, 7, 786-794; Koynova, R. et al. Mol. Pharmaceutics 2009, 6, 951-958). The present findings demonstrate for the first time that such a transfection enhancing influence of asymmetric hydrocarbon chains observed in pure synthetic cationic amphiphiles also works for cationic amphiphiles designed with natural, asym- metric fatty acyl chains of a food-grade oil. Herein, we demon- strate that cationic amphiphiles designed with the natural fatty acyl chain asymmetry of food-grade coconut oil are less cytotoxic and deliver genes selectively to mouse lung. Despite lauroyl chains being the major fatty acyl chains of coconut oil, both the in vitro and In vivo gene transfer efficiencies of such cationic amphiphiles were found to be remarkably superior (>4-fold) to those of their pure dilauroyl analogue. Mechanistic studies involving the technique of fluorescence resonance energy transfer (FRET) revealed higher biomembrane fusibility of the cationic liposomes of the coconut amphiphiles than that of the symmetric dilauroyl analogue. AFM study revealed pronounced fusogenic nonlamellar structures of the liposomes of coconut amphiphiles. Findings in the FRET and cellular uptake study, taken together, support the notion that the higher cellular uptake resulting from the more fusogenic nature of the liposomes of coconut amphiphiles 1 are likely to play a dominant role in making the coconut amphiphiles transfection competent.